title: PanCircBase: An online resource for the exploration of circular RNAs in pancreatic islets

abstract: Circular RNAs (circRNAs) are a novel class of covalently closed RNA molecules that recently emerged as a critical regulator of gene expression in development and diseases. Recent research has highlighted the importance of novel circRNAs in the biosynthesis and secretion of insulin from {beta}-cells of pancreatic islets. However, all circRNAs expressed in pancreatic islets or {beta}-cells are not readily available in the database. In this study, we analyzed publicly available RNA-sequencing datasets of the pancreatic islets to catalog all circRNAs expressed in pancreatic islets to construct the PanCircBase (www.pancircbase.net/) database that provides the following resources: (i) pancreatic islet circRNA annotation details (genomic position, host gene, exon information, splice length, sequence, other database IDs, cross-species conservation), (ii) divergent primers for PCR analysis of circRNAs, (iii) siRNAs for silencing of target circRNAs, (iv) miRNAs associated with circRNAs, (v) possible protein-coding circRNAs and their polypeptides. In summary, this is a comprehensive online resource for exploring circRNA expression and its possible function in pancreatic {beta}-cells.


title: A DNA segregation module for synthetic cells

abstract:
The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made towards reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid-liquid phase separation (LLPS). Entropy-driven DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, could represent a strategy towards an entropy-driven DNA segregation module within bottom-up assembled synthetic cells.

Table of Contents O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY An entropy-driven DNA segregation module for bottom-up assembled synthetic cells is realized. It is based on DNA droplets that are engineered to segregate upon enzymatic or photocleavage inside giant unilamellar lipid vesicles (GUVs). The segregation kinetics is altered by the confinement, as confirmed by lattice-based numerical simulations. DNA segregation is further controlled by temperature, ionic strengths and nucleobase sequence.

C_FIG_DISPLAY

category: biophysics


title: The Breast Cancer Epigenomics Track Hub

abstract:
BackgroundPioneering research has shown that high-throughput epigenomics assays such as ChlP-seq and ATAC-seq are applicable to patient-derived breast tumor samples. A host of public data has been accumulated since then, which are potentially of high value for basic research as well as personalized medicine. Such data sets constitute encyclopedias of biological knowledge. However, their impact has so far been limited by access obstacles, especially with regard to extraction and visualization of small portions of data that could potentially answer specific questions arising in a research context.

ResultsWe developed the breast cancer epigenomics track hub (BC hub), a resource intended to make it easy for occasional users to find, access and view data of their interest. The BC hub harbors ChIP-seq, ATAC-seq and copy number data from breast tumors, normal breast cells, patient-derived xenografts and breast cancer cell lines in a genome browsable track format. The tracks can be accessed via hyperlinks that automatically configure customized views for different interest groups. Here, we present a detailed description of the resource and informative use cases illustrating its potential in answering specific biological questions.

ConclusionsWe show that track hubs constitute a powerful way of bringing epigenomics data to the user who could benefit from them. The examples presented highlight the added-value of joint visualization of breast cancer data from different sources. The proof-of-concept provided here exemplifies and underscores the importance of efforts to make biological data FAIR (findable, accessible, interoperable and reusable), and may serve as an encouragement of similar bottom-up initiatives in other research fields. The BC hub is freely accessible at https://bchub.epfl.ch.

category: cancer biology


title: Dynamic tripartite construct of interregional engram circuits underlies forgetting of extinction memory

abstract: Fear extinction allows for adaptive control of learned fear responses but often fails, resulting in a renewal or spontaneous recovery of the extinguished fear, i.e., forgetting of the extinction memory readily occurs. Using an activity-dependent neuronal labeling strategy, we demonstrate that engram neurons for fear extinction memory are dynamically positioned in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC), which constitute an engram construct in the term of directional engram synaptic connectivity from the BLA or vHPC to mPFC, but not that in the opposite direction, for retrieval of extinction memory. Fear renewal or spontaneous recovery switches the extinction engram construct from an accessible to inaccessible state, whereas additional extinction learning or optogenetic induction of long-term potentiation restores the directional engram connectivity and prevents the return of fear. Thus, the plasticity of engram construct underlies forgetting of extinction memory.

category: neuroscience


title: Goal-driven optimization of single-neuron properties in artificial networks reveals regularization role of neural diversity and adaptation

abstract: Individual neurons, and the circuits they collectively form in the brain, have been subject to joint evolutionary pressure to produce system-level functions. Considerable effort has been invested in understanding the impact of single-neuron input-output mechanisms, such as diversity in f-I curves and spike frequency adaptation, on network computations. Yet, how goal-driven requirements at the network level influence single-neuron coding properties remains largely unexplored. Toward addressing this, we systematically investigate single-neuron input-output adaptive mechanisms, optimized in an end-to-end fashion in artificial recurrent neural networks. This is achieved by interconnected Adaptive Recurrent Units (ARU), which perform online control of a novel two-parameter family of activation functions mimicking the diversity of f-I curves found in common neural types in the brain. Our network of ARUs shows much-improved robustness to noise and changes in input statistics. Importantly, we find that ARUs recover precise biological coding strategies such as gain scaling and fractional order differentiation. Using tools from dynamical systems theory, we elucidate the role of these emergent single neuron properties and argue that neural diversity and adaption likely play an active regularization role that enables neural circuits to optimally propagate information across time. In doing so, we discuss how goal-driven optimization approaches, while not biologically plausible themselves, reveal neural mechanisms that are consistent with evolutionary pressures on the brain.


title: Differential activation of NLRP3 inflammasome by  Acinetobacter baumannii  strains

abstract: Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A. baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1{beta} and IL-18, the recruitment of innate immune effectors against A. baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the hosts innate response and activation of the inflammasome during A. baumannii infection. In this study, we compared nine A. baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death pathway in primary macrophages and mice. We found a striking variability in survival outcomes of mice and bacterial dissemination in organs among three ATCC A. baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found a stark contrast in activation of the NLRP3 inflammasome pathway, the non-canonical caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokines IL-1{beta} and IL-18 between A. baumannii strains. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with differential virulence to investigate the innate immune response to A. baumannii infection.

category: microbiology


title: Genomic, transcriptomic and proteomic depiction of iPSC-derived smooth muscle cells as emerging cellular models for arterial diseases

abstract:
BackgroundVascular smooth muscle cells (VSMCs) plasticity is a central mechanism in cardiovascular health and disease. We aimed at providing deep cellular phenotyping, epigenomic and proteomic depiction of SMCs derived from induced pluripotent stem cells (iPSCs) and evaluating their potential as cellular models in the context of complex genetic arterial diseases.

MethodsWe differentiated 3 human iPSC lines using either RepSox (R-SMCs) or PDGF-BB and TGF-{beta} (TP-SMCs), during the second half of a 24-days-long protocol. In addition to cellular assays, we performed RNA-Seq and assay for transposase accessible chromatin (ATAC)-Seq at 6 time-points of differentiation. The extracellular matrix content (matrisome) generated by iPSCs derived SMCs was analyzed using mass spectrometry.

ResultsBoth iPSCs differentiation protocols generated SMCs with positive expression of SMC markers. TP-SMCs exhibited greater capacity of proliferation, migration and lower calcium release in response to contractile stimuli compared to R-SMCs. RNA-Seq data showed that genes involved in the contractile function of arteries were highly expressed in R-SMCs compared to TP-SMCs or primary SMCs. Matrisome analyses supported an overexpression of proteins involved in wound repair in TP-SMCs and a higher secretion of basal membrane constituents by R-SMCs. Open chromatin regions of R-SMCs and TP-SMCs were significantly enriched for variants associated with coronary artery disease and blood pressure, while only TP-SMCs were enriched for variants associated with peripheral artery disease.

ConclusionsOur study portrayed two iPSCs derived SMCs models presenting complementary cellular phenotypes of high relevance to SMC plasticity. In combination with genome-editing tools, our data supports high relevance of the use of these cellular models to the study of complex regulatory mechanisms at genetic risk loci involved in several arterial diseases.

Graphical Abstract

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category: cell biology


title: Genetic architecture of spatially complex color patterning in hybrid Mimulus

abstract: Interspecies hybridization generates inter-genomic interactions, which may result in unique traits not seen in either parent species. Here we explore the genetic basis of two types of floral pigment phenotypes, in hybrids between monkeyflower species Mimulus cupreus and M. luteus var. variegatus. Mimulus cupreus has abundant yellow carotenoid pigmentation in its petal lobes, while M. l. variegatus has a derived reduction in carotenoid intensity. Thus, as expected, carotenoid intensity segregates in an F2 hybrid population. More surprisingly, both species appear to have petal lobes solidly and identically covered in magenta anthocyanin pigment (leading to an orange color in the high-carotenoid species M. cupreus), yet F1 and F2 hybrids exhibit novel and complex spatial patterns of anthocyanin spotting. A rare yellow morph of M. cupreus, which lacks petal anthocyanins, also generates spatially patterned offspring when hybridized with M. l. variegatus. We use this cross, together with newly developed genomic and image analysis tools, to investigate the genetic architecture of color and pattern variation in an F2 hybrid population. We report that the non-patterned carotenoid reduction in M. l. variegatus is genetically simple, and is explained by a single QTL which contains the Beta-carotene hydroxylase-1 (BCH1) gene. HPLC results show that beta-carotene content differs between dark yellow and light yellow petals, which supports a causal role for BCH1. The hybrid-specific anthocyanin patterning phenotypes are more complex, with one QTL of large effect and four detectable QTLs of small effect. These results illustrate how different types of traits may have predictably distinct genetic architectures, and provide candidate genomic regions for investigating the molecular mechanisms of both simple and complex floral color patterning.

category: genetics


title: Unifying genomics and transcriptomics in single cells with ResolveOME amplification chemistry to illuminate oncogenic and drug resistance mechanisms

abstract:
Discovering genomic variation in the absence of information about transcriptional consequence of that variation or, conversely, a transcriptional signature without understanding underlying genomic contributions, hinders understanding of molecular mechanisms of disease. To assess this genomic and transcriptomic coordination, we developed a new chemistry and method, ResolveOME, to extract this information out of the individual cell. The workflow unifies template-switching full-transcript RNA-Seq chemistry and whole genome amplification (WGA), followed by affinity purification of first-strand cDNA and subsequent separation of the RNA/DNA fractions for sequencing library preparation. In the ResolveOME methodology we leverage the attributes of primary template-directed amplification (PTA)1 to enable accurate assessment of single-nucleotide variation as a DNA feature--not achieved with existing workflows to assess DNA + RNA information in the same cell.

We demonstrated the validity of the technique in the context of two major phenomena in oncology: tumor heterogeneity (leading to cancer progression) and treatment resistance. Material from a primary patient breast cancer and an acute myeloid leukemia (AML) cell line, MOLM-13, was used to highlight multiomic biomarker paradigms enabled by this chemistry. Performance of the PTA-enabled genome amplification was largely unaffected by addition of RNA enrichment, with control WGS results showing > 95% genome coverage, precision > 0.99 and allele drop out < 15%. In the RNA fraction of the chemistry, we were able to routinely retrieve full-length transcripts that demonstrate a ratio of 1 for 5/3 bias, with increased coverage of intronic regions and 5 regions that are indicative of novel transcripts, showing strength of the template switching mechanism to capture isoform information with sparsity rates < 75%. We find remarkable cellular variability of revealed biomarkers at both in the genome and transcriptome despite employing a relatively small number of individual cells. In our primary patient sample of ductal carcinoma in situ (DCIS)/invasive ductal carcinoma (IDC) we observed oncogenic PIK3CA driver mutations and prototypical DCIS copy number alterations binned into heterogenous single-cell classes of genomic lesions. Within our quizartinib-treated MOLM-13 cells, we identified multiple potential mechanisms of resistance within seemingly sporadic changes and were able to associate specific mutation, copy number and expression significantly correlated to treatment. In this latter scenario, the DNA arm of our combined workflow uncovered a secondary FLT3 (non-internal tandem duplication (ITD)) mutation as a candidate primary driver of resistance to drug while the RNA arm showed matched transcript upregulation of AXL signal transduction as well as enhancer factor modulation. Importantly, proximal candidate regulatory SNVs, outside of the CDS, were identified and associated to upregulated transcripts in cis. The study highlights that both the genome and transcriptome are dynamic, leading to a set of combinatorial alterations that affect cellular evolution and that fate can be identified through ResolveOME application to individual cells.

category: genomics


title: Activation Of Transposable Elements Upon Statin Treatment

abstract: High cholesterol levels have been associated with cardiovascular diseases, and lowering them has been a key focus in the treatment of such diseases. Statins are drugs used with that aim, and can be divided in the lipophilic Simvastatin and the hydrophilic Rosuvastatin. Regardless of the statin type, a high proportion ([~]70%) of patients stop using statins due to suffering from side effects on skeletal muscle, such as myalgia, and muscle cramps. Thus, there has been a considerable effort in understanding how statins contribute to these side effects. A catalogue of genes and molecular pathways that change upon statin treatment has been recently published, allowing further understanding how the side effects occur. However, Transposable Elements (TEs) were not studied. TEs can move within a genome, and they are highly repetitive, representing about half of the human genome. Currently, most TEs in the human genome are inactive, but it has been shown that TEs can still transcribe, and that either via their transposition or their transcriptional activity, can influence gene expression. Here, using novel computational tools to accurately estimate TE expression, we studied their activity and predicted their potential impact on gene expression. We developed a catalogue of TEs expressed upon statin treatment, and the putative genes whose expression might be influenced by TEs. Overall, we speculate that based on our findings, TEs might be a key target in order to understand statin-mediated side effects.

category: bioinformatics


title: Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

abstract: VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5- carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions provides evidence that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters.


title: Generic solving of physiologically-based kinetic models in support of next generation risk assessment due to chemicals

abstract:
O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/490045v1_ufig1.gif" ALT="Figure 1">
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This paper proposes an innovative and unified modelling framework, in both the writing of PBK equations as matrix ordinary differential equations (ODE), and in its exact solving simply expressed with matrix products. This generic PBK solution allows to consider as many as state-variables as needed to quantify chemical absorption, distribution, metabolism and excretion processes within living organisms when exposed to chemical substances. This generic PBK model makes possible any compartmentalisation of the model to be considered, as well as all appropriate inter-connections between compartments and with the external medium.

We first introduce our PBK modelling framework, with all intermediate steps from the matrix ODE until the exact solution. Then we apply this framework to bioaccumulation testing, before illustrating its concrete use through complementary case studies in terms of species, compound and model complexity. Upon our findings, we finally propose to consider this approach to eventually be part of a future revision of the current regulation.

category: pharmacology and toxicology


title: Synthetic Mechanotransduction Using Engineered SynNotch Receptors

abstract: Cells can sense and interpret mechanical stimuli from their environment, but the ability to engineer customized mechanosensing capabilities has remained a synthetic biology challenge. Here, we introduce a set of synthetic Notch (SynNotch)-derived proteins that can be used to convert extracellular tensile forces into specifiable gene expression changes. By elevating the tension levels needed to induce SynNotch activation, in combination with structure-guided mutagenesis, we designed tunable mechanoreceptors with sensitivities in the physiologically relevant picoNewton (pN) range. Cells expressing these receptors could distinguish between varying levels of ligand-mediated tension and enact customizable transcriptional responses in turn. The utility of these tools was demonstrated by the design of a decision-making circuit, through which fibroblasts could be made to differentiate into myoblasts in response to mechanostimulation with tensile forces of distinct magnitudes. This work provides insight regarding how mechanically-induced structural alterations in proteins can be used to transduce physical forces into biochemical signals, and the system should facilitate further programming of force-related phenomena in biological systems.

category: synthetic biology


title: A role for the anterior hippocampus in autobiographical memory construction regardless of temporal distance

abstract: Mounting evidence suggests distinct functional contributions of the anterior and posterior hippocampus to autobiographical memory retrieval, but how these subregions function under different retrieval demands as memories age is not yet understood. Specifically, autobiographical memory retrieval is not a homogenous process, rather, it is thought to consist of multiple stages: an early stage of memory construction and a later stage of detailed elaboration, which may differently engage the hippocampus over time. In the present study, we analyzed data from 40 participants who constructed and overtly elaborated upon recent and remote memories in response to picture cues in the fMRI scanner. We previously reported a temporal gradient in the posterior hippocampus during the elaboration period of autobiographical retrieval, with posterior hippocampal activation observed for recent but not remote timepoints. Here, we consider the previously unanalyzed construction stage of retrieval, where participants searched for and selected a memory. We found no evidence of a temporal gradient during memory construction, instead observing strong anterior hippocampus activity regardless of memory remoteness. Our findings suggest a unique contribution of the anterior hippocampus to the construction process of autobiographical retrieval over time. These findings highlight that retrieval processes, which have yet to be considered in current models of systems consolidation, offer novel insights to hippocampal subregion function over time.


title: Characterization of Extensive Diversity In Immunoglobulin Light Chain Variable Germline Genes Across Biomedically Important Mouse Strains

abstract: The light chain immunoglobulin genes of biomedically relevant mouse strains are poorly documented in current germline gene databases. We previously showed that IGH loci of wild-derived mouse strains representing the major mouse subspecies contained 247 germline IGHV sequences not curated in the international ImMunoGeneTics (IMGT) information system, which is the most commonly used database that curates the germline repertoires used for sequence alignment in AIRR-seq analysis. Despite containing levels of polymorphism similar to the IGH locus, the germline gene content and diversity of the light chain loci have not been comprehensively cataloged. To explore the extent of germline light chain repertoire diversity across mouse strains commonly used in the biomedical sciences, we performed AIRR-seq analysis and germline gene inference for 18 inbred mouse strains, including the four wild-derived strains with diverse sub-species origins. We inferred 1582 IGKV and 63 IGLV sequences, representing 459 and 22 unique IGKV and IGLV sequences. Of the unique inferred germline IGKV and IGLV sequences, 67.8% and 59%, respectively, were undocumented in IMGT. Across strains we observed germline IGKV sequences shared by three distinct IGK haplotypes and a more conserved IGLV germline repertoire. In addition, J gene inference indicated a novel IGK2 allele shared between PWD/PhJ and MSM/MsJ and a novel IGLJ1 allele for LEWES/EiJ and IGLJ2 allele for MSM/MsJ. Finally, a combined IGHV, IGKV, and IGLV phylogenetic analysis of wild-derived germline repertoires displayed reduced germline diversity for the light chain repertoire compared to the heavy chain repertoire, suggesting potential evolutionary differences between the two chains.

category: immunology


title: Metabarcoding metacommunities: time, space, and land use interact to structure aquatic macroinvertebrate communities in streams

abstract: There is an increasing need to move beyond evaluating the effect of land use on stream communities by only studying local variables, and instead incorporate a metacommunity perspective which integrates environmental and spatial factors across larger spatial scales. The use of molecular tools (DNA metabarcoding) to identify bioindicator groups, such as aquatic macroinvertebrates, can provide greater taxonomic resolution to explore patterns in stream metacommunities. In this study, we collected aquatic macroinvertebrates from streams in southern Ontario which spanned a gradient of agricultural disturbance and used DNA metabarcoding to identify the species composition from these samples. We address a significant knowledge gap in previous stream aquatic macroinvertebrate metacommunity studies by incorporating molecular identification as well as a temporal component. We observed that a combination of local habitat conditions, regional agricultural land use, and spatial position influenced aquatic macroinvertebrate community composition, suggesting there is an interaction between environmental filtering and dispersal processes that structures these communities. However, aquatic macroinvertebrate communities were also highly dissimilar between streams and composed of many rare species, and a large percentage of unexplained variation suggests that there is a strong stochastic component to community assembly. We also observed that there is a seasonal component to metacommunity dynamics, with different water quality variables being significant to community composition in each sampling month. While we expected that an increased percentage of surrounding agricultural land use would result in more homogenous macroinvertebrate communities, we only detected this relationship in May and found evidence that a larger riparian buffer width can mitigate the effects of agricultural land use. We demonstrate the utility of DNA metabarcoding for revealing patterns in metacommunity dynamics that may not be detectable using coarse taxonomic identifications, and reveal the importance of incorporating a seasonal component when evaluating the influence of land use on community composition.

category: ecology


title: Role of aIF5B in archaeal translation initiation

abstract: In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNAiMet, aIF5B and aIF1A at 2.7 [A] resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed.

category: biochemistry


title: One-Pot Endonucleolytically Exponentiated Rolling Circle Amplification by CRISPR-Cas12a Affords Sensitive, Expedited Isothermal Detection of MicroRNAs

abstract: MicroRNAs (miRNAs) are a class of short non-coding RNAs that play essential roles in gene expression regulation. While miRNAs offer a promising source for developing potent cancer biomarkers, the progress towards clinical utilities remains largely limited, due in part to the long-standing challenge in sensitive, specific, and robust detection of miRNAs in human biofluids. Emerging next-generation molecular technologies, such as the CRISPR-based methods, promise to transform nucleic acid testing. The prevailing strategy used in existing CRISPR-based methods is to hyphenate two separate reactions for pre-amplification, e.g., rolling circle amplification (RCA), and amplicon detection by Cas12a/13a trans-cleavage in tandem. Thus, existing CRISPR-based miRNA assays require multiple manual steps and lack the analytical performance of the gold standard, RT-qPCR. Radically deviating from the existing strategies, we developed a one-step, one-pot isothermal miRNA assay termed "Endonucleolytically eXponenTiated Rolling circle Amplification with the dual-functional CRISPR-Cas12a" (EXTRA-CRISPR) to afford RT-PCR-like performance for miRNA detection. We demonstrated the superior analytical performance of our EXTRA-CRISPR assay to detect miRNAs (miR-21, miR-196a, miR-451a, and miR-1246) in plasma extracellular vesicles, which allowed us to define a potent EV miRNA signature for detection of pancreatic cancer. The analytical and diagnostic performance of our one-pot assay were shown to be comparable with that of the commercial RT-qPCR assays, while greatly simplifying and expediting the analysis workflow. Therefore, we envision that our technology provides a promising tool to advance miRNA analysis and clinical marker development for liquid biopsy-based cancer diagnosis and prognosis.

category: bioengineering


title: Microbial metabolite butyrate-prodrug polymeric micelles promote gut health and treat food allergies

abstract: The gut microbiome modulates the bodys response to food antigens1. Beneficial taxa, specifically butyrate-producing Clostridia, are depleted in food-allergic individuals2,3. Although butyrate is known to play important roles in regulating gut immunity and maintaining epithelial barrier function4-6, its clinical translation is challenging due to its offensive odor and quick absorption in the upper gut. Here, we developed two polymeric micelle systems, one with a neutral charge (NtL-ButM) and one with a negative charge (Neg-ButM), that release butyrate from their polymeric core in the ileum or the cecum, respectively. Treatment with NtL-ButM in germ-free (and thus butyrate-depleted) mice up-regulated genes expressing antimicrobial peptides in the ileal epithelium. We show that these butyrate-containing micelles, used in combination, restore a barrier-protective response in mice treated with either dextran sodium sulfate or antibiotics. Treatment with the micelles protects peanut-allergic mice from an anaphylactic reaction to peanut challenge and rescues their dysbiosis by increasing the abundance of Clostridium Cluster XIVa. By restoring microbial and mucosal homeostasis, these butyrate-prodrug polymeric micelles may function as a new, antigen-agnostic approach to the treatment of food allergy.


title: Building a knowledge graph to enable precision medicine

abstract: Developing personalized diagnostic strategies and targeted treatments requires a deep understanding of disease biology and the ability to dissect the relationship between molecular and genetic factors and their phenotypic consequences. However, such knowledge is fragmented across publications, non-standardized research repositories, and evolving ontologies describing various scales of biological organization between genotypes and clinical phenotypes. Here, we present PrimeKG, a precision medicine-oriented knowledge graph that provides a holistic view of diseases. PrimeKG integrates 20 high-quality resources to describe 17,080 diseases with 4,050,249 relationships representing ten major biological scales, including disease-associated protein perturbations, biological processes and pathways, anatomical and phenotypic scales, and the entire range of approved and experimental drugs with their therapeutic action, considerably expanding previous efforts in disease-rooted knowledge graphs. In addition, PrimeKG supports artificial intelligence analyses of how drugs might target disease-associated molecular perturbations by containing an abundance of 'indications', 'contradictions', and 'off-label use' drug-disease edges lacking in other knowledge graphs. We accompany PrimeKG's graph structure with text descriptions of clinical guidelines to enable multimodal analyses.


title: Building a Knowledge Graph to Enable Precision Medicine


title: Lithium orotate is more potent, effective, and less toxic than lithium carbonate in a mouse model of mania

abstract:
BackgroundLithium carbonate (LiCO) is a mainstay treatment option for the prevention of mood-episode recurrences in bipolar disorder (BD). Unfortunately, the narrow therapeutic index of LiCO is associated with common thyroid and kidney complications that lead to high rates of non-compliance. Lithium orotate (LiOr) is an alternative compound that is suggested to possess uptake properties that would allow for reduced dosing to mitigate toxicity and compliance concerns.

MethodsDose responses were established for LiOr and LiCO in male and female mice using an amphetamine-induced hyperlocomotion (AIH) murine (C57BL/6) model (n = 5-11); AIH captures manic elements of BD and is sensitive to a dose-dependent lithium blockade. These studies were followed by an examination of the relative toxicities of LiOr and LiCO over 14 consecutive daily administrations (n = 5-7).

ResultsA partial block of AIH was maintained by LiCO at doses of 15 mg/kg or greater in males and 20 mg/kg or greater in females. In contrast, a near-complete blockade was observed for LiOr concentrations equal to or exceeding 1.5 mg/kg in both males and females, indicating improved efficacy and potency. Prior application of polyethylene glycol-400 completely blocked the effects of LiOr on AIH while sparing LiCO, suggesting differences in transport between the two compounds. LiCO, but not LiOr, elicited polydipsia in both sexes, elevated serum creatinine levels in males, and increased serum TSH expression in females.

ConclusionsLiOr demonstrates superior efficacy, potency, and tolerability to LiCO in both male and female mice as a result of select transport-mediated uptake.


title: Impatiens banen sp. nov. & I. etugei sp. nov. (Balsaminaceae), threatened lowland species of the Cross-Sanaga Interval, Cameroon

abstract:
We describe two range-restricted new species to science from the genus Impatiens (Balsaminaceae), both threatened, from the Cross-Sanaga Interval of western Cameroon. The first, Impatiens banen, appears to be restricted to an open seepage microhabitat on granitic inselbergs in the lowland-submontane forest zone of the Ebo Forest in Littoral Region and is provisionally assessed using the 2012 IUCN standard as Vulnerable. Sharing characters with Impatiens burtonii and I. mannii, it differs from both, and appears to be unique in Cameroon inter alia 1) in the bicolored united lateral petals, the upper petals being white, while the lower petals are an intense pink-purple, 2) the hairy, filamentous spur, purple with a white apex, is curved along its length (through nearly 360{degrees}), almost describing a circle. Inselberg-specific species are unusual in Impatiens.

The second species, Impatiens etugei, of the I. macroptera aggregate, is restricted to rocks in the Mutel River of the Kom Wum Forest Reserve of NorthWest Region and is assessed as Critically Endangered. Having similarities with I. mackeyana and I. letouzeyi, it differs from other species in the aggregate inter alia by having opposite leaves (vs always alternate), flower exterior white (vs pink or pink-purple), and in the dorsal petal having a pair of lateral projections (vs projections absent).

category: plant biology


title: Light-dependent modulation of protein localization and function in living bacteria cells

abstract: Most bacteria lack membrane-enclosed organelles to compartmentalize cellular processes. In lieu of physical compartments, bacterial proteins are often recruited to macromolecular scaffolds at specific subcellular locations to carry out their functions. Consequently, the ability to modulate a proteins subcellular location with high precision and speed bears the potential to manipulate its corresponding cellular functions. Here we demonstrate that the CRY2/CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations inside live E. coli cells including the nucleoid, the cell pole, membrane, and the midcell division plane. We further show that such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. Finally, we demonstrate that the CRY2/CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system.


title: Coordinated control of neuronal differentiation and wiring by a sustained code of transcription factors

abstract: The enormous diversity of cell types in nervous systems presents a challenge in identifying the genetic mechanisms that encode it. Here, we report that nearly 200 distinct neurons in the Drosophila visual system can each be defined by unique combinations of [~]10 transcription factors that are continuously expressed by them. We show that targeted modifications of this  selector code induce predictable conversions of cell fates between neurons in vivo. These conversions appear morphologically and transcriptionally complete, arguing for a conserved gene regulatory program that jointly instructs both the type-specific development and the terminal features of neurons. Cis-regulatory sequence analysis of open chromatin links one of these selectors to an upstream patterning gene in stem cells that specifies neuronal fates. Experimentally validated network models show that selectors interact with ecdysone signaling to regulate downstream effectors controlling brain wiring. Our results provide a generalizable framework of how specific fates are initiated and maintained in postmitotic neurons.


title: Fc-engineered antibodies leverage neutrophils to drive control of Mycobacterium tuberculosis

abstract: Novel vaccination and therapeutic strategies are urgently needed to mitigate the tuberculosis (TB) epidemic. While extensive efforts have focused on potentiating cell-mediated immunity to control Mycobacterium tuberculosis (Mtb) infection, less effort has been invested in exploiting the humoral immune system to combat Mtb. Emerging data point to a role for antibodies in microbial control of Mtb, however the precise mechanism(s) of this control remain incompletely understood. Here we took an antibody Fc-engineering approach to determine whether Fc-modifications could improve the ability of antibodies to restrict Mtb, and to define Fc-mediated mechanism(s) antibodies leverage for this restriction. Using an antibody specific to the capsular polysaccharide -glucan, we engineer a panel of Fc variants to augment or dampen select antibody effector functions, rationally building antibodies with enhanced capacity to promote Mtb restriction in a human whole blood model of infection. Surprisingly, restrictive Fc-engineered antibodies drive Mtb control in a neutrophil, not monocyte, dependent manner. Using single cell RNA sequencing, we show that restrictive antibodies promote neutrophil survival and expression of cell intrinsic antimicrobial programs. These data provide a roadmap for exploiting Fc-engineered antibodies as a novel class of TB therapeutics able to harness the protective functions of neutrophils to achieve disease control.


title: A transcriptional mechanism involving R-loop, m6A modification and RNA abasic sites regulates an enhancer RNA of APOE.

abstract:
The DNA genetic code and the RNA regulatory code determine phenotypes from gene expression to disease susceptibility. DNA sequence variants lead to phenotypic differences among individuals, while within an individual, RNA dynamically confers cell identity and responds to cellular and environmental signals. To provide regulation for different cell types and conditions, the nucleotides of RNA are modified by hundreds of chemical reactions, and RNA folds into innumerable shapes. To decipher the RNA regulatory code is to understand how RNA sequence and structure respond to cellular needs. Here, we deciphered one part of the RNA code where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We uncovered an enhancer RNA, AANCR, that regulates the transcription and expression of APOE. When AANCR is folded into an R-loop, which is then modified by N6-adenine methylation and N-glycosidic cleavage, it is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in some cell types and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. By genetic analysis, we confirmed that AANCR regulates APOE expression. DNA sequence variants in AANCR are associated with APOE expression and also with Alzheimers disease. Our data show that DNA and RNA sequence and structure jointly regulate gene expression that influence disease risk.

HighlightsO_LIm6A, RNA abasic sites and R-loops jointly regulate transcription by pausing RNA Polymerase II.
C_LIO_LIAn enhancer RNA regulates APOE expression.
C_LIO_LIEnhancer RNA of APOE modifies susceptibility to Alzheimers disease.
C_LI

category: molecular biology


title: RNA exosome ribonuclease DIS3 degrades Pou6f1 to promote mouse pre-implantation cell differentiation

abstract:
Mammalian development is precisely controlled by cell differentiation. Discovery of new regulators and investigating their crosstalk provide insights into genetic networks defining pre-implantation development. To identify novel developmental repressors, we established a knockout mouse model of Dis3, an RNA exosome associated RNase. Homozygous Dis3 null embryos arrest at the morula-to-blastocyst transition. Using single embryo RNA-seq, we discovered persistence of Pou6f1 mRNA in homozygous null Dis3 embryos and determined that the cognate protein represses transcription of Nanog and Cdx2. The resultant defects in cell differentiation disrupted the morula-to-blastocyst transition and was embryonic lethal. To overcome the paucity of embryos, homozygous Dis3 null mouse embryonic stem cells were derived to identify additional gene targets of POU6F1. While microinjection of the Dis3 cRNA into zygotes rescued the morula-to-blastocyst block, point mutations of Dis3 in individual blastomeres transformed the cell and prevented incorporation into embryos. Our findings uncover a not heretofore reported regulatory pathway of DIS3-POU6F1 in pre-implantation mammalian embryogenesis.

In BriefMammalian pre-implantation development is regulated by master transcription factors and their crosstalk. Wu and Dean report that an RNA exosome associated RNase, DIS3, degrades Pou6f1 mRNA to de-repress transcription of Nanog and Cdx2 genes. In the absence of DIS3, POU6F1 protein persists and embryos arrest as morulae unable to become blastocysts due to lack of cell differentiation.

HighlightsO_LIDis3 knockout mice have morula arrest due to lack of cell differentiation.
C_LIO_LIDIS3 binds and degrades Pou6f1 mRNA before the morula stage.
C_LIO_LIPOU6F1 globally occupies promoters to regulate gene transcription.
C_LIO_LIDIS3 mutation results in cell transformation in embryonic development.
C_LI

category: developmental biology


title: Staining-Free, In-Flow Enumeration of Tumor Cells in Blood Using Digital Holographic Microscopy and Deep Learning

abstract: Currently, detection of circulating tumor cells (CTCs) in cancer patient blood samples relies on immunostaining, which does not provide access to live CTCs, limiting the breadth of CTC-based applications. As a first step to address this limitation, here, we demonstrate staining-free enumeration of tumor cells spiked into lysed blood samples using digital holographic microscopy (DHM), microfluidics and machine learning (ML). A 3D-printed module for laser assembly was developed to simplify the optical set up for holographic imaging of cells flowing through a sheath-based microfluidic device. Computational reconstruction of the holograms was performed to localize the cells in 3D and obtain the plane of best focus images to train deep learning models. First, we evaluated the classification performance of two convolutional neural networks (CNNs): ResNet-50 and a custom-designed shallow Network dubbed s-Net. The accuracy, sensitivity and specificity of these networks were found to range from 97.08% and 99.32%. Upon selecting the s-Net due to its simple architecture and low computational burden, we formulated a decision gating strategy to significantly lower the false positive rate (FPR). By applying an optimized decision threshold to mixed samples prepared in silico, the FPR was reduced from 1x10-2 to 2.77x10-4. Finally, the developed DHM-ML framework was successfully applied to enumerate spiked MCF-7 breast cancer cells from lysed blood samples containing a background of white blood cells (WBCs). We conclude by discussing the advances that need to be made to translate the DHM-ML approach to staining-free enumeration of CTCs in cancer patient blood samples.


title: How to Best Represent Proteins in Machine Learning-based Prediction of Drug/Compound-Target Interactions

abstract: The identification of drug/compound-target interactions (DTIs) constitutes the basis of drug discovery, for which computational predictive approaches have been applied. As a relatively new data-driven paradigm, proteochemometric (PCM) modeling utilizes both protein and compound properties as a pair at the input level and processes them via statistical/machine learning. The representation of input samples (i.e., proteins and their ligands) in the form of quantitative feature vectors is crucial for the extraction of interaction-related properties during the artificial learning and subsequent prediction of DTIs. Lately, the representation learning approach, in which input samples are automatically featurized via training and applying a machine/deep learning model, has been utilized in biomedical sciences. In this study, we performed a comprehensive investigation of different computational approaches/techniques for protein featurization (including both conventional approaches and the novel learned embeddings), data preparation and exploration, machine learning-based modeling, and performance evaluation with the aim of achieving better data representations and more successful learning in DTI prediction. For this, we first constructed realistic and challenging benchmark datasets on small, medium, and large scales to be used as reliable gold standards for specific DTI modeling tasks. We developed and applied a network analysis-based splitting strategy to divide datasets into structurally different training and test folds. Using these datasets together with various featurization methods, we trained and tested DTI prediction models and evaluated their performance from different angles. Our main findings can be summarized under 3 items: (i) random splitting of datasets into train and test folds lead to near-complete data memorization and produce highly over-optimistic results, as a result, it should be avoided, (ii) learned protein sequence embeddings work well in DTI prediction and offer high potential, even though no information related to protein structures, interactions or biochemical properties is utilized during their generation, and (iii) PCM models tend to learn from compound features and leave out protein features, mostly due to the natural bias in DTI data, indicating the requirement for new and unbiased datasets. We hope this study will aid researchers in designing robust and high-performing data-driven DTI prediction systems that have real-world translational value in drug discovery.


title: How to Approach Machine Learning-based Prediction of Drug/Compound-Target Interactions


title: Differential regulation of GUV mechanics via actin network architectures

abstract: Actin networks polymerize and depolymerize to construct highly organized structures, thereby, endowing the mechanical phenotypes found in a cell. It is generally believed that the amount of filamentous actin and actin network architecture determine cytoplasmic viscosity and elasticity of the whole cell. However, the intrinsic complexity of a cell and numerous other endogenous cellular components make it difficult to study the differential role of distinct actin networks in regulating cell mechanics. Here, we model a cell by using giant unilamellar vesicles (GUVs) encapsulating actin filaments and networks assembled by various actin crosslinker proteins. Perturbation of these cytoskeletal vesicles using AC electric fields revealed that deformability depends on lumenal viscosity and actin network architecture. While actin-free vesicles exhibited large electromechanical deformations, deformations of GUVs encapsulating actin filaments were significantly dampened. The suppression of electrodeformation of actin-GUVs can be similarly recapitulated by using aqueous PEG 8000 solutions at different concentrations to modulate viscosity. Furthermore, alpha actinin-crosslinked actin networks resulted in decreased GUV deformability in comparison to actin filament-encapsulating GUVs, and membrane-associated actin networks through the formation of dendritic actin cortex greatly dampened electrodeformation of GUVs. These results highlight the organization of actin networks regulates the mechanics of GUVs and shed insights into the origin of differential deformability of cells.

category: biophysics


title: Immune defense in Drosophila melanogaster depends on diet, sex, and mating status

abstract: Post-mating immunosuppression has been widely accepted as a female trait in Drosophila melanogaster. Our results challenge this notion by presenting a mating-immunity trade-off in males as well as in females. When inoculated with the fungal pathogen, Beauveria bassiana, both males and females die faster compared to inoculated virgins, and survival is lower when inoculated flies are continuously mated compared to a single day of mating. Past studies with Beauveria bassiana have shown females to be more susceptible to infection than males. Our results challenge this finding as well, showing that the direction of sexual dimorphism in immune defense depends on mating status, specific Beauveria bassiana strain, and fly genotype. Moreover, we show that survival after fungal infection is largely influenced by diet, and that post-infection dietary improvements can help enhance survival. Post-mating suppression in Drosophila survival of B. bassiana infection presents study opportunities with potential applications for biological control of insect vectors of human disease and insect crop pests.

category: physiology


title: The capsid precursor protein of astrovirus VA1 is proteolytically processed intracellularly

abstract:
Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients, and its recent propagation in cell culture has opened the possibility to study its biology. Unlike classical human astroviruses, VA1 growth was found to be independent of trypsin during virus replication in vitro. In this work, we show that despite its independence on trypsin activation for cell infection, the VA1 capsid precursor protein, of 86 kDa (VP86), is processed intracellularly, and this proteolytic processing is important for astrovirus VA1 infectivity. Antibodies raised against different regions of the capsid precursor showed that the polyprotein can be processed starting at either its amino-or carboxy-terminal end, and they allowed us to identify that proteins of about 33 (VP33) and 38 (VP38) kDa constitute the core and the spike proteins of the mature infectious virus particles, respectively. The amino-terminal end of the spike protein was found to be Thr-348. Whether the protease involved in intracellular cleavage of the capsid precursor is of viral or cellular origin remains to be determined, but the cleavage is independent of caspases. Also, trypsin is able to degrade the capsid precursor but has no effect on VP34 and VP38 proteins when assembled into virus particles. These studies provide the basis for advancement of the knowledge of astrovirus VA1 cell entry and replication.

IMPORTANCEHuman astrovirus VA1 has been associated with neurological disease in immunocompromised patients. Its recent propagation in cell culture has facilitated the study of its biology. In this work, we show that despite the ability of this virus to grow in the absence of trypsin, a marked feature of human classical astroviruses, the capsid precursor protein of astrovirus VA1 is cleaved intracellularly to yield the mature infectious particles, formed by two polypeptides, VP33 that constitutes the core domain of the virus particle, and V38 that forms the spike of the virus. These studies provide a platform to advance our knowledge on astrovirus VA1 cell entry and replication.


title: CORK1, a LRR-Malectin Receptor Kinase for Cellooligomer Perception in Arabidopsis thaliana

abstract: Cell wall integrity (CWI) maintenance is central for plant cells. Mechanical or chemical distortions, pH changes, or breakdown products of cell wall polysaccharides activate plasma membrane-localized receptors and induce appropriate downstream responses. Microbial interactions alter or destroy the structure of the plant cell wall, connecting CWI maintenance to immune responses. Cellulose is the major polysaccharide in the primary and secondary cell wall. Its breakdown generates short-chain cellooligomers which induce Ca2+-dependent CWI responses. We have shown here that these responses require the malectin domain-containing CELLOOLIGOMER-RECEPTOR KINASE 1 (CORK1) in Arabidopsis. CORK1 is required for cellooligomer-induced cytoplasmic Ca2+ elevation, reactive oxygen species (ROS) production, mitogen associated protein kinase (MAPK) activation, cellulose synthase phosphorylation, and the regulation of CWI-related genes including those involved in biosynthesis of cell wall material, secondary metabolites and tryptophan. Phosphoproteome analyses identified early targets involved in signaling, cellulose synthesis, the endoplasmatic reticulum/Golgi secretory pathway, cell wall repair and immune responses. Two conserved phenylalanine residues in the malectin domain are crucial for CORK1 function. We propose that cellulose breakdown products bind to the malectin domain in CORK1, indicating its role as a novel receptor kinase for CWI maintenance.


title: Decoding violated sensory expectations from the auditory cortex of anaesthetized mice: Hierarchical recurrent neural network depicts separate 'danger' and 'safety' units

abstract: The ability to respond appropriately to sensory information received from the external environment is among the most fundamental capabilities of central nervous systems. In the auditory domain, processes underlying this behaviour are studied by measuring auditory-evoked electrophysiology during sequences of sounds with predetermined regularities. Identifying neural correlates of ensuing auditory novelty responses is supported by research in experimental animals. In the present study, we reanalysed epidural field potential recordings from the auditory cortex of anaesthetised mice during frequency and intensity oddball stimulation. Multivariate pattern analysis (MVPA) and hierarchical recurrent neural network (RNN) modelling were adopted to explore these data with greater resolution than previously considered using conventional methods. Time-wise and generalised temporal decoding MVPA approaches revealed previously underestimated asymmetry between responses to sound-level transitions in the intensity oddball paradigm, in contrast with tone frequency changes. After training, the cross-validated RNN model architecture with four hidden layers produced output waveforms in response to simulated auditory inputs that were strongly correlated with grand-average auditory-evoked potential waveforms (r2 > 0.9). Units in hidden layers were classified based on their temporal response properties and characterised using principal component analysis and sample entropy. These demonstrated spontaneous alpha rhythms, sound onset and offset responses, and putative  safety and  danger units activated by relatively inconspicuous and salient changes in auditory inputs, respectively. The hypothesised existence of corresponding biological neural sources is naturally derived from this model. If proven, this would have significant implications for prevailing theories of auditory processing.

category: neuroscience


title: Human Lung-resident Mucosal-Associated Invariant T cells are Abundant, Express Antimicrobial Proteins, and are Cytokine Responsive

abstract: Mucosal-associated Invariant T (MAIT) cells are an innate-like T cell subset that recognize a broad array of microbial pathogens, including respiratory pathogens. Here we investigate the transcriptional profile of MAIT cells localized to the human lung, and postulate that MAIT cells may play a role in maintaining homeostasis at this mucosal barrier. Using the MR1/5-OP-RU tetramer, we identified MAIT cells and non-MAIT CD8+ T cells in lung tissue not suitable for transplant from human donors. We used RNA-sequencing of MAIT cells compared to non-MAIT CD8+ T cells to define the transcriptome of MAIT cells in the human lung. We show that, as a population, lung MAIT cells are polycytotoxic, secrete the directly antimicrobial molecule IL-26, express genes associated with persistence, and selectively express cytokine and chemokine-related molecules distinct from other lung-resident CD8+ T cells, such as interferon-{gamma}- and IL-12-receptors. These data highlight MAIT cells predisposition to rapid pro-inflammatory cytokine responsiveness and antimicrobial mechanisms in human lung tissue, concordant with findings of blood-derived counterparts, and support a function for MAIT cells as early sensors in the defense of respiratory barrier function.


title: Decreased in vitro dihydroartemisinin sensitivity in malaria parasites infecting sickle cell disease patients

abstract:
BackgroundPartial ACTs treatment failure in Plasmodium falciparum malaria has been previously reported in sickle cell patients. The main purpose of this study was to investigate the in vitro susceptibility of clinical isolates to DHA to find out hypothesis backing up the reason of this poor therapeutic response.

ResultsA total of 134 clinical isolates from patients attending health centers in Abidjan with uncomplicated Plasmodium falciparum malaria were selected. Hemoglobin HbAS, HbSS, HbAC, HbSC and HbAA were identified. Parasitemia and hemoglobin level at inclusion were lower in sickle cell patients with major forms than in patients with normal phenotype. A significant number of parasites with survival rates ranging from 14.68 to 33.75% were observed in clinical isolates from the SS phenotype. At inclusion, these resistant clinical isolates showed lower parasite densities, and patients had lower red blood cell count and hematocrit levels compared to those with susceptible clinical isolates. A low rate of parasitic growth has more often occurred with AS sickle cell phenotype. However, the decrease in in vitro sensitivity to DHA was not associated with Kelch 13-Propeller gene polymorphism.

ConclusionThis study highlights an in vitro decreased sensitivity to DHA, for clinical isolates collected from sickle cell SS patients living in Abidjan (Cote dIvoire), which is not related to the Pfkelch13 gene mutations. These clinical isolates may represent a health threat for sickle cell disease patients especially during crisis. Moreover, these results could suggest additional mechanisms of artemisinin resistance that need to be explored.

category: microbiology


title: Shock or jump: deimatic behaviour is repeatable and polymorphic in a yellow-bellied toad

abstract: Inter-individual variation in antipredatory strategies has long attracted curiosity among scientists. Deimatisms is a complex and time-structured antipredatory strategy consisting in prey suddenly unleashing unexpected defences to frighten predators and stop their attack. Being deimatism traditionally considered as a stereotyped antipredatory response, the inter-individual variation in phenotypic traits related to deimatic displays is almost unexplored. In this study, we employed common garden experiments on 71 yellow-bellied toad Bombina pachypus to investigate the extent and pattern of inter-individual variation in the unken-reflex behaviour, a deimatic display performed by some amphibians. Results show that deimatic displays consistently differ among individuals. Only about half of the individuals reacted to the predation stimuli by exhibiting the display, which varied in responsiveness, duration and intensity. All the investigated descriptors were repeatable (R > 0.50, p < 0.01). Finally, we found significant correlations between the measured parameters, defining two alternative behavioural profiles: individuals quickly doing unken-reflex, with high intensity and long duration of the display, and individuals avoiding unken-reflex but rather escaping. Such dichotomy resembles respectively the proactive and reactive coping styles. Such an unexpected variation in deimatic behaviour raises intriguing questions on the evolutionary processes shaping multiple adaptive responses to predation within populations.


title: Interphotoreceptor matrix proteoglycans IMPG1 and IMPG2 proteolyze in the SEA domain and reveal localization mutual dependency

abstract: The interphotoreceptor matrix (IPM) is a specialized extracellular mesh of molecules surrounding the inner and outer segments of the photoreceptor neurons. The interphotoreceptor matrix proteoglycan 1 and 2 (IMPG1 and IMPG2) are major components of the IPM. Both proteoglycans possess SEA (Sperm protein, Enterokinase and Agrin) domains, which are described to support protein proteolysis. Interestingly, humans with mutations in the SEA domains of IMPG1 and IMPG2 are associated with retinitis pigmentosa (RP) vision disease. This work investigates the SEA-mediated proteolysis of IMPG1 and IMPG2 and its significance to IPM physiology. Western blot relative molecular mobility analysis of IMPG1 from retina confirmed protein proteolysis. Point mutations in the SEA domain of IMPG1 inhibit protein proteolysis, as shown in cell culture assays. Likewise, IMPG2 also proteolyzes at the SEA domain generating two subunits: a membrane-attached and an extracellular peptide, as seen by western blot and immunohistochemical assays. Importantly, the extracellular portion of IMPG2 traffics toward the outer segment IPM by a mechanism dependent on IMPG1. These results demonstrate that proteolysis is part of the maturation of IMPG1 and IMPG2, which is important for protein localization and linked to vision diseases. Moreover, the results indicate interdependency between IMPG1 and IMPG2, which helps understand the molecular mechanisms involved in the development of vision diseases in patients with defective IMPG molecules.


title: The Medicines for Malaria Venture Malaria Box contains inhibitors of protein secretion in Plasmodium falciparum blood stage parasites

abstract:
Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current anti-malarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria box of 400 anti-malarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs - modifications which are both mediated by parasite-derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future anti-malarial that rapidly inhibits parasite virulence before eliminating parasites from the host.

SynopsisPlasmodium falciparum exports proteins into its host cell to perform a myriad of functions required for survival. We adapted an assay to screen for small molecules that inhibit protein secretion and export. Screening the 400-compound Medicines for Malaria Venture (MMV) Malaria Box uncovered several potential export inhibitors. The most promising of these compounds, MMV396797, blocked protein export at the parasite and reduced host rigidification and cytoadherence, two functions which are mediated by exported proteins.


title: A Simulator for Somatic Evolution Study Design

abstract:
MotivationSomatic evolution plays a key role in development, cell differentiation, and normal aging, but also diseases such as cancer, which is now mainly thought of as a disease of genetic and epigenetic modification. Understanding mechanisms of somatic mutability -- variant types and frequencies, phylogenetic structure, mutational signatures, and clonal heterogeneity -- and how they can vary between cell lineages will likely play a crucial role in biological discovery and medical applications. This need has led to a proliferation of new technologies for profiling single-cell variation, each with distinctive capabilities and limitations that can be leveraged alone or in combination with other technologies. The enormous space of options for assaying somatic variation, however, presents unsolved informatics problems with regards to selecting optimal combinations of technologies for designing appropriate studies for any particular scientific questions. Versatile simulation tools are needed to make it possible to explore and optimize potential study designs if researchers are to deploy multiomic technologies effectively.

ResultsIn this paper, we present a simulator allowing for the generation of synthetic data from a wide range of clonal lineages, variant classes, and sequencing technology choices, intended to provide a platform for effective study design in somatic lineage analysis. Our simulation framework allows for the assessment of study design setups and their statistical validity in determining different ground-truth cancer mechanisms. The user is able to input various properties of the somatic evolutionary system, mutation classes (e.g., single nucleotide polymorphisms, copy number changes, and classes of structural variation), and biotechnology options (e.g., coverage, bulk vs single cell, whole genome vs exome, error rate, number of samples) and can then generate samples of synthetic sequence reads and their corresponding ground-truth parameters for a given study design. We demonstrate the utility of the simulator for testing and optimizing study designs for various experimental queries.

Contactrussells@andrew.cmu.edu

Availabilityhttps://github.com/CMUSchwartzLab/MosaicSim


title: Genomic characteristics and clinical significance of CD56+ Circulating Tumor Cells in Small Cell Lung Carcinoma

abstract:
IntroductionCirculating Tumor Cells (CTC) have been studied in various solid tumors. However, clinical utility of CTC in Small Cell Lung Cancer (SCLC) remains to be demonstrated. Most published studies are based on Cellsearch(R) isolation methods that did not capture CTC undergoing epithelial-mesenchymal transition. The aim of the CTC-CPC study was to develop an EpCAM-independent CTC isolation method allowing isolation of a broader range of living CTC from SCLC and decipher their genomic and biological characteristics.

MethodWe initiated a monocentric prospective non-interventional study (CTC-CPC) from April 2016 including only treatment-naive newly diagnosed SCLC. CD56+CTC were isolated from whole blood samples using an immunofluorescence-based method, at diagnosis and relapse after first-line treatment. Whole-exome-sequencing (WES) was performed on isolated CTC, matched PBMCs and tumor biopsies for genomic characterization, mutational signature classification and biological altered pathways identification in four patients. In addition, CTC Derived Xenografts (CDXs) were generated and submitted to RNA-sequencing.

ResultsPhenotypic study confirms tumor lineage and tumorigenic properties of isolated cells for the 4 patients analyzed with WES. Notably, CDX model derived from CD56+CTC was successfully classified with an ASCL1 transcriptomic profile. WES of CD56+CTC and matched tumor biopsy reveal common genomic alteration frequently impaired in SCLC. Moreover, CD56+CTC were characterized by a high mutation load and a distinct mutational profile compared to match tumors biopsies. The BioInfoMiner (BIM) web platform allowed the identification of a genomic signature for the CD56+CTC at the time of diagnosis. In addition to classical pathways altered in SCLC related to neural cell adhesion, extracellular matrix organization, hypoxia, signaling kinase cascaded and immune response, we found new biological processes specifically affected in CD56+CTC at diagnosis, sumoylation and transcriptional regulation by AP-2 and RUNX2. High numeration of CD56+CTC (>7/mL) at diagnosis was associated with ES-SCLC in a cohort of 33 patients. Finally, comparing genomic features of diagnosis and relapse CD56+CTC, we identify reminiscent altered oncogenic pathways (e.g. DLL3 or MAPK pathway) that are currently investigated in targeted therapy trials.

ConclusionsWe report a versatile and easy-to-use method of CD56+CTC detection for newly diagnosed SCLC. Numeration of CD56+CTC at diagnosis is correlated with disease extension. Isolated CD56+CTC conserve tumorigenic properties and show a distinct mutational profile. We report a minimal gene set as a unique signature of CD56+CTC and identify new affected biological pathways enriched in EpCAM-independent isolated CTC in SCLC. Finally, our results suggest that studying CD56+CTC at relapse might help to identify the oncologic pathways that seeds tumor resistance to first-line treatment.

category: cancer biology


title: Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology

abstract: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1-MPNST), but can occur sporadically. Through a multi-institution collaboration, we have developed 13 NF1-associated MPNST patient-derived xenografts (PDX). Genomic analysis of the PDX-tumor pairs identified somatic mutations in NF1 (61%), SUZ12 (61%), EED (15%), and TP53 (15%), and chromosome 8 (Chr8) gain (77%), consistent with published data. Pre-clinical models that capture this molecular heterogeneity are needed to identify and prioritize effective drug candidates for clinical translation. Here, we describe the successful development of a medium-throughput ex vivo 3D microtissue model with several advantages over 2D cell line growth, which can be utilized to predict drug response in vivo. Herein, we present proof-of-principle of this PDX-to-microtissue system, using four genomically representative MPNST and three drugs. This work highlights the development of a novel ex vivo to in vivo preclinical platform in MPNST that successfully captures the genomic diversity observed in patients and represents a resource to identify future therapeutic strategies.


title: Repeated birth injuries lead to pelvic floor muscle dysfunction and impairment in regeneration

abstract:
ObjectivesChildbirth is a key risk factor for pelvic floor muscle (PFM) injury and dysfunction, and subsequent pelvic floor disorders (PFDs). Multiparity further exacerbates these risks. Using the pre-clinical rat model of simulated birth injury (SBI), we previously identified that an SBI leads to PFM atrophy and fibrosis. We hypothesized that multiple SBIs further overwhelm PFM regenerative capacity, leading to functionally relevant pathological alterations long-term.

Study DesignRats underwent SBI and were allowed to recover for 8 weeks to undergo another SBI. Animals were sacrificed at acute, subacute, and long-term time points post-second injury (N=3-6/time point), and pubocaudalis (PCa) was harvested to assess ex vivo muscle function, histomorphological properties and gene expression.

ResultsAcutely following the 1st SBI, PCa force was decreased relative to controls. At 4 weeks, PCa force was recovered and remained unchanged at 8 weeks. Similarly, lower PCa force was observed immediately after repeated SBI. In contrast to functional recovery after 1st SBI, PCa force remained lower at 4 weeks post-2nd SBI and continued to be decreased even after 12 weeks after repeated injury. Fiber size was smaller at the long-term time points after 2nd SBI compared to controls and single SBI groups. As opposed to the resolution of centralized nuclei at 8 weeks post-1st SBI, regenerating myofibers persisted even at 12 weeks post-2nd SBI. In contrast to the peak of collagen content at 4 weeks post-1st SBI, this parameter raised progressively over 12 weeks after repeated SBIs. Prolonged inflammatory response, impairment in muscle anabolism, and sustained expression of ECM remodeling genes were observed after repeated SBIs.

ConclusionsRepeated birth injuries delay PFM regeneration and impair function in the pre-clinical rat model.


title: Biomechanics and hydraulics of Opuntia ficus-indica roots exposed to extreme drought

abstract: Succulent plants possess traits that allow them to complete physiological functions under extreme environments and root are at the frontline of the stress: the drying soil. Previous works in succulent plants have reported the extraordinary reversible mechanism of root shrinkage that disconnects plants from drying soils, reestablishing the hydraulic connection when water availability is restored. Yet, this rectifier-like mechanism would require complex biomechanical and hydraulic control at organ, tissue, and cell level. In here we evaluated the changes in hydraulic and mechanical behavior of Opuntia fine roots under extreme drought stress. Using a combination of techniques, we found that fine roots get more elastic as drought stress gets more extreme, allowing cells to modify their shape while preventing permanent damage. Furthermore, we found abrupt decreases in Lpr, that coincided with increased root shrinkage, suberin deposition and structural damage inside the endodermis via lacunae formation and possibly cell wall folding. Our data suggest that, in drought stressed succulent plants, the biomechanics of organs, tissues, and possibly cell walls are deeply coupled with belowground hydraulics, highlighting the need to continue working on deciphering the physiological mechanism that governs the interplay between mechanics and hydraulics at cell level in fine roots during drought.


abstract:
Strict iron regulation is essential for normal brain function. The main iron compounds responsible for iron homeostasis, transferrin and ferritin, are distributed heterogeneously across the brain and are implicated in aging, neurodegenerative diseases and cancer. However, non-invasive discrimination between iron compounds, such as transferrin and ferritin, remains a challenge. We present a novel magnetic resonance imaging (MRI) technology for mapping of iron compounds in the living brain (the r1-r2* relaxivity). The specificity of the r1-r2* relaxivity to the presence of ferritin and transferrin is validated by both bottom-up and top-down approaches, incorporating in vitro, in vivo and ex vivo analyses. In vitro, our MRI approach reveals the distinct paramagnetic properties of ferritin and transferrin. In the in vivo human brain, we validate our approach against ex vivo iron compounds quantification and gene expression. Our approach predicts the transferrin-ferritin fraction across brain regions and in aging. It reveals brain tumors iron homeostasis, and enhances the distinction between tumor tissue and non-pathological tissue without contrast agents. Therefore, our approach may allow for non-invasive research and diagnosis of iron homeostasis in living human brains.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/490254v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1ffbb4org.highwire.dtl.DTLVardef@1c5fc07org.highwire.dtl.DTLVardef@1253cc9org.highwire.dtl.DTLVardef@1b9baf7_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Uncovering molecular iron compounds in the living human brain.

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title: Longitudinal 7T MRI reveals volumetric changes in subregions of human medial temporal lobe to sex hormone fluctuations

abstract: The hippocampus and surrounding medial temporal lobe (MTL) are critical for memory processes, with local atrophy linked to memory deficits. Animal work shows that MTL subregions densely express sex hormone receptors and exhibit rapid structural changes synchronized with hormone fluctuations. Such transient effects in humans have thus far not been shown. By combining a dense-sampling protocol, ultra-high field neuroimaging and individually-derived segmentation analysis, we demonstrate how estradiol and progesterone fluctuations affect MTL subregion volumes across the human menstrual cycle. Twenty-seven healthy women (19-34 years) underwent 7T MRI at six timepoints to acquire T1-weighted and T2-weighted images. Linear mixed-effects modeling showed positive associations between estradiol and parahippocampal cortex volume, progesterone and subiculum and perirhinal Area 35 volumes, and an estradiol*progesterone interaction with CA1 volume. We confirmed volumetric changes were not driven by hormone-related water (cerebral spinal fluid) or blood-flow (pulsed arterial spin labeling) changes. These findings suggest that sex hormones alter structural brain plasticity in subregions that are differentially sensitive to hormones. Mapping how endogenous endocrine factors shape adult brain structure has critical implications for womens health during the reproductive years as well as later in life, such as increased dementia risk following perimenopause, a period of pronounced sex hormone fluctuations.


title: Decoding of Speech Information using EEG in Children with Dyslexia: Less Accurate Low-Frequency Representations of Speech, Not "Noisy" Representations

abstract: The amplitude envelope of speech carries crucial low-frequency acoustic information that assists linguistic decoding. The sensory-neural Temporal Sampling (TS) theory of developmental dyslexia proposes atypical encoding of speech envelope information <10 Hz, leading to atypical phonological representations. Here a backward linear TRF model and story listening were employed to estimate the speech information encoded in the electroencephalogram in the canonical delta, theta and alpha bands by 9-year-old children with and without dyslexia. TRF decoding accuracy provided an estimate of how faithfully the childrens brains encoded low-frequency envelope information. Between-group analyses showed that the children with dyslexia exhibited impaired reconstruction of speech information in the delta band. However, when the quality of speech encoding for each child was estimated using child-by-child decoding models, then the dyslexic children did not differ from controls. This suggests that children with dyslexia encode neither "noisy" nor "normal" representations of the speech signal, but different representations.


title: Angiotensin II regulates the neural expression of subjective fear in humans

abstract: There has been growing interest in using pharmacological fMRI to identify novel treatments for fear, yet these models are limited with respect to determining process-specific effects on the actual subjective experience of fear which represents the key symptom why patients seek treatment. Combining a selective angiotensin II type 1 receptor (ATR1) antagonist with a fear vigilance fMRI paradigm and neurofunctional decoding in n = 87 individuals we demonstrate that AT1R blockade selectively reduces the neurofunctional reactivity to fear-inducing visual oddballs in terms of attenuating dorsolateral prefrontal activity and amygdala-ventral anterior cingulate (vACC) communication, and further show that the observed effect is due to reduced subjective fear, but not threat or non-specific negative affect, using a decoding analysis. Our findings demonstrate a highly specific role of AT1R in subjective fear experience and demonstrate the feasibility of a precision approach to the affective characterization of novel receptor targets for fear in humans.


title: Optimal Control Costs of Brain State Transitions in Linear Stochastic Systems

abstract: The brain is a system that performs numerous functions by controlling its states. Quantifying the cost of this control is essential as it reveals how the brain can be controlled based on the minimization of the control cost, and which brain regions are most important to the optimal control of transitions. Despite its great potential, the current control paradigm in neuroscience uses a deterministic framework and is therefore unable to consider stochasticity, severely limiting its application to neural data. Here, to resolve this limitation, we propose a novel framework for the evaluation of control costs based on a linear stochastic model. Following our previous work, we quantified the optimal control cost as the minimal Kullback-Leibler divergence between the uncontrolled and controlled processes. In the linear model, we established an analytical expression for minimal cost and showed that we can decompose it into the cost for controlling the mean and covariance of brain activity. To evaluate the utility of our novel framework, we examined the significant brain regions in the optimal control of transitions from the resting state to seven cognitive task states in whole-brain imaging data. We found that, in realizing the different transitions, the lower visual areas played a commonly significant role in controlling the means, while the posterior cingulate cortex played a commonly significant role in controlling the covariances.


title: Temporal changes in the microglial proteome of male and female mice after a diffuse brain injury using label-free quantitative proteomics

abstract: Traumatic brain injury (TBI) triggers neuroinflammatory cascades mediated by microglia, which promotes tissue repair in the short-term. These cascades may exacerbate TBI-induced tissue damage and symptoms in the months to years post-injury. However, the progression of the microglial function across time post-injury and whether this differs between biological sexes is not well understood. In this study, we examined the microglial proteome in the days (3- and 7-days) to 1 month (28 days) after a midline fluid percussion injury (mFPI) in male and female mice using label-free quantitative proteomics. We identified a reduction in microglial proteins involved with clearance of neuronal debris via phagocytosis at 3- and 7-days post-injury. At 28 days post-injury pro-inflammatory proteins were decreased and anti-inflammatory proteins were increased in microglia. These results indicate a reduction in microglial clearance of neuronal debris in the days post-injury with a shift to anti-inflammatory function by 1 month. The changes in the microglial proteome that occurred across time post-injury did not differ between biological sexes. However, we did identify an increase in microglial proteins related to pro-inflammation as well as insulin and estrogen signalling in males compared with female mice that occurred with or without a brain injury. Although microglial response was similar between males and females up to 1 month following TBI, biological sex differences in the basal microglial proteome has implications for the efficacy of treatment strategies targeting the microglial response post-injury.


title: Minute-scale oscillatory sequences in medial entorhinal cortex

abstract: The medial entorhinal cortex (MEC) hosts many of the brains circuit elements for spatial navigation and episodic memory, operations that require neural activity to be organized across long durations of experience1. While location is known to be encoded by a plethora of spatially tuned cell types in this brain region2-6, little is known about how the activity of entorhinal cells is tied together over time. Among the brains most powerful mechanisms for neural coordination are network oscillations, which dynamically synchronize neural activity across circuit elements7-10. In MEC, theta and gamma oscillations provide temporal structure to the neural population activity at subsecond time scales1,11-13. It remains an open question, however, whether similarly powerful coordination occurs in MEC at behavioural time scales, in the second-to-minute regime. Here we show that MEC activity can be organized into a minute-scale oscillation that entrains nearly the entire cell population, with periods ranging from 10 to 100 seconds. Throughout this ultraslow oscillation, neural activity progresses in periodic and stereotyped sequences. This activity was elicited while mice ran at free pace on a rotating wheel in darkness, with no change in its location or running direction and no scheduled rewards. The oscillation sometimes advanced uninterruptedly for tens of minutes, transcending epochs of locomotion and immobility. Similar oscillatory sequences were not observed in neighboring parasubiculum or in visual cortex. The ultraslow oscillation of activity sequences in MEC may have the potential to couple its neurons and circuits across extended time scales and to serve as a scaffold for processes that unfold at behavioural time scales, such as navigation and episodic memory formation.


title: GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma

abstract:
BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of CAR-T cell therapy have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T cell therapy also remain limited.

MethodsWe profiled an extensive biobank of patients biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR-T cells from peripheral blood of glioblastoma and diffuse midline glioma patients and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control.

ResultsHere we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite glioblastoma patients having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an IL-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Markers associated with tumor control included those related to T-cell homing, infiltration, and cytotoxicity.

ConclusionsTargeting GD2 using a clinically deployed CAR-T therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.

What is already known on this topicGD2 is a tumor antigen of significant interest for targeting immunotherapy. A single preclinical study has shown the effectiveness of GD2-CAR-T cell therapy in an orthotopic xenograft model of diffuse midline glioma. Similarly, there is one previous preclinical study of GD2-CAR-T therapy in a orthotopic glioblastoma xenograft model but tumor control was achieved only following intracranial injection of CAR-T cells. Given that GD2-CAR-T therapy is already being evaluated clinically for other tumor indications, it is important to establish whether there is an acceptable rationale for its use in brain tumors.

What this study addsThis is the first description of a GD2-targeted CAR-T cell therapy that shows antitumor effectiveness in a preclinical model of human glioblastoma following intravenous administration. It is also the first study to investigate the potential effects that the immune profile of glioblastoma patients may have on the feasibility of CAR-T cell manufacturing.

How this study might affect research, practice, or policyThe results of this study have led to the initiation of an Australian phase 1 clinical trial program aiming to test GD2-specific CAR-T cells for the treatment of childhood and adult primary brain tumors. The study provides valuable insights into the microenvironmental factors that influence the effectiveness of CAR-T cell therapy for this type of tumor, paving the way for further optimization of CAR-T cell technology for treatment of aggressive primary brain tumors such as glioblastoma.


title: Inferring the T-cells repertoire dynamics of healthy individuals

abstract: The adaptive immune system is a diverse ecosystem that responds to pathogens by selecting cells with specific receptors. While clonal expansion in response to particular immune challenges has been extensively studied, we do not know the neutral dynamics that drive the immune system in absence of strong stimuli. Here we learn the parameters that underlie the clonal dynamics of the T-cell repertoire in healthy individuals of different ages, by applying Bayesian inference to longitudinal immune repertoire sequencing (RepSeq) data. Quantifying the experimental noise accurately for a given RepSeq technique allows us to disentangle real changes in clonal frequencies from noise. We find that the data are consistent with clone sizes following a geometric Brownian motion, and show that its predicted steady state is in quantitative agreement with the observed power-law behaviour of the clone-size distribution. The inferred turnover time scale of the repertoire increases substantially with patient age, and depends on the clone size in some individuals.


title: Spatial, Quantitative and Functional Deconstruction of Virus and Host Protein Interactions Inside Intact Cytomegalovirus Particles

abstract: Herpesviruses assemble large enveloped particles that are difficult to characterize structurally due to their size, fragility and complex proteome with partially amorphous nature. Here we use cross-linking mass spectrometry and quantitative proteomics to derive a spatially resolved interactome map of intact human cytomegalovirus virions. This enabled the de novo allocation of 32 viral proteins into four spatially resolved virion layers, each organized by a dominant viral scaffold protein. The viral protein UL32 engages with all layers in an N-to-C-terminal radial orientation bridging nucleocapsid to viral membrane. In addition, we observed the layer-specific recruitment of 82 host proteins, a subset of which are constitutively and selectively incorporated via specific host-virus interactions. We uncover how the recruitment of PP1 phosphatase and 14-3-3 proteins by UL32 affects early and late steps during viral biogenesis. Collectively, this study provides global structural insights into the native configuration of virus and host protein interactions inside herpesvirus particles.


title: Accelerated aging induced by stress in experimental murine ocular hypertension.

abstract: Aging, a universal process that affects all cells in an organism, is a major risk factor for a group of neuropathies called glaucoma, where elevated intraocular pressure is one of the known stresses affecting the tissue. Our understanding of molecular impact of aging on response to stress in retina is very limited, therefore we developed a new mouse model to approach this question experimentally. Here we show that susceptibility to response to stress increases with age and is primed on epigenetic level. We demonstrate that program activated by hypertension is similar to natural aging, and that one of the earliest pathways activated upon stress is senescence. Finally, we show that multiple instances of pressure elevation cause accelerated aging of young retina as measured on transcriptional and epigenetic level. Our work emphasizes the importance of early diagnosis and prevention as well as age-specific management of age-related eye-diseases, including glaucoma.