title: Vectofusin-1 based T-cell transduction approach for generating murine chimeric antigen receptor T-cells for cancer.


title: Cap analysis of gene expression (CAGE) sequencing reveals alternative promoter usage in complex disease

abstract: The role of alternative promoter usage in tissue specific gene expression has been well established, however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) tag sequencing from the left ventricle (LV) of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, the Brown Norway (BN) to understand role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites (TSS) in the rat LV, including 1,970 novel cardiac TSS resulting in new transcripts. We identified 27 genes with alternative promoter usage between SHR and BN which could lead to protein isoforms differing at the amino terminus between two strains. Additionally, we identified 475 promoter switching events where a shift in TSS usage was within 100bp between SHR and BN, altering length of the 5 UTR. Genomic variants located in the shifting promoter regions showed significant allelic imbalance in F1 crosses, confirming promoter shift. We found that the insulin receptor gene (Insr) showed a switch in promoter usage between SHR and BN in heart and liver. The Insr promoter shift was significantly associated with insulin levels and blood pressure within a panel of BXH/HXB recombinant inbred (RI) rat strains. This suggests that the hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.

category: bioinformatics


title: Quantification of circadian interactions and protein abundance defines a mechanism for operational stability of the circadian clock

abstract: The mammalian circadian clock exerts substantial control of daily gene expression through cycles of DNA binding. Understanding of mechanisms driving the circadian clock is hampered by lack of quantitative data, without which predictive mathematical models cannot be developed. Here we develop a quantitative understanding of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We have used fluorescent correlation spectroscopy (FCS) to track dynamic changes in CRISPR-modified fluorophore-tagged proteins in time and space in single cells across SCN and peripheral tissues. We determine the contribution of multiple rhythmic processes in coordinating BMAL1 DNA binding, including the roles of cycling molecular abundance, binding affinities and two repressive modes of action. We find that nuclear BMAL1 protein numbers determine corresponding nuclear CLOCK concentrations through heterodimerization and define a DNA residence time of 2.6 seconds for this complex. Repression of CLOCK:BMAL1 is in part achieved through rhythmic changes to BMAL1:CRY1 affinity as well as a high affinity interaction between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling of these data reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

category: systems biology


title: Systems analysis of the familial cardiomyopathy signaling network

abstract: Familial cardiomyopathy is a precursor of heart failure and sudden cardiac death. Over the past several decades, researchers have discovered numerous gene mutations primarily in sarcomeric and cytoskeletal proteins causing two different disease phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathies. However, molecular mechanisms linking genotype to phenotype remain unclear. Here, we employ a systems approach by integrating experimental findings into a cohesive signaling network to scrutinize genotype to phenotype mechanisms. We developed a predictive model of the HCM/DCM signaling network utilizing a logic-based differential equations approach and evaluated model performance in predicting experimental data from four contexts (HCM, DCM, pressure overload, and volume overload). The model has an overall prediction accuracy of 83.8% with higher accuracy in the HCM context (90%) compared with DCM (75%). Global sensitivity analysis identified key reactions of the signaling network, with calcium activation of myofilament force development and calcium-calmodulin kinase signaling ranking the highest. We performed a structural revision analysis to identify missing interactions in HCM/DCM signaling and found some potential reactions controlling calcium regulatory proteins. Combination pharmacotherapy analysis predicted that downregulation of signaling components such as calcium, titin and its associated proteins, growth factor receptors, and PI3K-AKT could inhibit myocyte growth in HCM. In DCM, PI3K-AKT-NFAT downregulation combined with upregulation of Ras/ERK12 or titin or Gq protein could attenuate cardiac remodeling. The influence of existing medications on cardiac growth in HCM and DCM contexts was simulated by the model. This HCM/DCM signaling model demonstrates utility in investigating genotype to phenotype mechanisms in familial cardiomyopathy.


title: Intrinsic regulators of the action potential waveform control dopamine release to shape behavior

abstract:
Despite the widely known role of dopamine in reinforcement learning, how the patterns of dopamine release that are critical to the acquisition, performance, and extinction of conditioned responses are generated is poorly resolved. Here, we demonstrate that the coordinated actions of two ion channels, Kv4.3 and BKCa1.1, control the pattern of dopamine release on different time scales to regulate separate phases of reinforced behavior in mice. Inactivation of Kv4.3 in VTA dopamine neurons increases pacemaker activity and excitability that is associated with increased ramping prior to lever press in a learned instrumental response paradigm. Loss of Kv4.3 enhanced performance of the learned response and facilitated extinction. In contrast, loss of BKCa1.1 increased burst firing and phasic dopamine release that enhanced learning of an instrumental response. Inactivation of BKCa1.1 increased the reward prediction error that was associated with an enhanced extinction burst in early extinction training. These data demonstrate that temporally distinct patterns of dopamine release are regulated by the intrinsic properties of the cell to shape behavior.

One sentence summaryWe show that ion channels in midbrain dopamine neurons are critical for patterning neurotransmitter release to regulate reinforcement learning.

category: neuroscience


title: Nuclear soluble cGAS senses DNA virus infection

abstract: The cytosolic DNA sensor cGAS detects foreign DNA from pathogens or self-DNA from cellular damage and instigates type I interferon (IFN) expression. Recent studies find that cGAS also localizes in the nucleus and binds the chromatin. Despite how cGAS is inhibited in the nucleus is well elucidated, whether nuclear cGAS participates in DNA sensing is not clear. Here, we report that herpes simplex virus 1 (HSV-1) infection caused the release of cGAS from the chromatin into the nuclear soluble fraction. Like its cytosolic counterpart, the leaked nuclear soluble cGAS could sense viral DNA, produce cGAMP, and induce mRNA expression of type I IFN and interferon-stimulated genes. Furthermore, the nuclear cGAS limited HSV-1 infection. Taken together, our study demonstrates that HSV-1 infection releases cGAS from the chromatin tethering and, in turn, the nuclear soluble cGAS activates type I IFN production.

category: immunology


title: Cerebrospinal fluid outflow through skull channels instructs cranial hematopoiesis

abstract:
Interactions between the immune and central nervous systems strongly influence brain health. Although the blood-brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues, particularly dural lymphatic vessels that allow cerebrospinal fluid outflow, facilitate intersystem communication. Here we observe that cerebrospinal fluid exits into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria usurp this perivascular route to infect the skulls hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. Because skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in cerebrospinal fluid by regional marrow has broad implications for neurological disorders.

One-Sentence SummarySkull channels transport cerebrospinal fluid from the subarachnoid space to the cranial bone marrow via a perivascular route, which bacteria use during meningitis.


title: The adaptive role of cell death in yeast communities stressed with macrolide antifungals

abstract:
Microorganisms cooperate with each other to protect themselves from environmental stressors. An extreme case of such cooperation is regulated cell death for the benefit of other cells. Dying cells can provide surviving cells with nutrients or induce their stress-response by transmitting an alarm signal; however, the role of dead cells in microbial communities is unclear. Here we searched for types of stressors the protection from which can be achieved by death of a subpopulation of cells. Thus, we compared the survival of Saccharomyces cerevisiae cells upon exposure to various stressors in the presence of additionally supplemented living versus dead cells. We found that dead cells contribute to yeast community resistance against macrolide antifungals (e.g. amphotericin B [AmB] and filipin) to a greater extent than living cells. Dead yeast cells absorbed more macrolide filipin than control cells because they exposed intracellular sterol-rich membranes. We also showed that, upon the addition of lethal concentrations of AmB, supplementation with AmB-sensitive cells but not with AmB-resistant cells enabled the survival of wild-type cells. Together, our data suggests that cell-to-cell heterogeneity in sensitivity to AmB can be an adaptive mechanism helping yeast communities to resist macrolides, which are naturally occurring antifungal agents.

ImportanceEukaryotic microorganisms harbour elements of programmed cell death (PCD) mechanisms that are homologous to the PCD of multicellular metazoa. However, it is still debated whether microbial PCD has an adaptive role or the processes of cell death are an aimless operation in self-regulating molecular mechanisms. Here, we demonstrated that dying yeast cells provide an instant benefit for their community by absorbing macrolides, which are bacteria-derived antifungals. Our results illustrate the principle that the death of a microorganism can contribute to the survival of its kin and suggest that early plasma membrane permeabilization improves community-level protection. The latter makes a striking contrast to the manifestations of apoptosis in higher eukaryotes, the process by which plasma membranes maintain integrity.

category: microbiology


title: Cardiomyocyte GSK-3β deficiency induces cardiac progenitor cell proliferation in the ischemic heart through paracrine mechanisms

abstract:
Cardiomyopathy is an irreparable loss and novel strategies are needed to induce resident cardiac progenitor cell (CPC) proliferation in situ to enhance the possibility of cardiac regeneration. Here we sought to identify the potential roles of glycogen synthase kinase-3{beta} (GSK-3{beta}), a critical regulator of cell proliferation and differentiation, in CPC proliferation post-myocardial infarction (MI).

Cardiomyocyte-specific conditional GSK-3{beta} knockout (cKO) and littermate control mice were employed and challenged with MI. Though cardiac left ventricular chamber dimension (LVID) and contractile functions were comparable at two-week post-MI, cKO mice displayed significantly preserved LV chamber and contractile function vs. control mice at four-weeks post-MI. Consistent with protective phenotypes, an increased percentage of c-kit-positive cells (KPCs) were observed in the cKO hearts at four and six-weeks post-MI which was accompanied by increased levels of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts was mainly from a cardiac lineage as the majority of identified KPCs were negative for the hematopoietic lineage marker, CD45. Mechanistically, cardiomyocyte-GSK-3{beta} profoundly suppresses the expression and secretion of growth factors, including basic-FGF angiopoietin-2, erythropoietin, stem cell factor (SCF), PDGF-BB, G-CSF, and VEGF, post-hypoxia.

In conclusion, our findings strongly suggest that loss of cardiomyocyte-GSK-3{beta} promotes cardiomyocyte and resident CPC proliferation post-MI. The induction of cardiomyocyte and CPC proliferation in the ischemic cKO hearts is potentially regulated by autocrine and paracrine signaling governed by dysregulated growth factors post-MI. A strategy to inhibit cardiomyocyte GSK-3{beta} could be helpful for the promotion of in situ cardiac regeneration post-ischemic injury.

category: molecular biology


title: Drone honey bees (Apis mellifera) are disproportionately sensitive to abiotic stressors despite expressing high levels of stress response proteins

abstract: Drone honey bees (haploid males) are the obligate sexual partners of queens, and the availability of healthy, high-quality drones directly affects a queens fecundity and productivity of her subsequent colony. Yet, our understanding of how stressors affect drone fecundity and physiology is presently limited. We investigated sex biases in susceptibility to abiotic stressors (cold stress, topical imidacloprid exposure, and topical exposure to a realistic cocktail of pesticides), and we found that drones were more sensitive to cold and imidacloprid exposure but the cocktail was not toxic at the concentrations tested. We corroborated this lack of apparent toxicity with in-hive cocktail exposures via pollen feeding. We then used quantitative proteomics to investigate protein expression profiles in the hemolymph of topically exposed workers and drones, and we show that drones express surprisingly high levels of putative stress response proteins relative to workers. Drones apparently invest in strong constitutive expression of damage-mitigating proteins for a wide range of stressors, yet they are still sensitive to stress when challenged. The robust expression of stress-response proteins suggests that drone stress tolerance systems are fundamentally rewired relative to workers, and their susceptibility to stress depends on more than simply gene dose or deleterious recessive alleles.

category: cell biology


title: Cohesin is essential for the timely activation of condensin and anaphase segregation

abstract:
Chromosome segregation requires the separation of sister chromatids and the sustained condensation of chromatids during anaphase. Yeast cohesin has a dual function on metaphase chromosomes, holding sister chromatids together and organising individual chromatids into loops. Cleavage by separase is thought to remove all cohesin from chromosomes at the anaphase onset. This fulfils the requirement for chromatid separation, but it is unclear how the structure of segregating chromatids is maintained following cleavage. Here we demonstrate that auxin-mediated degradation of cohesins kleisin in anaphase, causes catastrophic chromosome mis-segregation demonstrating that cohesin affects post-metaphase processes. We identify a pool of cohesin bound to anaphase/telophase chromosomes and demonstrate that its inactivation causes defects in the organisation of centromeric regions and condensin function. Our data thus uncovers an unsuspected role for cohesin on anaphase/telophase chromosomes that is essential for the fidelity of segregation.

One sentence summaryCohesin complexes on segregating chromosomes provide centromere structure and regulate condensin activity, demonstrating that cohesin has active roles after metaphase.


title: Local mechanical stimuli shape tissue growth in vertebrate joint morphogenesis

abstract: The correct formation of synovial joints is essential for proper motion throughout life. Movement-induced forces are critical to creating correctly shaped joints, but it is unclear how cells sense and respond to these mechanical cues. To determine how mechanical stimuli drive joint morphogenesis, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a TRPV4 agonist to impair chondrocyte mechanosensitivity during joint morphogenesis. We quantified growth and shape in regrown humeri from whole mount light sheet fluorescence images of the regenerated limbs. Results revealed statistically significant differences in morphology and cell proliferation between groups, indicating that mechanical stimuli play a role in the shaping of joints. We simulated local tissue growth in a finite element model with a biological contribution to growth proportional to chondrocyte density, and a mechanical contribution to growth proportional to fluid pressure. Computational predictions agreed with experimental outcomes, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.

category: developmental biology


title: Endothelial cell signature in muscle stem cells showed by VEGFA-FLT1-AKT1 axis for muscle stem cell survival

abstract: Endothelial and skeletal muscle lineages arise from common embryonic progenitors. Despite their shared developmental origin, adult endothelial cells (ECs) and muscle stem cells (MuSCs) (satellite cells) have been thought to possess distinct gene signatures and signaling pathways. Here we shift this paradigm by uncovering how adult MuSC behavior is affected by the expression of a subset of EC transcripts. We used several computational analyses including single-cell RNAseq to show that MuSCs express low levels of canonical EC markers. We demonstrate that MuSC survival is regulated by one such prototypic endothelial signaling pathway (VEGFA-FLT1). Using pharmacological and genetic gain- and loss-of-function studies, we identify the FLT1-AKT1 axis as the key effector underlying VEGFA-mediated regulation of MuSC survival. All together, our data support that the VEGFA-FLT1-AKT1 pathway promotes MuSC survival during muscle regeneration, and highlights how the minor expression of select transcripts is sufficient for affecting cell behavior.


title: Endothelial cell signature in muscle stem cells validated by VEGFA-FLT1-AKT1 axis promoting survival of muscle stem cell


title: The growth and expansion of meningeal lymphatic networks are affected in craniosynostosis

abstract: Congenital skull malformations are associated with vascular anomalies that can impair fluid balance in the central nervous system. We previously reported that humans with craniosynostosis and mutations in TWIST1 have dural venous sinus malformations. It is still unknown whether meningeal lymphatic networks, which are patterned alongside the venous sinuses, are also affected. Using a novel skull flat mounting technique, we show that the growth and expansion of meningeal lymphatics are perturbed in Twist1 craniosynostosis models. Changes to the local meningeal environment, including hypoplastic dura and venous malformations, affect the ability of lymphatic networks to sprout and remodel. Dorsal networks along the transverse sinus are hypoplastic with reduced branching. By contrast, basal networks closer to the skull base are more variably affected, showing exuberant growth in some animals suggesting they are compensating for vessel loss in dorsal networks. Injecting molecular tracers into cerebrospinal fluid reveals significantly less drainage to the deep cervical lymph nodes, indicative of impaired lymphatic function. Collectively, our results show that meningeal lymphatic development is hindered in craniosynostosis, suggesting central nervous system waste clearance may be impeded.


title: Protein-Metabolite Interactomics Reveals Novel Regulation of Carbohydrate Metabolism

abstract: Metabolism is highly interconnected and also has profound effects on other cellular processes. However, the interactions between metabolites and proteins that mediate this connectivity are frequently low affinity and difficult to discover, hampering our understanding of this important area of cellular biochemistry. Therefore, we developed the MIDAS platform, which can identify protein-metabolite interactions with great sensitivity. We analyzed 33 enzymes from central carbon metabolism and identified 830 protein-metabolite interactions that were mostly novel, but also included known regulators, substrates, products and their analogs. We validated previously unknown interactions, including two atomic-resolution structures of novel protein-metabolite complexes. We also found that both ATP and long-chain fatty acyl-CoAs inhibit lactate dehydrogenase A (LDHA), but not LDHB, at physiological concentrations in vitro. Treating cells with long-chain fatty acids caused a loss of pyruvate/lactate interconversion, but only in cells reliant on LDHA. We propose that these regulatory mechanisms are part of the metabolic connectivity that enables survival in an ever-changing nutrient environment, and that MIDAS enables a broader and deeper understanding of that network.

category: biochemistry


title: SampleQC: robust multivariate, multi-celltype, multi-sample quality control for single cell data

abstract: Quality control (QC) is a critical component of single-cell RNA-seq (scRNA-seq) processing pipelines. Current approaches to QC implicitly assume that datasets are comprised of one celltype, potentially resulting in biased exclusion of rare celltypes. We introduce SampleQC, which robustly fits a Gaussian mixture model across multiple samples, and improves sensitivity and reduces bias compared to current approaches. We show via simulations that SampleQC is less susceptible to exclusion of rarer celltypes. We also demonstrate SampleQC on a complex real dataset (867k cells over 172 samples). SampleQC is general, is implemented in R, and could be applied to other data types.


title: Human transcriptional gene regulatory network compiled from 14 data resources

abstract: Transcriptional regulatory network (TRN) orchestrates spatio-temporal expression of genes to generate cellular responses for survival. The transcription factors (TF) regulating expression of their target genes (TG) are the fundamental units of TRN. Several databases have been developed to catalogue human TRN based on low- and high-throughput experimental and computational studies considering their importance in understanding cellular physiology. However, literature lacks comparative assessment on the strength and weakness of each database. In this study, we compared over 2.2 million regulatory pairs between 1,379 TF and 22,518 TG assembled from 14 data resources. Our study reveals that the TF and TG were common across data resources but not their regulatory pairs. We observed that the TF and TG of the regulatory pairs showed weak expression correlation, significant gene ontology overlap, co-citations in PubMed and low numbers of TF-TG pairs representing transcriptional repression relationships. Furthermore, each TF-TG regulatory pair assigned a combined confidence score reflecting its reliability based on its presence in multiple databases and co-expression. The TRN containing 2,246,598 TF-TG pairs, of which, 44,284 with the information on TFs activating or repressing effects on their TG is available upon request. This study brings the information about transcriptional regulation scattered over the literature and databases at one place in the form of one of the most comprehensive and complete human TRNs assembled to date, which will be a valuable resource for benchmarking TRN prediction tools, and to the scientific community working in functional genomics, gene expression and gene regulation analysis.


title: Membrane cholesterol interferes with tyrosine phosphorylation but facilitates the clustering and signal transduction of EGFR

abstract: Epidermal growth factor receptor (EGFR) activates major cell signaling pathways that regulate various cell responses. Its dimerization and clustering coupled with its lateral mobility are critical for EGFR function, but the contribution of the plasma membrane environment to EGFR function is unknown. Here we show, using single-molecule analysis, that EGFR mobility and clustering are altered by the depletion of cholesterol or sphingomyelin, major lipids of membrane subdomains, causing significant changes in EGFR signaling. When cholesterol was depleted, the subdomain boundary in EGFR diffusion disappeared, the fraction of EGFR pre-dimers was increased, and the ligand-induced phosphorylation of EGFR was enhanced. In addition, the depletion of either lipid prevented the formation of immobile clusters after EGF association and decreased the phosphorylation of downstream proteins. Our results revealed that cholesterol plays dichotomous roles in the signaling pathway of EGFR and that clustering in the membrane subdomains is critical for EGFR signal transduction.

category: biophysics


title: Experimental and mathematical models of intracellular calcium dynamics for evaluating combined anticancer effects of Afatinib and RP4010 in esophageal cancer

abstract: Targeting dysregulated Ca2+ signaling in cancer cells is an emerging chemotherapy approach. We previously reported that store-operated Ca2+ entry (SOCE) blockers, such as RP4010, are promising antitumor drugs for esophageal cancer. As a tyrosine kinase inhibitor (TKI), afatinib received FDA approval to be used in targeted therapy for patients with EGFR mutation-positive cancers. While preclinical studies and clinical trials have shown that afatinib has benefits for esophageal cancer patients, it is not known whether combination of afatinib and RP4010 could achieve better anticancer effects. Since TKI can alter intracellular Ca2+ dynamics through EGFR/phospholipase C-{gamma} pathway, in this study, we evaluated the inhibitory effect of afatinib and RP4010 on intracellular Ca2+ oscillations in esophageal cancer cells using both experimental and mathematical simulations. Our mathematical simulation of Ca2+ oscillations could fit well with experimental data responding to aftinib or RP4010, separately or in combination. The results showed that combination of afatinib and RP4010 presented synergistic anticancer effect. This intracellular Ca2+ dynamic-based mathematical simulation approach could be useful for a rapid and cost-effective evaluation of combined targeting therapy drugs.

category: cancer biology


title: Balancing growth amidst salinity stress-lifestyle perspectives from the extremophyte model Schrenkiella parvula

abstract:
Schrenkiella parvula, a leading extremophyte model in Brassicaceae, can grow and complete its life cycle under multiple environmental stresses, including high salinity. While foundational genomic resources have been created for S. parvula, a comprehensive physiological or structural characterization of its salt stress responses is absent. We aimed to identify the influential traits that lead to stress-resilient growth of this species. We examined salt-induced changes in the physiology and anatomy of S. parvula throughout its lifecycle across multiple tissues. We found that S. parvula maintains or even exhibits enhanced growth during various developmental stages at salt stress levels known to inhibit growth in Arabidopsis and most crops. The resilient growth of S. parvula was associated with key traits that synergistically allow continued primary root growth, expansion of xylem vessels across the root-shoot continuum, and a high capacity to maintain tissue water levels by developing larger and thicker leaves while facilitating continued photosynthesis during salt stress. These traits at the vegetative phase were followed by a successful transition to the reproductive phase via early flowering, development of larger siliques, and production of viable seeds during salt stress. Additionally, the success of self-fertilization during early flowering stages was dependent on salt-induced filament elongation in flowers that aborted in the absence of salt. Our results suggest that the maintenance of leaf water status and enhancement of selfing in early flowers to ensure reproductive success, are among the most influential traits that contribute to the extremophyte lifestyle of S. parvula in its natural habitat.

One sentence summarySchrenkiella parvula salt-resilient growth is facilitated by uncompromised primary root growth, expansion of xylem vessels, maintenance of leaf water status and photosynthesis, and early flowering.

category: plant biology


title: Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

abstract:
Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse {beta}-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug- resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

IMPACT STATEMENTDisruption of disulfide bond formation sensitizes resistant Gram- negative bacteria expressing {beta}-lactamases and mobile colistin resistance enzymes to currently available antibiotics.


title: Analyses of the 5-prime Ends of Escherichia coli ORFs

abstract: Sequence biases at 5 ends of coding sequences differ from those of the remainder of ORFs, reflecting differences in function. Internal sequence biases promote translational efficiency by several mechanisms including correlating codon usage and tRNA concentration. However, the early region may also facilitate translational initiation, establishment of the reading frame, and polypeptide processing. Here we examine the beginnings of the ORFs of an Escherichia coli K12 reference genome. The results extend previous observations of A-richness to include an overabundance of the AAA triplet in all reading frames, consistent with the hypothesis that the beginnings of ORFs contribute to initiation site accessibility. Results are also consistent with the idea that the first two amino acids are under selection because they facilitate solvation of the amino-terminus at the end of the ribosomal exit channel. Moreover, serine is highly overrepresented as the second amino acid, possibly because it can facilitate removal of the terminal formylmethionine. Non-AUG initiation codons are known to be less efficient than AUG at directing initiation, presumably because of relatively weak base pairing to the initiator-tRNA. But non-UAG initiation codons are not followed by unusual 3 nearest neighbor codons. Moreover, the four NUG initiation codons do not differ in their propensity to frameshift in an assay known to be sensitive to base pair strength. Altogether, these data suggest that the 5 ends of ORFs are under selection for several functions, and that initiation codon identity may not be critical beyond its role in initiation.


title: Tip growth in the brown alga Ectocarpus is controlled by a RHO-GAP-BAR domain protein independently from F-actin organisation

abstract: The brown alga Ectocarpus is a filamentous seaweed that grows by tip growth and branching. In the morphometric mutant etoile, tip growth is slower than in the WT and eventually stops. In this paper, we show that the causal etoile mutation is a null mutation in a bi-domain BAR-RhoGAP gene. By quantitative RT-PCR, we showed that ETOILE is ubiquitously expressed in prostrate filaments of the Ectocarpus sporophyte, and is downregulated in the etoile mutant. We immunolocalised both domains of the protein in WT and etoile, as well as RAC1, the known target of Rho-GAP enzymes. Thus, ETOILE would be localised at the apical cell dome where it would control the localisation of EsRAC1 to the plasma membrane. Actin staining showed that the mutant is not affected in F-actin structures. Overall, these results suggest that in Ectocarpus, BAR-RhoGAP controls tip growth by controlling RAC1 localization and through an actin-independent mechanism.


title: The rise of grasslands is linked to atmospheric CO2 decline in the late Palaeogene

abstract: Grasslands are predicted to experience a major biodiversity change by the year 2100 in part due to recent and projected increases in atmospheric CO2 concentration. A better understanding of how grasslands have responded to past environmental changes will help predict the outcome of current and future environmental changes. Here, we explore the relationship between past atmospheric CO2 and temperature fluctuations and the shifts in diversification rate of grasses (Poaceae) and daisies (Asteraceae), two exceptionally species-rich grassland families ([~]11,000 and [~]23,000 species, respectively). To this end, we developed a novel Bayesian approach that simultaneously estimates diversification-rates through time from time-calibrated phylogenies and correlations between environmental variables and diversification rates. Additionally, we developed a new statistical approach that incorporates the information of the distribution of missing species in the phylogeny. We found strong evidence supporting a simultaneous increase in diversification rates for daisies and grasses after the most significant reduction of atmospheric CO2 in the Cenozoic ([~]34 Mya). The fluctuations of paleo-temperatures, however, appear not to have had a significant relationship with the diversification of these grassland families. Overall, our results shed new light on our understanding of the origin of grasslands in the context of past environmental changes.

category: evolutionary biology


title: Tailored extracellular electron transfer pathways enhance the electroactivity of Escherichia coli

abstract: Extracellular electron transfer (EET) engineering in Escherichia coli holds great potential for bioremediation, energy and electrosynthesis applications fueled by readily available organic substrates. Due to its vast metabolic capabilities and availability of synthetic biology tools to adapt strains to specific applications, E. coli is of advantage over native exoelectrogens, but limited in electron transfer rates. We enhanced EET in engineered strains through systematic expression of electron transfer pathways differing in cytochrome composition, localization and origin. While a hybrid pathway harboring components of an E. coli nitrate reductase and the Mtr complex from the exoelectrogen Shewanella oneidensis MR-1 enhanced EET, the highest efficiency was achieved by implementing the complete Mtr pathway from S. oneidensis MR1 in E. coli. We show periplasmic electron shuttling through overexpression of a small tetraheme cytochrome to be central to the electroactivity of this strain, leading to enhanced degradation of the pollutant methyl orange and significantly increased electrical current to graphite electrodes.

category: bioengineering


title: Dual avatars of E. coli grxB encoded Glutaredoxin 2 perform ascorbate recycling and ion channel activities

abstract: Glutaredoxins (Grxs) are single-domain redox enzymes of the thioredoxin superfamily, and primarily function as glutathione (GSH) dependent disulphide reductases. Whereas, the E. coli Glutaredoxin 2 (EcGrx2) encoded by grxB has two conserved GST-fold domains, it still lacks a classical Grx-like functions. In this study, we show for the first time, that EcGrx2 exists in both soluble and membrane integrated forms. The soluble form associates with a previously unidentified GSH dependent dehydroascrobate (DHA) reductase, and the membrane integrated form possesses ion channel activities. Using enzyme kinetic data and structural data we unequivocally demonstrate that EcGrx2 recycles ascorbate (AsA) from DHA. This ability to recycle AsA is inhibited by Zinc (Zn2+). We also show that both wildtype and the E. coli grxB deletion mutant can be rescued from H2O2-induced oxidative stress using ascorbate as an antioxidant, which otherwise is only known as a carbon source in bacteria. Moreover, the grxB- mutant is susceptible to intracellular killing by ROS producing macrophages. We further discovered that EcGrx2 integrates into the native E. coli membrane and show that the purified soluble protein readily inserts into artificial lipid bilayer membrane and conducts ions in vitro. Our data demonstrates a highly conserved functional similarity among EcGrx2-orthologs and highlights that the utilization and subsequent recycling of ascorbate as an antioxidant by grxB harbouring gram-negative bacteria, including human pathogens, may provide a survival advantage under hostile oxidative environments.


title: One-step Isolation of Protein C-terminal Peptides from V8 Protease-digested Proteins by Metal Oxide-based Ligand Exchange Chromatography

abstract: We have developed a one-step method to isolate protein C-terminal peptides from V8 protease-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, affording a digested peptide with two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one -carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the non-terminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 g of peptides (10 g each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 {micro}g of HeLa peptides was further divided into six fractions by high-pH reversed-phase LC prior to nanoLC/MS/MS, 2202 protein C-termini were identified with less than 3% contamination with internal peptides. We believe this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.


title: Pathway profiling of a novel SRC inhibitor, AZD0424, in combination with MEK inhibitors.

abstract: A more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments allows us anticipate how cells adapt to targeted therapy enabling more informed prediction of rational drug combinations. The non-receptor tyrosine kinase SRC regulates many cellular signalling processes and pharmacological inhibition has long been a target of drug discovery projects for the treatment of cancer. Here we describe the in vitro and in vivo characterisation of the small molecule SRC inhibitor, AZD0424. We show that AZD0424 potently inhibits the phosphorylation of tyrosine-416 of SRC (IC50 [~] 100 nM) in many cancer cell lines; however inhibition of cell viability, via a G1 cell cycle arrest, was observed only in a sub-set of cancer cell lines in the low (on target) micromolar range. We profiled the changes in intracellular pathway signalling in cancer cells following exposure to AZD0424 and other targeted therapies using Reverse Phase Protein Array analysis. We demonstrate that SRC is activated in response to MEK inhibitor (trametinib or AZD6244)-treatment of KRAS mutant colorectal cell lines (HCT116 and DLD1) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 revealed reduction of EGFR, FAK and SRC compensatory activation, and, synergistically inhibits cell viability in vitro. In vivo, trametinib-treatment of mice bearing HCT116 tumours increased phosphorylation of SRC on Tyr416, and when combined with AZD0424, inhibition of tumour growth is greater than trametinib alone. We also demonstrate that drug-induced resistance to trametinib is not re-sensitised by AZD0424 treatment in vitro, likely as a result of multiple compensatory signalling mechanisms; however inhibition of SRC remains an effective way to block invasion of trametinib resistant tumour cells. These data imply that inhibiting SRC may offer a useful addition to MEK inhibitor combination strategies.


title: Cholinergic interneurons mediate cocaine extinction through similar plasticity across medium spiny neuron subtypes

abstract:
Cholinergic interneurons (ChINs) in the nucleus accumbens (NAc) have been implicated in the acquisition and extinction of drug associations, as well as related plasticity in medium spiny neurons (MSNs). However, since most previous work has relied on artificial manipulations, if and how endogenous patterns of cholinergic signaling relate to drug associations is unknown. Moreover, despite great interest in the opposing effects of dopamine on MSN subtypes, whether ChIN-mediated effects are similar or different across MSN subtypes is also unknown. Here, we find that endogenous acetylcholine event frequency during extinction negatively correlates with the strength and persistence of cocaine-context associations across individuals, consistent with effects of artificial manipulation of ChIN activity during extinction. Moreover, ChIN activation during extinction produces a reduction in excitatory synaptic strength on both MSN subtypes, similar to the effect of multiple extinction sessions in the absence of ChIN manipulations. Together, our findings indicate that natural variation in NAc acetylcholine may contribute to individual differences in drug-context extinction by modulating glutamatergic presynaptic strength similarly at both D1R and D2R MSN subtypes.

Graphical abstract

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title: Grid-cell modules remain coordinated when neural activity is dissociated from external sensory cues

abstract: The representation of an animals position in the medial entorhinal cortex (MEC) is distributed across several modules of grid cells, each characterized by a distinct spatial scale. The population activity within each module is tightly coordinated and preserved across environments and behavioral states. Little is known, however, about the coordination of activity patterns across modules. We analyzed the joint activity patterns of hundreds of grid cells simultaneously recorded in animals that were foraging either in the light, when sensory cues could stabilize the representation, or in darkness, when such stabilization was disrupted. We found that the states of different grid modules are tightly coordinated, even in darkness, when the internal representation of position within the MEC deviates substantially from the true position of the animal. These findings suggest that internal brain mechanisms dynamically coordinate the representation of position in different modules, to ensure that grid cells jointly encode a coherent and smooth trajectory of the animal.


title: Harnessing Matrix Completion to Unify and Extend Viral Serology Studies

abstract:
The development of new vaccine strategies, as well as our understanding of key processes that shape viral evolution and host antibody repertoires, rely on measuring multiple antibody responses against large panels of viruses. Given the enormous diversity of circulating virus strains and antibody responses, exhaustive testing of all antibody-virus interactions is infeasible. Even within individual studies with limited panels, exhaustive testing is not always done, and there is no common framework for combining information across studies with partially overlapping panels, especially when the assay type or host species differ. Results from prior studies have demonstrated that virus-antibody interactions can be characterized in a vastly simpler and lower-dimensional space, suggesting that relatively few measurements could accurately predict unmeasured antibody-virus interactions. Here, we apply matrix completion to several of the largest-scale studies for both influenza and HIV-1. We explore how prediction accuracy evolves as the number of available measurements changes and approximate the number of additional measurements necessary in several highly incomplete datasets (suggesting [~]250,000 measurements could be saved). In addition, we show how the method can combine disparate datasets, even when the number of available measurements is below the theoretical limit that guarantees successful prediction. This approach can be readily generalized to other viruses or more broadly to other low-dimensional biological datasets.

SignificanceOne of the central problems in immunology is to characterize how our vast array of antibodies inhibits the diverse pathogens we encounter in our lives. In this work, we apply a well-studied mathematical technique called matrix completion that leverages patterns in partially-observed antibody-virus inhibition data to infer unmeasured interactions. We predict the results of tens of thousands of missing experiments in influenza and HIV-1 studies and quantify the expected error of our estimates. By harnessing matrix completion, future experiments could be designed that only collect a fraction of measurements, saving time and resources while maximizing the information gained.


title: Stochastic microbiome assembly depends on context

abstract:
Observational studies reveal substantial variability in microbiome composition across individuals. While some of this variability can be explained by external factors like environmental, dietary, and genetic differences between individuals, in this paper we show that for the model organism Drosophila melanogaster the process of microbiome assembly is inherently stochastic and contributes a baseline level of microbiome variability even among organisms that are identically reared, housed, and fed. In germ-free flies fed known combinations of bacterial species, we find that some species colonize more frequently than others even when fed at the same high concentration. We develop a new ecological technique that infers the presence of interactions between bacterial species based on their colonization odds in different contexts, requiring only presence/absence data from two-species experiments. We use a progressive sequence of probabilistic models, in which the colonization of each bacterial species is treated as an independent stochastic process, to reproduce the empirical distributions of colonization outcomes across experiments. We find that incorporating context-dependent interactions substantially improves the performance of the models. Stochastic, context-dependent microbiome assembly underlies clinical therapies like fecal microbiota transplantation and probiotic administration, and should inform the design of synthetic fecal transplants and dosing regimes.

Significance StatementIndividuals are constantly exposed to microbial organisms that may or may not colonize their gut microbiome, and newborn individuals assemble their microbiomes through a number of these acquisition events. Since microbiome composition has been shown to influence host physiology, a mechanistic understanding of community assembly has potentially therapeutic applications. In this paper we study microbiome acquisition in a highly-controlled setting using germ-free fruit flies inoculated with specific bacterial species at known abundances. Our approach revealed that acquisition events are stochastic, and the colonization odds of different species in different contexts encode ecological information about interactions. These findings have consequences for microbiome-based therapies like fecal microbiota transplantation that attempt to modify a persons gut microbiome by deliberately introducing foreign microbes.


title: Adaptive nanopore sequencing on miniature flow cell detects extensive antimicrobial resistence

abstract: Rapid screening of hospital admissions to detect asymptomatic carriers of resistant bacteria can prevent pathogen outbreaks. However, the resulting isolates rarely have their genome sequenced due to cost constraints and long turn-around times to get and process the data, limiting their usefulness to the practitioner. Here we use real-time, on-device target enrichment ("adaptive") sequencing on a new type of low-cost nanopore flow cell as a highly multiplexed assay covering 1,147 antimicrobial resistance genes. Using this method, we detected three types of carbapenemase in a single isolate of Raoultella ornithinolytica (NDM, KPC, VIM). Further investigation revealed extensive horizontal gene transfer within the underlying microbial consortium, increasing the risk of resistance spreading. From a technical point of view, we identify two important variables that can increase the enrichment of target genes: higher nucleotide identity and shorter read length. Real-time sequencing could thus quickly inform how to monitor this case and its surroundings.


title: HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration

abstract:
ABSTRACT

HIV-1 replication is durably controlled in certain untreated HIV-1-infected individuals expressing particular human leukocyte antigens (HLA). These HLAs tag infected cells for elimination by presenting specific viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). In individuals expressing HLA-B27, CTLs primarily target the capsid protein (CA)-derived KK10 epitope. Selection of CA mutation R264K helps HIV-1 escape the CTL response but severely diminishes virus infectivity. Here we report that the R264K mutation-associated infectivity defect arises primarily from impaired viral DNA integration. Strikingly, selection of the compensatory CA mutation S173A or depletion of host cyclophilin A largely rescues the R264K-associated integration and infectivity defects. Collectively, our study reveals novel mechanistic insights into the fitness defect incurred by an HIV-1 variant escaping a CA-directed CTL response.


title: Rethinking symbiotic metabolism: trophic strategies in the microbiomes of different sponge species

abstract: In this study we describe the major lithoheterotrophic and autotrophic processes in 21 microbial sponge-associated phyla using novel and existing genomic and transcriptomic datasets. We show that a single gene family, molybdenum-binding subunit of dehydrogenase (coxL), likely evolved to benefit both lithoheterotrophic and organoheterotrophic symbionts, through adaptation to different inorganic and organic substrates. We show the main microbial carbon fixation pathways in sponges are restricted to specialized symbiotic lineages within five phyla. We also propose that sponge symbionts, in particular Acidobacteria, are capable of assimilating carbon through anaplerotic processes. However, the presence of symbionts genomically capable of autotrophy does not inform on their actual contribution to light and dark carbon fixation. Using radioisotope assays we identified variability in the relative contributions of chemosynthesis to total carbon fixation in different sponge species. Furthermore, the symbiosis of sponges with two closely related Cyanobacteria results in outcomes that are not predictable by analysis of -omics data alone: Candidatus Synechococcus spongiarum contributes to the holobiont carbon budget by transfer of photosynthates, while Candidatus Synechococcus feldmannii does not. Our results highlight the importance of combining sequencing data with physiology to gain a broader understanding of carbon metabolism within holobionts characterized by highly diverse microbiomes.


title: Transcriptional organization and regulation of the Pseudomonas putida flagellar system

abstract:
A single region of the Pseudomonas putida genome, designated the flagellar cluster, includes 59 genes potentially involved in the biogenesis and function of the flagellar system. Here we combine bioinformatics and in vivo gene expression analyses to clarify the transcriptional organization and regulation of the flagellar genes in the cluster. We have identified eleven flagellar operons and characterized twenty-two primary and internal promoter regions. Our results indicate that synthesis of the flagellar apparatus and core chemotaxis machinery is regulated by a three-tier cascade in which fleQ is a Class I gene, standing at the top of the transcriptional hierarchy. FleQ- and{sigma} 54-dependent Class II genes encode most components of the flagellar structure, part of the chemotaxis machinery and multiple regulatory elements, including the flagellar{sigma} factor FliA. FliA activation of Class III genes enables synthesis of the filament, one stator complex and completion of the chemotaxis apparatus. Accessory regulatory proteins and an intricate operon architecture add complexity to the regulation by providing feedback and feed-forward loops to the main circuit. Because of the high conservation of the gene arrangement and promoter motifs, we believe that the regulatory circuit presented here may also apply to other environmental pseudomonads.

ORIGINALITY-SIGNIFICANCE STATEMENTThis is the first integrative study of the flagellar transcriptional cascade in Pseudomonas putida. Our results provide a new transcriptional organization featuring several operons with a nested architecture, detailed regulatory characterization of twenty-two flagellar promoters and a novel hierarchy for the regulatory circuit of the flagellar transcriptional cascade. The results presented represent a significant departure from previous models for other related bacteria. High conservation of flagellar gene organization and promoter sequences suggest that our observations may be relevant to other pseudomonads.


title: Implication of the cellular factor CTCF in the regulation of Bovine Leukemia Virus latency and tridimensional chromatin organization

abstract: Bovine Leukemia Virus (BLV)-induced tumoral development is a multifactorial phenomenon which remains largely unelucidated. Here, we highlighted the critical role of the cellular CCCTC-binding factor (CTCF) both in the regulation of BLV transcriptional activities and in the deregulation of the tridimensional (3D) chromatin architecture surrounding the BLV integration site. We demonstrated the in vivo recruitment of CTCF to three conserved CTCF binding motifs along the BLV provirus. Next, we showed a critical role for CTCF in delimitating the epigenetic landscape along the BLV provirus as well as to repress the 5Long Terminal Repeat (LTR) promoter activity, thereby contributing to viral latency, while favoring the 3LTR promoter activity. Finally, we demonstrated that BLV integration deregulated host cellular 3D chromatin organization through the formation of abnormal viral/host chromatin loops. Altogether, our results highlight CTCF as a new critical effector of BLV transcriptional regulation and BLV-induced physiopathology.


title: Mitochondrial mRNA is a stable and convenient reference for the normalization of RNA-seq data

abstract: The normalization of high-throughput RNA sequencing (RNA-seq) data is needed to accurately analyze gene expression levels. Traditional normalization methods can either correct the differences in sequencing depth, or correct both the sequencing depth and other unwanted variations introduced during sequencing library preparation through exogenous spike-ins1-4. However, the exogenous spike-ins are prone to variation5,6. Therefore, a better normalization approach with a more appropriate reference is an ongoing demand. In this study, we demonstrated that mitochondrial mRNA (mRNA encoded by mitochondria genome) can serve as a steady endogenous reference for RNA-seq data analysis, and performs better than exogenous spike-ins. We also found that using mitochondrial mRNA as a reference can reduce batch effects for RNA-seq data. These results provide a simple and practical normalization strategy for RNA-seq data, which will serve as a valuable tool widely applicable to transcriptomic studies.


title: Interactions between RNA m6A modification, alternative splicing, and poly(A) tail revealed by MePAIso-seq2

abstract: RNA post-transcriptional regulation involves 5'-end capping, 3' poly(A) tailing (including polyadenylation sites, tail length, and non-A residues1, 2), alternative splicing, and chemical modifications including N6-methyladenosine (m6A)3. Studying the interplay of m6A, alternative splicing, alternative polyadenylation sites, poly(A) tail length, and non-A residues in poly(A) tails requires monitoring them simultaneously on one transcript, however strategies to achieve this are lacking. Therefore, we developed a new method, combining m6A-specific methylated RNA immunoprecipitation and the PacBio-based, tail-included, full-length RNA sequencing approach PAIso-seq2, which weve named m6A and poly(A) inclusive RNA isoform sequencing 2 (MePAIso-seq2). Using MePAIso-seq2, we revealed that m6A promotes and inhibits a similar number of alternative splicing events in mouse cell lines, showing that m6A does affect alternative splicing. In contrast, no correlation was detected between m6A and alternative polyadenylation sites choice. Surprisingly, we found that m6A-modified RNAs possess longer poly(A) tails and a lower proportion of poly(A) tails containing non-A residues, especially in mouse embryonic stem cells. Together, we developed a new method to detect full-length m6A-modified RNAs to comprehensively study the relationships between m6A, alternative splicing, and poly(A) tailing, laying a foundation for further exploration of the functional coordination of different RNA post-transcriptional modifications.


title: Direct multiplex recombinase polymerase amplification for rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa in food

abstract: Foodborne illness undermines human health by causing fever, stomachache and even lethality. Among foodborne bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa are of extraordinary significance which drive reasons of food and beverage poisoning in numerous cases. Today, PCR has been widely used to examine the presence of different foodborne pathogens. However, PCR requires specialized equipment and skillful personnel which limit its application in the field. Recently, there is an emerging of isothermal PCR methods in which the reactions occur at low and constant temperature, allowing their application in restricted-resource settings. In this work, multiplex Recombinase Polymerase Amplification (RPA) was used to simultaneously detect S. aureus and P. aeruginosa with high sensitivity and specificity. The limit detection of multiplex RPA was 10 and 30 fg/reaction of genomic DNAs of S. aureus and P. aeruginosa, respectively. Besides, the reaction time was reduced to only 25 minutes with a low incubation temperature of 39 {degrees}C. Markedly, multiplex RPA reactions succeeded to directly detect as low as 1 and 5 CFU/reaction of S. aureus and P. aeruginosa cells, respectively without the requirement of extracting DNA genome. Moreover, the multiplex RPA reliably detected the two foodborne bacteria in milk, fruit juice and bottled water samples. In general, the direct multiplex RPA described in this study is a rapid, simple, sensitive and efficient alternative tool that could be used to detect the presence of S. aureus and P. aeruginosa without the necessity of costly devices and high-trained staff.


title: Comprehensive analysis of mRNA poly(A) tail reveals complex and conserved regulation

abstract: Non-templated poly(A) tails are added to the 3'-end of most mRNAs, which have important roles in post-transcriptional regulation1-3. Recent studies have revealed that poly(A) tails are not composed purely of A residues, but also contain U, C and G residues internally and at their 3'-ends4-6, revealing new levels of complexity. However, no method is able to analyze these internal and terminal non-A residues simultaneously. Here, we developed a new method called unbiased capture of 3-terminal followed by full-length RNA sequencing (UCTF-seq) which captures RNA 3'-ends by direct 3' adaptor ligation and rRNA removal by CRISPR/Cas9. This method allows simultaneous evaluation of the poly(A) tail length and 5'-end, internal, and 3'-end non- A residues together with the full-length cDNA for a transcript. Applying this method, we achieved the first complete transcriptome-wide 3' tail map of mRNA within the nuclear and cytoplasmic compartments of mammalian cells, uncovering differences in poly(A) tail length and non-A residues between these two mRNA populations. A survey of diverse eukaryotic species revealed the conservation of a subset of poly(A) tails containing consecutive U residues in the internal positions, whereas those with consecutive C or G residues were of much lower abundance. Together, we established the first method to be able to comprehensively analyze poly(A) tail 5'-end, internal and 3'-end non-A residues in addition to the length simultaneously, and reveal the first complete mRNA 3' tail map, providing rich insights into the regulatory roles of poly(A) tails.


title: Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

abstract: Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.


title: Cell tip growth underlies injury response of marine macroalgae

abstract: Regeneration is a widely observed phenomenon by which the integrity of an organism is recovered after damage. So far, studies on the molecular and cellular mechanisms of regeneration have been limited to a handful of model multicellular organisms. Here, we systematically surveyed the regeneration ability of marine macroalgae (Rhodophyta, Phaeophyceae, Chlorophyta) after thallus severing and applied live cell microscopy on them to uncover the cellular response to the damage. We observed three types of responses - budding, rhizoid formation and/or sporulation - in 25 species among 66 examined, demonstrating the high potential of regeneration of macroalgae. In contrast, callus formation, which often accompanies plant regeneration, was never observed. We monitored the cellular and nuclear dynamics during cell repair or rhizoid formation of four phylogenetically diverged Rhodophyta and Chlorophyta species (Colaconema sp., Dasya sessilis, Cladophora albida, Codium fragile). We observed tip growth of the cells near the damaged site as a common response, despite the difference in the number of nuclei and cells across species. Nuclear translocation follows tip growth, enabling overall uniform distribution of multinuclei (Dasya sessilis, Cladophora albida, Codium fragile) or central positioning of the mononucleus (Colaconema sp.). In contrast, the control of cell cycle events, such as nuclear division and septation, varied in these species. In Dasya sessilis, the division of multinuclei was synchronised, whereas it was not the case in Cladophora albida. Septation was tightly coupled with nuclear division in Colaconema and Dasya but not in others. These observations show that marine macroalgae utilise a variety of regeneration pathways, with some common features. This study also provides a novel methodology of live cell biology in macroalgae, offering a foundation for the future of this under-studied taxon.


title: Adipocyte-Specific Ablation Of PU.1 Promotes Energy Expenditure and Ameliorates Metabolic Syndrome In Aging Mice

abstract:
ObjectiveAlthough PU.1/Spi1 is known as a master regulator for macrophage development and function, we have reported previously that it is also expressed in adipocytes and is transcriptionally induced in obesity. Here, we investigated the role of adipocyte PU.1 in the development of age-associated metabolic syndrome.

MethodsWe generated mice with adipocyte specific PU.1 knockout, assessed metabolic changes in young and aged PU.1fl/fl (control) and AdipoqCre PU.1fl/fl(aPU.1KO) mice, including body weight, body composition, energy expenditure and glucose homeostasis. We also performed transcriptional analyses using RNA-Sequencing of adipocytes from these mice.

ResultsaPU.1KO mice have elevated energy expenditure at a young age and decreased adiposity and increased insulin sensitivity in later life. Corroborating these observations, transcriptional network analysis indicated the existence of validated, aPU.1-modulated regulatory hubs that direct inflammatory and thermogenic gene expression programs.

ConclusionsOur data provide evidence for a previously uncharacterized role of PU.1 in the development of age-associated obesity and insulin resistance.


title: Unique Microglial Transcriptomic Signature within the Hippocampal Neurogenic Niche

abstract: Microglia, the resident immune cells of the brain, are crucial in the development of the nervous system. Recent evidence demonstrates that microglia modulate adult hippocampal neurogenesis by inhibiting cell proliferation of neural precursors and survival both in vitro and in vivo, thus maintaining a balance between cell division and cell death in the neural stem cell pool. There are increasing reports suggesting these microglia found in neurogenic niches differ from their counterparts in non-neurogenic areas. Here, we present evidence that microglia in the hippocampal neurogenic niche are a specialized population that express genes known to regulate neurogenesis. By comprehensively profiling myeloid lineage cells in the hippocampus using single cell RNA-sequencing, we resolve transcriptomic differences in microglia originating from the subgranular zone. These cells have lower expression of genes associated with homeostatic microglia and increased expression of genes associated with phagocytosis. Intriguingly, this small yet distinct population expresses a gene signature with substantial overlap with previously characterized phenotypes, including disease associated microglia (DAM), a particularly unique and compelling microglial state.


title: Re-polyadenylation occurs predominantly on maternal mRNA degradation intermediates during mammalian oocyte-to-embryo transition

abstract: The nascent mRNA transcribed in the nucleus is cleaved and polyadenylated before it is transported to the cytoplasm for translation1. Polyadenylation can also occur in the cytoplasm for post-transcriptional regulation, especially in neurons, oocytes and early embryos1,2. Recently, we revealed transcriptome-wide maternal mRNA cytoplasmic re-polyadenylation during the mammalian oocyte-to-embryo transition (OET)3-6. However, the mechanism of re-polyadenylation during mammalian OET, including the sites to be re-polyadenylated and the enzymes involved, is still poorly understood. Here, by analyzing the PAIso-seq1 and PAIso-seq2 poly(A) inclusive transcriptome data during OET in mice, rats, pigs, and humans, we reveal conserved re-polyadenylation of mRNA degradation intermediates. These re-polyadenylated mRNA degradation intermediates account for over half of the polyadenylated mRNA during OET in all four species. We find that mRNA degradation intermediates for re-polyadenylation are generated through Btg4-mediated deadenylation in both mouse and human. Interestingly, the poly(A) tails on the re-polyadenylated mRNA degradation intermediates are of different lengths and contain different levels of non-A residues compared to regular polyadenylation sites, suggesting specific regulation and function of these poly(A) tails in mammalian OET. Together, our findings reveal the maternal mRNA degradation intermediates as substrates for conserved cytoplasmic dominant re-polyadenylation during mammalian OET, and uncover the mechanism of production of these mRNA degradation intermediates. These findings provide new insights into mRNA post-transcriptional regulation, and a new direction for the study of mammalian OET.


title: The Need for Transfer Learning in CRISPR-Cas Off-Target Scoring

abstract:
MotivationThe scalable design of safe guide RNA sequences for CRISPR gene editing depends on the computational "scoring" of DNA locations that may be edited. As there is no widely accepted benchmark dataset to compare scoring models, we present a curated "TrueOT" dataset that contains thoroughly validated datapoints to best reflect the properties of in vivo editing. Many existing models are trained on data from high throughput assays. We hypothesize that such models may suboptimally transfer to the low throughput data in TrueOT due to fundamental biological differences between proxy assays and in vivo behavior. We developed new Siamese convolutional neural networks, trained them on a proxy dataset, and compared their performance against existing models on TrueOT.

ResultsOur simplest model with a single convolutional and pooling layer surprisingly exhibits state-of-the-art performance on TrueOT. Adding subsequent layers improved performance on a proxy dataset while compromising performance on TrueOT. We demonstrate improved generalization on TrueOT with a Siamese model of higher complexity when we apply transfer learning techniques. These results suggest an urgent need for the CRISPR community to agree upon a benchmark dataset such as TrueOT and highlight that various sources of CRISPR data cannot be assumed to be equivalent.

Availability and ImplementationOur code base and datasets are available on GitHub at github.com/baolab-rice/CRISPR_OT_scoring.


title: ATM phosphorylates PP2A subunit A resulting in nuclearexport and spatiotemporal regulation of the DNA damageresponse

abstract: Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phosphomimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.


title: Conserved secreted effectors determine endophytic growth and multi-host plant compatibility in a vascular wilt fungus

abstract: Fungal interactions with plant roots, either beneficial or detrimental, have a major impact on agriculture and ecosystems. The soil inhabiting ascomycete Fusarium oxysporum (Fo) constitutes a species complex of worldwide distribution causing vascular wilt in more than a hundred different crops. Individual isolates of the fungus exhibit host-specific pathogenicity, determined by proteinaceous effectors termed secreted in xylem (SIX). However, such isolates can also colonize roots of non-host plants asymptomatically as endophytes, or even protect them against pathogenic isolates. The molecular determinants of multi-host plant colonization are currently unknown. Here, we identified a set of fungal effectors termed ERCs (Early Root Compatibility effectors), which are secreted during early biotrophic growth of Fo on both host and non-host plants. In contrast to the strain-specific SIX effectors, which are encoded on accessory genomic regions, ERCs are encoded on core regions and are found across the entire Fo species complex as well as in other phytopathogens, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence on the host plant and rapid activation of plant immune responses, while in a non-pathogenic isolate it led to impaired root colonization and loss of biocontrol ability. Strikingly, some ERCs also contribute to Fo infection on the non-vascular land plant Marchantia polymorpha. Our results reveal an evolutionarily conserved mechanism for multi-host colonization by root infecting fungi.


title: Structured proteins are abundant in unevolved sequence space

abstract:
Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution.

category: synthetic biology


title: Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles


title: Deep brain stimulation in the subthalamic nucleus for Parkinson's disease can restore dynamics of striatal networks

abstract: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is highly effective in alleviating movement disability in patients with Parkinsons disease (PD). However, its therapeutic mechanism of action is unknown. The healthy striatum exhibits rich dynamics resulting from an interaction of beta, gamma and theta oscillations. These rhythms are at the heart of selection, initiation and execution of motor programs, and their loss or exaggeration due to dopamine (DA) depletion in PD is a major source of the behavioral deficits observed in PD patients. Interrupting abnormal rhythms and restoring the interaction of rhythms as observed in the healthy striatum may then be instrumental in the therapeutic action of DBS. We develop a biophysical networked model of a BG pathway to study how abnormal beta oscillations can emerge throughout the BG in PD, and how DBS can restore normal beta, gamma and theta striatal rhythms. Our model incorporates STN projections to the striatum, long known but understudied, that were recently shown to preferentially target fast spiking interneurons (FSI) in the striatum. We find that DBS in STN is able to normalize striatal medium spiny neuron (MSN) activity by recruiting FSI dynamics, and restoring the inhibitory potency of FSIs observed in normal condition. We also find that DBS allows the re-expression of gamma and theta rhythms, thought to be dependent on high DA levels and thus lost in PD, through cortical noise control. Our study shows how BG connectivity can amplify beta oscillations, and delineates the role of DBS in disrupting beta oscillations and providing corrective input to STN efferents to restore healthy striatal dynamics. It also suggests how gamma oscillations can be leveraged to enhance or supplement DBS treatment and improve its effectiveness.


title: Proteomic phenotyping of stimulated Mueller cells uncovers profound pro-inflammatory signaling and antigen-presenting capacity

abstract: Muller cells are the main macroglial cells of the retina exerting a wealth of functions to maintain retinal homoeostasis. Upon pathological changes in the retina, they become gliotic with both protective and detrimental consequences. Accumulating data also provide evidence for a pivotal role of Muller cells in the pathogenesis of diabetic retinopathy (DR). While microglial cells, the resident immune cells of the retina are considered as main players in inflammatory processes associated with DR, the implication of activated Muller cells in chronic retinal inflammation remains to be elucidated. In order to assess the signaling capacity of Muller cells and their role in retinal inflammation, we performed in-depth proteomic analysis of Muller cell proteomes and secretomes after stimulation with INF{gamma}, TNF, IL-4, IL-6, IL-10, VEGF, TGF{beta}1, TGF{beta}2 and TGF{beta}3. We used both, primary porcine Muller cells and the human Muller cell line MIO-M1 for our hypothesis generating approach. Our results point towards an intense signaling capacity of Muller cells, which reacted in a highly discriminating manner upon treatment with different cytokines. Stimulation of Muller cells resulted in a primarily pro-inflammatory phenotype with secretion of cytokines and components of the complement system. Furthermore, we observed evidence for mitochondrial dysfunction, implying oxidative stress after treatment with the various cytokines. Finally, both MIO-M1 cells and primary porcine Muller cells showed several characteristics of atypical antigen-presenting cells, as they are capable of inducing MHC class I and MHC class II with co-stimulatory molecules. In line with this, they express proteins associated with formation and maturation of phagosomes. Thus, our findings underline the importance of Muller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation underlying the pathogenesis of diabetic retinopathy.


title: Proteomic phenotyping of stimulated Müller cells uncovers profound pro-inflammatory signaling and antigen-presenting capacity


title: A novel regulatory gene promotes novel cell fate by suppressing ancestral fate in the sea anemone Nematostella vectensis

abstract:
Cnidocytes ("stinging cells") are an unequivocally novel cell type used by cnidarians (corals, jellyfish, and their kin) to immobilize prey. Although they are known to share a common evolutionary origin with neurons, the developmental program that promoted the emergence of cnidocyte fate is not known. Using functional genomics in the sea anemone, Nematostella vectensis, we show that cnidocytes evolved by suppression of neural fate in a subset of neurons expressing RFamide. We further show that a single regulatory gene, a C2H2-type zinc finger transcription factor (ZNF845), coordinates both the gain of novel (cnidocyte-specific) traits and the inhibition of ancestral (neural) traits during cnidocyte development and that this gene arose by domain shuffling in the stem cnidarian. Thus, we uncover a mechanism by which a truly novel regulatory gene (ZNF845) promoted the origin of a truly novel cell type (cnidocyte) through duplication of an ancestral cell lineage (neuron) and inhibition of its ancestral identity (RFamide).

SignificanceIn this study, we demonstrate how new cell types can arise in animals through duplication of an ancestral (old) cell type followed by functional divergence of the new daughter cell. Specifically, we show that stinging cells in cnidarians (jellyfish and corals) evolved by duplication of an ancestral neuron followed by inhibition of the RFamide neuropeptide it once secreted. This is the first evidence that stinging cells evolved from a specific subtype of neurons and suggests some neurons may be easier to co-opt for novel functions than others.


title: The transcription factor ELT-2 positively and negatively impacts direct target genes to modulate the Caenorhabditis elegans intestinal transcriptome

abstract: The transcription factor ELT-2 is the major factor required for Caenorhabditis elegans intestinal development. ELT-2 expression initiates in embryos, promoting development, but persists after hatching through larval and adult stages, where it contributes to diverse intestinal functions. How ELT-2s regulatory role changes over development is largely unexplored. We sought to determine whether ELT-2 target gene preference changes over developmental time and whether those changes underlie transcriptome dynamics. We analyzed stage-specific ELT-2 ChIP-seq assays to identify sets of dynamically changing ELT-2 peaks. Embryo-specific and larval-specific ELT-2 peaks were less common than peaks that accumulated in the L3 stage or progressively throughout development. To assess how ELT-2 occupancy related to transcriptional output, we compared patterns of ELT-2 occupancy over developmental time to intestinal expression dynamics and sets of ELT-2-dependent genes. ELT-2 occupancy predicted gene expression changes at larval but not embryonic stages, suggesting that ELT-2 promotes transcriptional output in some cases. However, we also found that ELT-2 negatively regulated a subset of its direct target genes. Repressed target genes were over-represented for neuronal and gonad functions, signifying that ELT-2 may protect the intestine from these tissue fates. Furthermore, we observed that ELT-2 repressed its own promoter in a negative feedback loop to stabilize the elt-2 genes expression to a set amount. These findings illustrate that ELT-2 exerts both positive and negative regulatory control - turning on some target genes to promote intestinal function while down-regulating others to prevent or reduce their transcriptional output.

category: genomics


title: Endothelial integration of mechanosensory signals by the spectrin cytoskeleton

abstract: Physiological blood flow induces the secretion of vasoactive compounds, notably NO, and promotes endothelial cell elongation and reorientation parallel to the direction of applied shear. How shear is sensed and relayed to intracellular effectors is incompletely understood. We demonstrate that an apical spectrin network is essential to convey the force imposed by shear to endothelial mechanosensors. By anchoring CD44, spectrin modulates the cell surface density of hyaluronan, which senses and translates shear into changes in plasma membrane tension. Spectrins also regulate the stability of apical caveolae, where the mechanosensitive Piezo1 channels are thought to reside. Accordingly, shear-induced Piezo1 activation and the associated calcium influx were absent in spectrin-deficient cells. As a result, cell realignment and flow-induced eNOS stimulation were similarly dependent on spectrin. We concluded that the apical spectrin network is not only required for shear sensing, but transmits and distributes the resulting tensile forces to mechanosensors that elicit protective and vasoactive responses.


title: Maternal mRNA deadenylation is defective in in vitro matured mouse and human oocytes

abstract: Oocyte in vitro maturation is a technique of assisted reproductive technology that was first introduced in patients with polycystic ovarian syndrome and is now used in most fertility clinics. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (in vitro MII) oocytes compared with in vivo maturated metaphase II (in vivo MII) oocytes in bovines, mice, and humans1-3. However, the underlying mechanisms of this abnormal expression are still poorly understood. In this study, we use PAIso-seq1 to reveal a transcriptome-wide expression profile of full-length transcripts containing entire poly(A) tails in in vivo and in vitro matured mouse and human oocytes. Our results indicate that more genes are up-regulated than down-regulated in in vitro MII oocytes in both mice and humans. Furthermore, we demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes in both mice and humans. We also found that the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes in mice and humans respectively, likely contributing to reduced translation and impaired global maternal mRNA deadenylation. Our findings highlight that impaired maternal mRNA deadenylation is a definite molecular defect in in vitro MII oocytes in both mice and humans. The findings here offer a new criterion for evaluating the quality of in vitro MII oocytes and a potential direction for improving in vitro maturation by fixing the dysregulated maternal mRNA deadenylation.


title: Systemic delivery of a CXCR4-CXCL12 signaling inhibitor encapsulated in synthetic protein nanoparticles for glioma immunotherapy

abstract:
Glioblastoma multiforme (GBM) is an aggressive primary brain tumor, with poor prognosis. Major obstacles hampering effective therapeutic response in GBM are tumor heterogeneity, high infiltration of immunosuppressive myeloid cells, and the presence of the blood-brain barrier. The C-X-C Motif Chemokine Ligand 12/ C-X-C Motif Chemokine Receptor 4 (CXCL12/ CXCR4) signaling pathway is implicated in GBM invasion and cell cycle progression. While the CXCR4 antagonists (AMD3100) has a potential anti-GBM effects, its poor pharmacokinetic and systemic toxicity had precluded its clinical application. Moreover, the role of CXCL12/ CXCR4 signaling pathway in anti-GBM immunity, particularly in GBM-mediated immunosuppression has not been elucidated. Here, we developed a synthetic protein nanoparticle (SPNPs) coated with the cell-penetrating peptide iRGD (AMD3100 SPNPs) to target the CXCR4/CXCL12 signaling axis in GBM. We showed that AMD3100 SPNPs effectively blocked CXCR4 signaling in mouse and human GBM cells in vitro as well as in GBM model in vivo. This results in inhibition of GBM proliferation and induction of immunogenic tumor cell death (ICD) leading to inhibition of GBM progression. Our data also demonstrate that blocking CXCR4 sensitizes GBM cells to radiation, eliciting enhanced release of ICD ligands. Combining AMD3100 SPNPs with radiotherapy inhibited GBM progression and led to long-term survival; with 60% of mice remaining tumor-free. This was accompanied by an anti-GBM immune response and sustained immunological memory that prevented tumor recurrence without further treatment. Finally, we showed that systemic delivery of AMD3100 SPNPs decreased the infiltration of CXCR4+ monocytic myeloid-derived suppressor cells to the tumor microenvironment. With the potent ICD induction and reprogrammed immune microenvironment, this strategy has significant potential for future clinical translation.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/457953v1_ufig1.gif" ALT="Figure 1">
View larger version (48K):
org.highwire.dtl.DTLVardef@17c9377org.highwire.dtl.DTLVardef@1b5a4dcorg.highwire.dtl.DTLVardef@5aa4eaorg.highwire.dtl.DTLVardef@1011a86_HPS_FORMAT_FIGEXP  M_FIG Immunological mechanism targeting Glioblastoma (GBM) upon blocking CXCR4 signaling pathway with AMD3100-conjugated nanoparticles (SPNPs).

(1) Radiotherapy induces glioma cell death, followed by Damage-associated molecular patterns (DAMPs) release. Dendritic cells (DC) are activated by DAMPs and migrate to the regional lymph node where they prime cytotoxic T lymphocyte immune response. Tumor-specific cytotoxic T cells infiltrate the tumor and attack glioma cells. (2) Glioma cells express CXCR4, as well its ligand CXCL12. CXCL12 induces glioma cell proliferation and, (3) as well as mobilization in the bone marrow of CXCR4 expressing myeloid MDSC, which will infiltrate the tumor, and inhibit tumor-specific cytotoxic T cells activity. GEMM of glioma when treated systemically with SPNPs AMD3100 SPNPs plus radiation, nanoparticles block the interaction between CXCR4 and CXCL12, thus (4) inhibiting glioma cell proliferation and (5) reducing mobilization in the bone marrow of CXCR4 expressing myeloid MDSC, (6) generating a reduced MDSC tumor infiltration, as well as releasing MDSC inhibition over tumor specific cytotoxic T cell response.

C_FIG


title: Metabolic regulation of species-specific developmental rates

abstract: Animals display significant inter-specific variation in the rate of embryonic development despite broad conservation of the overall sequence of developmental events. Differences in biochemical reaction speeds, including the rates of protein production and degradation, are thought to be responsible for distinct species-specific rates of development [1-3]. However, the cause of differential biochemical reaction speeds between species remains unknown. Using pluripotent stem cells, we have established an in vitro system that recapitulates the two-fold difference in developmental rate between early mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we showed that mass-specific metabolic rates scale with developmental rate and are therefore elevated in mouse cells compared to human cells. We further showed that reducing these metabolic rates by pharmacologically inhibiting the electron transport chain slows down the segmentation clock. The effect of the electron transport chain on the segmentation clock is mediated by the cellular NAD+/NADH redox balance independent of ATP production and, further downstream, by the global rate of protein synthesis. These findings represent a starting point for the manipulation of developmental rate, which would find multiple translational applications including the acceleration of human pluripotent stem cell differentiation for disease modeling and cell-based therapies.


title: Recurrent allopolyploidization events diversify eco-physiological traits in marsh orchids

abstract:
Whole-genome duplication, in particular allopolyploidy, has shaped the evolution of angiosperms and other organisms. Structural reorganization of chromosomes and repatterning of gene expression is frequently observed in early generation allopolyploids, with potential ecological consequences. The relative contributions of environmental and intrinsic drivers to these changes are unknown. We show here that in marsh orchids (Dactylorhiza, Orchidaceae), recurrently-formed allopolyploids are characterized by distinct eco-physiologies matching their respective environments, providing us with an excellent study system to address this question. Here we integrate eco-physiological and transcriptomic comparative studies to reveal a complex suite of intertwined, pronounced differences between sibling allopolyploids. We show that Dactylorhiza majalis that is distributed in Central and Southern Europe favors meadows with mesic soils. Its sibling allopolyploid D. traunsteineri occurs in fens, characterized by soils depleted by macro- and micronutrients, mainly in previously glaciated European areas. We further uncover distinct features in their nutrient transport, leaf elemental chemistry, light-harvesting, photoprotection, and stomata activity, that appear all linked to their distinct ecologies, in particular soil chemistry differences at their native sites. Recurrent polyploidization hence enriches biodiversity through eco-physiological diversification, providing the opportunity for sibling allopolyploids to evolve as distinct evolutionary units, despite pervasive interspecific gene flow.

Significance StatementWhole-genome duplication resulting in polyploidy has underpinned the evolution of flowering plants and other organisms, and is important for many crops. However, the ecological implications of polyploidy remain little understood. Here, we demonstrate that two sibling allopolyploid marsh orchid species prefer distinct habitats, and have evolved a suite of distinctive ecophysiological characters (e.g. nutrient transport, energy harvesting and photoprotection). We argue that the divergence of these characters in the nascent polyploids drove adaptation into distinct ecological niches (low nutrient fens versus meadows with mesic soils), generating ecological barriers that maintains distinct, independent lineages, even in the presence of interspecific gene flow.


title: To draw or not to draw: understanding the temporal organization of drawing behaviour using fractal analyses

abstract: Studies on drawing often focused on spatial aspects of the finished products. Here, the drawing behaviour was studied by analysing its intermittent process, between drawing (i.e. marking a surface) and interruption (i.e. a pause in the marking gesture). To assess how this intermittence develops with age, we collected finger-drawings on a touchscreen by 185 individuals (children and adults). We measured the temporal structure of each drawing sequence to determine its complexity. To do this, we applied temporal fractal estimators to each drawing time series before combining them in a Principal Component Analysis procedure. The youngest children (3 years-old) drew in a more stereotypical way with long-range dependence detected in their alternations between states. Among older children and adults, the complexity of drawing sequences increased showing a less predictable behaviour as their drawings become more detailed and figurative. This study improves our understanding of the temporal aspects of drawing behaviour, and contributes to an objective understanding of its ontogeny.

category: animal behavior and cognition


title: Differential Binding Kinetics for Evaluating Immune Checkpoint Inhibitors in Serum

abstract:
The ability to characterize the binding kinetics of drug-target interactions in a biologically relevant matrix, such as serum or plasma, remains a fundamental challenge in drug discovery. We apply a novel label-based giant magnetoresistance (GMR) biosensor platform to measure protein binding kinetics and affinities of drug-target pairs in buffer and different levels of serum. Specifically, we evaluate three well-established immune checkpoint inhibitors, pembrolizumab, nivolumab and atezolizumab and compare the results with label-free kinetic platforms: surface plasmon resonance (SPR) and bio-layer interferometry (BLI). Labeling of analytes does not affect their association and dissociation rates (on and off rates) from GMR biosensors which enables kinetic measurements in biologically relevant matrices. Only the GMR biossensors is consistently suitable for measuring binding kinetics in up to 80% serum. The faster and different off-rates of the three immune checkpoint inhibitors in the presence of serum should be considered when modeling their pharmacological performance.

TeaserWe reveal the effects of serum on binding kinetics of antibody drugs, relevant to the pharmacological performance of immunotherapeutic.