title: Pleiotropic effects on E. coli physiology of the AraC-like regulator from prophage origin, AppY

abstract: Bacterial genome diversity is largely due to prophages, which are viral genomes integrated into the genomes of bacteria. Most prophage genes are silent, but those that are expressed can provide unexpected properties to their host. Using as a model E. coli K-12, that carries 9 defective prophages in its genome, we aimed at highlighting the impact of viral genes on host physiology. We focused our work on AppY, a transcriptional regulator encoded on the DLP12 prophage. By performing RNA-Seq experiments, we showed that AppY production modulates the expression of more than 200 genes; among them, 13 were identified by ChIP-Seq as direct AppY targets. AppY directly and positively regulates several genes involved in the acid stress response including the master regulator gene gadE, but also nhaR and gadY, two genes involved in biofilm formation. Moreover, AppY indirectly and negatively impacts bacterial motility by favouring the degradation of FlhDC, the master regulator of the flagella biosynthesis. As a consequence of these regulatory effects, AppY increased acid stress resistance and biofilm formation while also causing a strong defect in motility. We therefore demonstrate here that AppY is a central regulator from phage origin that controls the expression of bacterial master regulators to provide benefits to E. coli under stress conditions. Our research shed light on the importance to consider the genetic dialogue occurring between prophages and bacteria to fully understand bacterial physiology.


title: First report of kdr L1014F and ace-1 G119S insecticide resistance in Belgian Culex mosquitoes

abstract:
The emergence of West Nile virus and Usutu virus in Europe is a significant risk to public health. In the absence of efficient antiviral therapy or vaccine candidates, the only strategy to control these arboviruses is to target the Culex mosquito vector. However, the selection pressure caused by exposure to insecticides in vector control or agricultural pest control can lead to the development of insecticide resistance reducing the efficacy of insecticide-based vector control interventions. In Culex mosquitoes, two of the most common amino acid substitutions associated with insecticide resistance are the kdr L1014F in the voltage gated sodium channel and G119S in the acetylcholinesterase.

In this study, Culex pipiens biotype pipiens, Culex torrentium, and Culex modestus were sampled from 2019 to 2021 in three distinct environmental habitats (urban, peri-urban, and agricultural) in and around the city of Leuven in Belgium. Two mutations resulting in L1014F and G119S amino acid substitutions were screened for in individual mosquitoes. Both mutations were observed in Culex pipiens and Culex modestus mosquitoes across the four collection sites, but not in Culex torrentium. Furthermore, multi-resistance or cross-resistance could be a developing threat, since both mutations were observed at low frequencies (resistant allele frequency <30%) and always in heterozygosity.

These results provide the first report of kdr L1014F and ace-1 G119S resistance in Culex pipiens and Culex modestus mosquitoes from Belgium, highlighting the importance of mosquito surveillance for the development of effective arbovirus control strategies in the event of an outbreak in Belgium.

category: molecular biology


title: A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

abstract: cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum {Delta}F/F0 of 1100% in HEK293T cells), relatively high brightness, appropriate affinity (a Kd of 2.17 {micro}M) and fast response kinetics (an association and dissociation half-time of 0.20 s and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted {beta}-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.


title: Integrative analysis of transcriptomics in human craniofacial development reveals novel candidate disease genes

abstract: Craniofacial disorders are among the most common of all congenital defects. A majority of craniofacial development occurs early in pregnancy and to fully understand how craniofacial defects arise, it is essential to observe gene expression during this critical time period. To address this we performed bulk and single-cell RNA-seq on human craniofacial tissue from embryonic development 4 to 8 weeks post conception. This data comprises the most comprehensive profiling of the transcriptome in the early developing human face to date. We identified 239 genes that were specifically expressed in craniofacial tissues relative to dozens of other human tissues and stages. We found that craniofacial specific enhancers are enriched within 400kb of these genes establishing putative regulatory interactions. To further understand how genes are organized in this program we constructed coexpression networks. Strong disease candidates are likely genes that are coexpressed with many other genes, serving as regulatory hubs within these networks. We leveraged large functional genomics databases including GTEx and GnomAD to reveal hub genes that are specifically expressed in craniofacial tissue and genes which are resistant to mutation in the normal healthy population. Our unbiased method revealed dozens of novel disease candidate genes that warrant further study.

category: genomics


title: Dual species transcriptomics reveals core metabolic and immunologic processes in the interaction between primary human neutrophils and Neisseria gonorrhoeae strains

abstract: Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe and lifelong clinical sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains that are resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs or neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing (RNA-seq) was used to define Gc and PMN transcriptional profiles alone and after infection. Three strains of Gc and three human donors transcriptional responses were assessed to characterize core host and bacterial responses. Comparative analysis of Gc transcripts revealed major overlap between the Gc response to PMNs, iron, and hydrogen peroxide; specifically, the TonB system and TonB dependent transporters (TDT) were upregulated in response to PMNs. We experimentally confirmed that induction of the iron-dependent TDT TbpB is responsive to the presence of PMNs and that tonB is required for Gc survival from PMNs. Pathway analysis of PMN transcripts induced by Gc infection revealed differential expression of genes driving pathways involved in cell adhesion and migration, inflammatory responses, and inflammation resolution. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and the anti-inflammatory prostaglandin PGE2 was confirmed to be induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of three isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease in humans.


title: An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

abstract: In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The DNA-bound state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (K9ac, K14ac, K18ac) is linked to increased engagement of H3K4me3 by the BPTF PHD finger, but it is unknown if this mechanism has broader extension. Here we show that cis H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and further extends to H3K4 writers, notably methyltransferase MLL1. This regulation is nucleosome-dependent and also observed in vivo, where H3 acetylation correlates with increased levels of cis H3K4me. These observations reveal an acetylation  chromatin switch on the H3 N-terminal tail that modulates the accessibility and function of H3K4 methylation in chromatin. Our findings also resolve the long-standing question of why H3K4me3 levels are linked with H3 acetylation.

category: biochemistry


title: Mechanistic insights into the functioning of GMP synthetase: a two-subunit, allosterically regulated, ammonia tunnelling enzyme

abstract:
Guanosine 5-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5-monophosphate (XMP) to GMP are comprised of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase and the ammonia thus generated is tunnelled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia tunnelling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channelling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure and cross-linking mass spectrometry guided integrative modelling. pH dependence of enzyme kinetics establish that ammonia is tunnelled across the subunits with the lifetime of the complex being [≤] 0.5 s. The crystal structure of XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.

Graphical Abstract

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title: Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly-dT termination signals

abstract: RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on poly-thymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryo-EM structures of the yeast Pol III PTC and complementary functional states at 2.7-3.9 [A] resolution. Pol III recognizes the poly-dT termination signal with subunit C128 that forms a hydrogen-bond network with the NT-strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly-dT signals.


title: Structural analysis of the bacterial amyloid curli

abstract: Two decades have passed since the initial proposition that amyloids are not only (toxic) byproducts of an unintended aggregation cascade, but that they can also be produced by an organism to serve a defined biological function. That revolutionary idea was borne out of the realization that a large fraction of the extracellular matrix that holds Gram-negative cells into a persistent biofilm is composed of protein fibers (curli; tafi) with cross-{beta} architecture, nucleation-dependent polymerization kinetics and classic amyloid tinctorial properties. The list of proteins shown to form so-called  functional amyloid fibers in vivo has greatly expanded over the years, but detailed structural insights have not followed at a similar pace, in part due to the associated experimental barriers. Here we combine extensive AlphaFold2 modelling and cryo-electron transmission microscopy to propose an atomic model of curli protofibrils, and their higher modes of organization. We uncover an unexpected structural diversity of curli building blocks and fibril architectures. Our results allow for a rationalization of the extreme physico-chemical robustness of curli, as well as earlier observations of inter-species curli promiscuity, and should facilitate further engineering efforts to expand the repertoire of curli-based functional materials.


title: Improvements on Sample Preparation and Peptide Separation for Reduced Peptide Mapping Based Multi-Attribute Method analysis of Therapeutic Monoclonal Antibodies Using Lys-C Digestion

abstract: The mass spectrometry based multi-attribute method (MAM) has gained popularity in the field of biopharmaceutical analysis as it promises a single method for comprehensive monitoring of multiple product quality attributes (PQAs) and product purity. Sample preparation for protein digestion and peptide separation are critical considerations for a reduced peptide mapping-based MAM. To avoid desalting steps required in tryptic protein digestion and in order to improve peptide separation for hydrophilic peptides, we developed an improved robust sample preparation using Lys-C protease for high-throughput MAM testing. Additionally, this method optimizes the peptide retention and separation of a stability-indicating VSNK peptide using a HSS T3 column for comprehensive PQA monitoring. A fully automated sample preparation had similar assay variations for PQAs monitoring compared to manual sample preparation. To the best of our knowledge, this is the first report of a high-resolution MS-based MAM using Lys-C digestion with enhanced PQA monitoring for hydrophilic peptides. The improved, robust MAM workflow for protein digestion and peptide separation will pave the way for broader MAM qualification and its applications for the characterization and quality control of therapeutic monoclonal antibodies.


title: Stress induced TDP-43 mobility loss independent of stress granules

abstract: TAR DNA binding protein 43 (TDP-43) is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS) and translocates to stress granules (SGs). The role of SGs as aggregation-promoting "bioreactors" for TDP-43, however, is still under debate. We analyzed TDP-43 mobility and localization under different stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides reduced mobility within SGs, a stress induced decrease of TDP-43 mobility in the cytoplasm and the nucleus was observed. Stress removal led to a recovery of TDP-43 mobility, which strongly depended on the stress duration.  Stimulated-emission depletion microscopy (STED) and  tracking and localization microscopy (TALM) revealed not only TDP-43 substructures within stress granules but also numerous patches of slow TDP-43 species throughout the cytoplasm. The data provide new insights into the aggregation of TDP-43 in living cells and provide evidence suggesting that TDP-43 oligomerization takes place in the cytoplasm separate from SGs.

category: biophysics


title: A cooperative switch within the KaiC hexamer revealed by cryo-EM

abstract: The circadian clock of cyanobacteria is based on an approximately 24h rhythm in the phosphorylation level of KaiC, a hexameric ATPase. This oscillation can be reconstituted in vitro by incubating three proteins together with ATP. Like all chemical oscillators, this system must include a nonlinear, or switch-like, feedback loop, whose nature has been unclear. Here, by using single particle cryo-EM at near-atomic resolution we identified two major conformational states of KaiC subunits, denoted as the exposed state and the buried state, which may provide a structural basis of how the KaiC hexamer changes its conformation during the (day-night) phosphorylation-dephosphorylation cycle. We classify the abundance and pattern of exposed and buried states within hexamers for more than 160,000 KaiC particles. The statistics of the spatial arrangement of the two states in hexamers can be quantitatively fit by a simple statistical physics model with an interaction energy between neighboring subunits and a local field that depends on phosphorylation state. Our study shows that phosphorylation shifts the probability of each conformation and reveals that there is substantial cooperativity between neighboring subunits, which can allow a KaiC hexamer to respond in an ultrasensitive, switch-like manner to changes in the phosphorylation level.


title: "Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAFV600E-Driven Lung Tumorigenesis."

abstract:
Mutationally-activated BRAFV600E is detected in ~2% of all human non-small cell lung cancers (NSCLC), and serves as a predictive biomarker for treatment of patients with FDA-approved pathway-targeted therapies that inhibit signaling by the BRAFV600E oncoprotein kinase. In genetically engineered mouse (GEM) models, expression of BRAFV600E in alveolar type 2 (AT2) pneumocytes initiates the development of benign lung tumors that, without additional genetic alterations, rarely progress to malignant lung adenocarcinomas. To identify genes that might cooperate with BRAFV600E for malignant lung cancer progression we employed Sleeping Beauty (SB)-mediated transposon mutagenesis, which dramatically accelerated the onset of lethal lung adenocarcinomas. Amongst the diverse group of genes identified by this in vivo screen was Rbms3 (RNA binding motif single-stranded interacting protein 3), an RNA-binding protein implicated as a possible tumor suppressor. Using CRISPR/CAS9 gene editing we confirmed that RBMS3 silencing cooperated with BRAFV600E to promote progression of malignant lung cancer with a distinct micropapillary architecture. Moreover, RBMS3 silencing also cooperated with BRAFV600E to promote the growth of lung organoids in vitro. BRAFV600E/RBMS3Null lung tumors displayed elevated expression of b-catenin (CTNNB1), suggesting that RBMS3 silencing may result in elevated signaling through the WNT>CTNNB1>c-MYC pathway. Finally, analyses of patient samples in The Cancer Genome Atlas (TCGA) revealed that the region of chromosome 3 encompassing RBMS3 is frequently lost in NSCLC and correlates with poor patient prognosis. Collectively, SB-mediated transposon mutagenesis has revealed the ability of a novel tumor suppressor, RBMS3, to cooperate with BRAFV600E to promote lung carcinogenesis, and suggests that RBMS3 silencing may contribute to malignant progression of numerous human lung cancers.

SIGNIFICANCEThe BRAFV600E oncoprotein kinase is a potent initiator of benign lung tumorigenesis, but is insufficient to elicit malignant lung adenocarcinoma without additional cooperating alterations. Sleeping Beauty-mediated transposon mutagenesis has revealed a number of genes that cooperate with BRAFV600E to promote lung cancer progression, in particular Rbms3, which encodes an RNA binding protein. Hence, this genetic screen provides a deeper understanding of the molecular mechanisms underlying BRAFV600E-driven lung carcinogenesis, and is an important step improving our ability to successfully target this disease.

category: cancer biology


title: Knowledge-guided deep learning models of drug toxicity improve interpretation

abstract: In drug development, a major reason for attrition is the lack of understanding of cellular mechanisms governing drug toxicity. The black-box nature of conventional classification models has limited their utility in identifying toxicity pathways. Here we developed DTox (Deep learning for Toxicology), an interpretation framework for knowledge-guided neural networks, which can predict compound response to toxicity assays and infer toxicity pathways of individual compounds. We demonstrate that DTox can achieve the same level of predictive performance as conventional models with a significant improvement in interpretability. Using DTox, we were able to rediscover mechanisms of transcription activation by three nuclear receptors, recapitulate cellular activities induced by aromatase inhibitors and PXR agonists, and differentiate distinctive mechanisms leading to HepG2 cytotoxicity. Virtual screening by DTox revealed that compounds with predicted cytotoxicity are at higher risk for clinical hepatic phenotypes. In summary, DTox provides a framework for deciphering cellular mechanisms of toxicity in silico.

category: pharmacology and toxicology


title: Structure of trimeric pre-fusion rabies virus glycoprotein in complex with two protective antibodies

abstract: Rabies virus (RABV) causes lethal encephalitis and is responsible for approximately 60,000 deaths per year. As the sole virion-surface protein, the rabies virus glycoprotein (RABV-G) mediates host-cell entry. RABV-Gs pre-fusion trimeric conformation displays epitopes bound by protective neutralizing antibodies which can be induced by vaccination or passively administered for post-exposure prophylaxis. We report a 2.8-[A] structure of a RABV-G trimer in the pre-fusion conformation, in complex with two neutralizing and protective monoclonal antibodies, 17C7 and 1112-1. One of these antibodies is a licensed prophylactic (17C7, Rabishield), which we show locks the protein in pre-fusion conformation. We demonstrate that targeted mutations can stabilize RABV-G in the pre-fusion conformation, a key step towards structure-guided vaccine design. These data reveal the higher-order architecture of a key therapeutic target and the structural basis of neutralization by antibodies binding two key antigenic sites, and will facilitate the development of improved vaccines and prophylactic antibodies.

category: microbiology


title: Extracellular vesicles and high-density lipoproteins: Exercise and estrogen-responsive small RNA carriers

abstract:
Decreased systemic estrogen levels (i.e., menopause) affect metabolic health. However, the detailed mechanisms underlying this process remain unclear. Both estrogens and exercise have been shown to improve metabolic health, which may be partly mediated by circulating microRNA (c-miR) signaling. In recent years, extracellular vesicles (EV) have increased interest in the field of tissue crosstalk. However, in many studies on EV-carried miRs, the co-isolation of high-density lipoprotein (HDL) particles with EVs has not been considered, potentially affecting the results. Here, we demonstrate that EV and HDL particles have distinct small RNA (sRNA) content, including both host and nonhost sRNAs. Exercise caused an acute increase in relative miR abundancy in EVs, whereas in HDL particles, it caused an increase in transfer RNA-derived sRNA. Furthermore, we demonstrate that estrogen deficiency caused by menopause blunts acute exercise-induced systemic miR-response in both EV and HDL particles.

HIGHLIGHTSO_LIExtracellular vesicles and HDL particles have a distinct sRNA content
C_LIO_LIExtracellular vesicles and HDL particles carry both host and nonhost sRNA cargo
C_LIO_LIEstrogen deficiency blunts the c-miR-response induced by acute exercise
C_LIO_LIExercise responsive miRs in HT users may regulate the choice of energy substrate
C_LI


title: ARID1A maintains transcriptionally repressive H3.3 associated with CHD4-ZMYND8 chromatin interactions

abstract: ARID1A is a signature subunit of the mammalian SWI/SNF (BAF) chromatin remodeling complex and is mutated at a high rate in malignancies and benign diseases originating from the uterine endometrium. Through genome-wide analysis of human endometriotic epithelial cells, we show that more than half of ARID1A binding sites are marked by the variant histone H3.3, including active regulatory elements. ARID1A loss leads to H3.3 depletion at ARID1A bound active regulatory elements and a concomitant redistribution of H3.3 towards genic elements. ARID1A interactions with the repressive chromatin remodeler CHD4 (NuRD) are associated with H3.3-containing chromatin regulation. ZMYND8, the CHD4-interacting acetyl-histone H4 reader, specifies ARID1A-CHD4-H3.3 target regulatory activity towards histone H4 lysine 16 acetylation (H4K16ac) to repress super-enhancers. ARID1A, H3.3, CHD4, and ZMYND8 co-repress the expression of genes governing extracellular matrix, motility, adhesion, and epithelial-to-mesenchymal transition. Moreover, these gene expression alterations are observed in human endometriomas. Altogether, these studies demonstrate that cooperation among a histone reader and different types of chromatin remodelers safeguards the endometrium through transcriptionally repressive H3.3.

category: genetics


title: Human UFSP1 is an active protease that regulates UFM1 maturation and UFMylation

abstract: An essential first step in the posttranslational modification of proteins with UFM1, UFMylation, is the proteolytic cleavage of pro-UFM1 to expose a C-terminal glycine. Of the two UFM1-specific proteases (UFSPs) identified in humans, only UFSP2 is reported to be active since the annotated sequence of UFSP1 lacks critical catalytic residues. Nonetheless, efficient UFM1 maturation occurs in cells lacking UFSP2 suggesting the presence of another active protease. We hereby identify a long isoform of UFSP1 to be this protease. Cells lacking both UFSPs show complete loss of UFMylation resulting from an absence of mature UFM1. While UFSP2, but not UFSP1, removes UFM1 from the ribosomal subunit RPL26, UFSP1 acts earlier in the pathway to mature UFM1 and cleave a potential auto-inhibitory modification on UFC1, thereby controlling activation of UFMylation. In summary, our studies reveal important distinctions in substrate specificity and localization-dependent functions for the two proteases in regulating UFMylation.


title: Aging-regulated TUG1 is dispensable for endothelial cell function

abstract: The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function.

category: cell biology


title: Connexin 43-mediated Neurovascular Interactions Regulate Neurogenesis in the Adult Brain Subventricular Zone

abstract: The subventricular zone (SVZ) is the largest neural stem cell (NSC) niche in the adult brain; herein, the blood-brain barrier is leaky, allowing direct interactions between NSCs and endothelial cells (ECs). Mechanisms by which direct NSC-EC interactions in the SVZ control NSC behavior are unclear. We found Cx43 to be highly expressed by both NSCs and ECs in the SVZ, and its deletion in either cell type leads to increased NSC proliferation and neuroblast generation, suggesting that Cx43-mediated NSC-EC interactions maintain NSC quiescence. This is further supported by in vitro studies showing co-culture with ECs decreases NSC proliferation and increases their expression of genes associated with quiescence in a Cx43-dependent manner. Cx43 mediates these effects in a channel-independent manner involving its cytoplasmic tail and ERK activation. Such insights further advance our understanding of NSC regulation in vivo and may inform NSC maintenance ex vivo for stem cell therapies for neurodegenerative disorders.

category: neuroscience


title: Influence of age and cognitive performance on resting-state functional connectivity of dopaminergic and noradrenergic centres

abstract: The process of ageing is associated with structural and functional changes in the brain, with a decline in cognitive functions observed as its inevitable symptom, even in the absence of neurodegenerative changes. A body of literature suggests dopamine and noradrenaline as prominent candidate molecules to mediate these effects, however, the knowledge regarding the underlying mechanisms is scarce. To fill this gap, we compared local and distant resting-state functional connectivity patterns of ventral tegmental area, substantia nigra pars compacta and locus coeruleus in healthy young (20-35 years; N = 37) and older adults (55-80 years; N = 27). Additionally, we sought long-range functional connectivity patterns of these structures associated with performance in tasks probing executive, attentional and reward functioning, as well as compared the functional coupling of the left and right substantia nigra. The results showed that each substantia nigra had stronger coupling with ipsilateral cortical and subcortical areas along with contralateral cerebellum, and that strength of functional connections of this structure with angular gyrus and lateral orbitofrontal cortex predicted the visuomotor search abilities. In turn, ageing was associated with increased local synchronisation in the ventral tegmental area, and differences in functional connectivity of the dopaminergic midbrain with numerous cortical and subcortical structures. Locus coeruleus functional coupling showed no differences between the groups and was not associated with any of the behavioural functions. To the best of our knowledge, this work is the first one to report the age-related effects on midbrain local synchronisation and its connectivity with key recipients of dopaminergic innervation, such as striatum, anterior cingulate and insula.


title: EphB1 controls proper long-range cortical axon guidance through a cell non-autonomous role in GABAergic cells

abstract: EphB1 is required for proper guidance of cortical long-range axon projections during brain development, but how EphB1 regulates this process remains unclear. To determine the relevant cell types in which EphB1 functions for proper cortical axon guidance, we generated a new floxed EphB1 mouse. We show here that EphB1 conditional knockout (cKO) in most of the developing cortical excitatory neurons (Emx1-Cre) failed to produce cortical axon guidance defects. However, EphB1 cKO in GABAergic cell populations (Vgat-Cre or dlx1/2-Cre) reproduced fully the cortical axon guidance defects observed in global EphB1-/- mice. Interestingly, in the EphB1 cKOVgat mice, the misguided axon bundles in the dorsal striatum contained comingled striatal GABAergic and cortical glutamatergic axons. Surprisingly, conditional loss of EphB1 in D1 or D2 dopamine receptor-expressing populations did not produce the cortical axon guidance defects, and no D1-or D2-SPN axons were detected within the misguided axon bundles of EphB1 KO mice. In contrast, parvalbumin-positive GABAergic cell axons were observed in the misguided axon bundles in EphB1 KO mice. Finally, a large fraction of the ectopic axons in EphB1 KO mice were in close apposition to the major blood vessels in the ventral telencephalon, suggesting that EphB1 expression in GABAergic cells normally promotes avoidance of these ectopic long-range axons from the developing cerebral vasculature. Together, our data reveal a new role for EphB1 in GABAergic cells to influence proper cortical glutamatergic long-range axon guidance during early brain development.


title: Path vectors: a neural code for sequential memory

abstract: While recalling lists of unrelated items is highly challenging we can recall much longer sequences structured as an episode or story. It is unknown why these patterns have such a striking influence on memory. We introduce a model where the experience of an episode or story is represented as a path through a pre-existing network of cognitive states. We demonstrate that by summing the neural representations of the visited states, this path can be transformed into a simple neural code: a path vector. We show how, by leveraging sparse connectivity and high dimensionality, path vectors provide robust codes for a large set of sequences and can be decoded mechanistically for memory retrieval. Fitting our model to data reveals how human free and serial recall may emerge from adapting coding mechanisms tuned for sequences aligned with existing network paths. We thus posit that sequences such as episodes or stories map more directly to existing cognitive network paths than arbitrary lists, with the latter eliciting paths that tend to interfere and impair recall. Our model suggests that mnemonic strategies like imposing narrative structure on a list act to improve recall by reducing such path interference. This work illuminates a simple bioplausible means for flexibly recruiting existing cognitive structures to encode new memories.


title: Label-free three-photon imaging of intact human cerebral organoids: tracking early events in brain development and deficits in Rett Syndrome

abstract: Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids ([~]2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.


title: Investigating relative contributions to psychiatric disease architecture fromsequence elements originating across multiple evolutionary time-scales

abstract:
It remains an open question when and why psychiatric and other disease evolved in human evolutionary history. We harness large genome-wide association studies and recent studies that have identified human-gained epigenetic marks, selective sweeps, archaic introgression, and accelerated evolution to help tackle this question by assessing the enrichment of heritability in these sequence elements for 41 complex traits. We find that human-gained epigenetic elements in the fetal brain harbor variants affecting skeletal, dermatological, and respiratory traits early and psychiatric traits later in development, that genomic sequences under selection since our divergence with Neanderthals are enriched in heritability for autism, and that regions depleted in ancestry from other hominids contribute more to heritability for psychiatric than other traits. Psychiatric disease may be a concomitant outcome of the evolutionary processes that made us uniquely human.

One-Sentence SummaryVariants that cause autism and other psychiatric disease today co-locate with sequence elements that have been important during human evolution.

category: evolutionary biology


title: LabNet: hardware control software for the Raspberry Pi

abstract: Single-board computers such as the Raspberry Pi make it easy to control hardware setups for laboratory experiments. GPIOs and expansion boards (HATs) give access to a whole range of sensor and control hardware. However, controlling such hardware can be challenging, when many experimental setups run in parallel and the time component is critical. LabNet is a C++ optimized control layer software to give access to the Raspberry Pi connected hardware over a simple network protocol. LabNet was developed to be suitable for time-critical operations, and to be simple to expand. It leverages the actor model to simplify multithreading programming and to increase modularity. The message protocol is implemented in Protobuf and offers performance, small message size, and supports a large number of programming languages on the client side. It shows good performance compared to locally executed tools like Bpod or pyControl, outperforms Autopilot and reaches sub-millisecond range in network communication latencies. LabNet itself does not provide support for the design of experimental tasks. This is left to the client. LabNet can be used for general automation in experimental laboratories with its control PC located at some distance. LabNet is open source and under continuing development.


title: Mucosal microbiomes and Fusobacterium genomics in Vietnamese colorectal cancer patients

abstract: Perturbations in the gut microbiome have been linked to the promotion and prognosis of colorectal cancer (CRC), with the colonic overabundance of Fusobacterium nucleatum shown as the most consistent marker. Despite the increasing health burden inflicted by CRC in low- and middle-income countries like Vietnam, the CRC-specific microbiome in these populations remains underexplored. Here we conducted a study in Vietnam to enrol 43 CRC patients (cases) and 25 patients with non-cancerous colorectal polyps (controls) between December 2018 and January 2020. Our study investigated the mucosal microbiome signature and genomic diversity of Fusobacterium in Vietnamese CRC patients, using a combination of 16S rRNA gene profiling, anaerobic microbiology, and whole genome sequencing. We found that several oral bacteria, including F. nucleatum and Leptotrichia, were significantly more abundant in the tumour mucosa, and these two bacteria were also more enriched in tumours of advanced CRC stages (III-IV). We obtained 53 Fusobacterium genomes from the saliva, tumour and non-tumour mucosa of six CRC patients. Isolates from the gut mucosa belonged to diverse F. nucleatum subspecies (nucleatum, animalis, vincentii, polymorphum) and a potential new subspecies of F. periodonticum. The Fusobacterium population within each individual was distinct and in many cases diverse, with minimal intra-clonal variation. Phylogenetic analyses showed that within each individual, tumour-associated Fusobacterium were clonal to those isolated from non-tumour mucosa, but distantly related to those isolated from saliva. Genes encoding major virulence factors (Fap2 and RadD) showed variability in length and evidence of horizontal gene transfer. Our work provides a framework to understand the genomic diversity of Fusobacterium within the CRC patients, which can be exploited for the development of CRC diagnostic and therapeutic options targeting this oncobacterium.


title: Repurposing Live Attenuated Trivalent MMR Vaccine as Cost-effective Cancer Immunotherapy

abstract: Despite its rising promise, cancer immunotherapy remains out of reach for many patients because of the extensive cost of manufacturing immunotherapy products. In this study, we show that intratumoral injections of the trivalent measles, mumps, and rubella (MMR) live attenuated viral vaccine (LAVs) modulates a potent cytotoxic T-cell antitumor immune response, resulting in tumor growth inhibition and improved survival in syngeneic mouse models of hepatocellular carcinoma and colorectal cancer. Using an integrated transcriptomic and proteomic approach, we demonstrated that mechanistically, MMR exerts its antitumor activity by priming innate and adaptive antitumor immune responses, leading to immunologically coordinated cancer cells death. Our findings highlight a promising potential for LAVs, such as MMR, to be repurposed as cost-effective cancer immunotherapy.

category: immunology


title: A hierarchical genotyping framework using DNA melting temperatures applied to adenovirus species typing

abstract:
Known genetic variation for a pathogen, in conjunction with post-PCR melting curve analysis, can be leveraged to provide increased taxonomic detail for pathogen identification in commercial molecular diagnostic tests. The increased taxonomic detail may be used by clinicians and public health decision makers to observe circulation patterns, monitor for outbreaks, and inform local testing practices. We propose a method for expanding the taxonomic resolution of PCR diagnostic systems by incorporating a priori knowledge of the assay design and publicly available sequence information into a genotyping classification model. For multiplexed PCR systems, this framework is generalized to incorporate information from multiple assays that react with different gene targets of the same pathogen to increase classification accuracy. To illustrate the method, a hierarchical classification model is developed for the BioFire(R) Respiratory 2.1 Panel and the BioFire(R) Respiratory 2 Panel (collectively the BioFire Respiratory Panels - highly multiplexed PCR diagnostic tests) to predict the species of human adenovirus (HAdV) from Adenovirus Detected test results. Performance of the classification model was characterized via a 10-fold cross-validation on a labeled dataset and exhibited 95% {+/-} 4% accuracy. The model was then applied to the BioFire(R) Syndromic Trends dataset, which contains deidentified patient test data from BioFire Respiratory Panels collected at over 100 sites across the globe since 2015. Adenovirus Detected test results in BioFire Syndromic Trends were classified to produce predicted prevalence of each HAdV species within the United States from 2018 through 2021. These results show a marked change in both the predicted prevalence for HAdV and the species makeup with the onset of the COVID-19 pandemic. In particular, HAdV-B decreased from a pre-pandemic predicted prevalence of up to 40% to less than 5% in 2021, while HAdV-A and HAdV-F species both increased in predicted prevalence.

Author SummaryIn the diagnosis of infectious disease there is often a trade-off between time to result, cost, and the amount of information gained about the pathogen. We develop a method that can provide increased genotypic information about a pathogen in a test result, with no additional time or cost. Our method is applied to the BioFire Respiratory(R) 2.1 Panel and the BioFire Respiratory(R) 2 Panel to speciate positive test results for adenovirus. The method shows robust performance in correctly identifying the adenovirus species from the test results. We estimate the prevalence of each adenovirus species in the United States by applying this method to adenovirus positive test results in the BioFire Syndromic Trends network. This information can be used in near real-time to augment the Centers for Disease Control and Prevention efforts to monitor adenovirus species circulation. Our mathematical framework can be generalized to other genotyping applications.


title: Hand Gesture Prediction via Transient-phase sEMG using Transfer Learning of Dilated Efficient CapsNet: Towards Generalization for Neurorobotics

abstract:
There has been an accelerated surge to utilize the deep neural network for decoding central and peripheral activations of the humans nervous system to boost up the spatiotemporal resolution of neural interfaces used in neurorobotics. Such algorithmic solutions are motivated for use in human-centered robotic systems, such as neurorehabilitation, prosthetics, and exoskeletons. These methods are proved to achieve higher accuracy on individual data when compared with the conventional machine learning methods but are also challenged by their assumption of having access to massive training samples.

ObjectiveIn this letter, we propose Dilated Efficient CapsNet to improve the predictive performance when the available individual data is very minimum and not enough to train an individualized network for controlling a personalized robotic system.

MethodWe proposed the concept of transfer learning using a new design of the dilated efficient capsular neural network to relax the need of having access to massive individual data and utilize the field knowledge which can be learned from a group of participants. In addition, instead of using complete sEMG signals, we only use the transient phase, reducing the volume of training samples to 20% of the original and maximizing the agility.

ResultsIn experiments, we validate our model performance with various amounts of injected personalized training data (25%-100% of transient phase) that is segmented once by time and once by repetition. The results of this paper support the use of transfer learning using a dilated capsular neural network and show that with the use of such a model, the knowledge domain learned on a small number of subjects can be utilized to minimize the need for new data of new subjects while focusing only on the transient phase of contraction (which is a challenging neural interfacing problem).

category: bioengineering


title: Statistical notes on false positive and negative error rates in the evaluation of long-term carcinogenicity bioassays

abstract: The appropriate interpretation of mortality-adjusted tumor incidences in long-term carcinogenicity bioassays depends substantially on the actual false positive and false negative error rates. These depend, among other things, on the type of analysis of multiple correlated tumor sites and the mode of dose-response dependence in relation to the design. Selected quantitative results, such as shape-to-design relationship and discreteness are presented and the influence of further issues is discussed qualitatively.

category: bioinformatics


title: Intraspecific diversification and mitonuclear discordance in native versus introduced areas: co-introduction of Dolicirroplectanum lacustre, a monogenean gill parasite of the invasive Nile perch Lates niloticus

abstract: The Nile perch (Lates niloticus) is a notorious invasive species. The introductions of Nile perch into several lakes and rivers in the Lake Victoria region led to the impoverishment of the trophic food webs, particularly well documented in Lake Victoria. Along with the introductions of the Nile perch, its parasites were co-introduced. Dolicirroplectanum lacustre (Monogenea, Diplectanidae) is a gill parasite of latid fishes (Lates spp.) inhabiting several major African freshwater systems. We examined the intra-specific diversification of D. lacustre from L. niloticus in Lake Albert (native range) and Lake Victoria (introduced range) by assessing morphological and genetic differentiation, and microhabitat preference. We expected reduced morphological and genetic diversity for D. lacustre in Lake Victoria compared to Lake Albert, as a result of the historical introductions. Dolicirroplectanum lacustre displays high morphological variability within and between African freshwaters. Mitonuclear discordance within the morphotypes of D. lacustre indicates an incomplete reproductive barrier between the morphotypes. The diversification in the mitochondrial gene portion is directly linked with the morphotypes, while the nuclear gene portions indicate conspecificity. Based on our results, we reported reduced genetic and morphological diversity, potentially being a result of a founder effect in Lake Victoria.


title: Multiscale mapping of in vivo 3D epidermal melanin distribution of human skin using a fast large-area multiphoton exoscope (FLAME)

abstract: Melanin plays a significant role in the regulation of epidermal homeostasis and photoprotection of human skin. The assessment of its epidermal distribution and overall content is of great interest due to its involvement in a wide range of physiological and pathological skin processes. Among several spectroscopic and optical imaging methods that have been reported for non-invasive quantification of melanin in human skin, the approach based on the detection of two-photon excited fluorescence lifetime distinguishes itself by enabling selective detection of melanin with sub-cellular resolution, thus facilitating its quantification while also resolving its depth-profile. A key limitation of prior studies on the melanin assessment based on this approach is their inability to account for the skin heterogeneity due to the reduced field of view of the images, which results in high dispersion of the measurement values. Pigmentation in both normal and pathological human skin is highly heterogeneous and its macroscopic quantification is critical for reliable measurements of the epidermal melanin distribution and for capturing melanin-related sensitive dynamic changes as a response to treatment. In this work, we employ a fast large-area multiphoton exoscope (FLAME), recently developed by our group for clinical skin imaging, that has the ability to evaluate the 3D distribution of epidermal melanin content in vivo macroscopically (millimeter scale) with microscopic resolution (sub-micron) and rapid acquisition rates (minutes). We demonstrate significant enhancement in the reliability of the melanin density and distribution measurements across Fitzpatrick skin types I to V by capturing the intra-subject pigmentation heterogeneity enabled by the large volumetric sampling. We also demonstrate the potential of this approach to provide consistent measurement results when imaging the same skin area at different times. These advances are critical for clinical and research applications related to monitoring pigment modulation as a response to therapies against pigmentary skin disorders, skin aging, as well as skin cancers.


title: DNA 5-methylcytosine detection and methylation phasing using PacBio circular consensus sequencing

abstract: It has been reported recently that DNA 5-methylcytosine (5mC) in CpG contexts can be detected using PacBio circular consensus sequencing (CCS). However, the accuracy and robustness of computational methods using long CCS reads still need to be improved. In this study, we present a deep learning method, ccsmeth, to detect DNA 5mCpGs from PacBio CCS subreads. ccsmeth utilizes attention-based bidirectional Gated Recurrent Unit (GRU) networks to infer DNA methylation states. Testing ccsmeth using CCS subreads of amplified DNA and M.SssI-treated DNA, we found that ccsmeth achieved higher performances than existing methods. We also compared the results of ccsmeth on long CCS reads with bisulfite sequencing and Nanopore sequencing. The results demonstrated that ccsmeth can accurately detect 5mCpGs from CCS data sequenced using >10 kb insert library. Moreover, using PacBio CCS data, we proposed a pipeline which can detect haplotype-aware methylation in human.


title: Lower limb in sprinters with larger relative mass but not larger normalized moment of inertia: novel insights into human plasticity

abstract:
Inhomogeneous muscle hypertrophy in response to daily motor executions is a morphological adaptation in humans. Inhomogeneous morphological adaptation would change the mass distribution, which in turn may affect the mechanical difficulty in moving from an inertia perspective; however, the morphological adaptation from the inertia perspective has not been examined. Here we show no corresponding differences in the normalized mass and normalized moment of inertia between the sprinters and the untrained non-sprinters. We analyzed the fat- and water-separated magnetic resonance images from the lower limbs of 11 male sprinters (100 m best time of 10.44-10.83 s) and 12 untrained non-sprinters. The lower-limb relative mass was significantly larger in sprinters (18.7{+/-}0.7% body mass) than in non-sprinters (17.6{+/-}0.6% body mass), while the normalized moment of inertia of the lower limb around the hip in the anatomical position was similar between them (0.044{+/-}0.002 vs. 0.042{+/-}0.002 [a. u.]). The thigh relative mass in sprinters (12.9{+/-}0.4% body mass) was significantly larger than that in non-sprinters (11.9{+/-}0.4% body mass), whereas the shank and foot relative masses were similar. We revealed that the adaptation to sprinting in humans does not relatively increase the mechanical difficulty to swing the lower limb in terms of inertia, even though the lower-limb mass increases, reflecting muscular hypertrophy. This is suggested to be one of the significant advantages of human morphology for adaptability to explosive motor tasks.

NEW & NOTEWORTHYWe compared the lower-limb inertia properties between male sprinters and untrained non-sprinters by using the fat- and water-separated magnetic resonance images. Sprinters had lower limbs with a larger relative mass but not a larger normalized moment of inertia than non-sprinters. We suggest one of the practical advantages of human morphological characteristics for their adaptation to explosive motor tasks, which provides a novel perspective to the literature for understanding human morphological adaptability.

category: physiology


title: Lower limb in sprinters with larger relative mass but not larger normalized moment of inertia


title: A Tensor Decomposition Uncovers the Effect of Ageing on Muscle and Grip-Load Force Couplings During Grasping

abstract: Do motor patterns of grasp-to-lift movements change as a result of ageing? Previous studies often relied on simple temporal and kinetic variables to unveil differences caused by ageing, yet their neuromuscular origins remain largely unknown. Here we employed a bimanual grasping protocol with younger and older adults and combined measurements of muscle activity with grip and load forces to provide a window into the neuromuscular strategies underlying effective grasping. We introduced a tensor decomposition to identify patterns of muscle activity and grip-load force ratios while also characterising their temporal profiles and relative activation across object weights and participants of different age groups. This approach extracted the motor components underpinning object grasping across participants. We then probed age-induced changes in these components. A classification analysis revealed two motor components that are differentially recruited between the two age groups. Linear regression analyses further showed that advanced age and poorer manual dexterity can be predicted by the coupled activation of forearm and hand muscles which is associated with high levels of grip force. Our findings suggest that ageing may induce stronger muscle couplings in distal aspects of the upper limbs, and a less economic grasping strategy to overcome age-related decline in manual dexterity.


title: Studies for searching factors relating rumination time around the heat season in Holstein milking cows

abstract: To identify factors affecting rumination time (RT), factorial analyses in a dairy farm were performed. In univariate analyses, differences in distribution were observed between low (< 45 kg/d) and high ([≥] 45 kg/d) milk yield (MY) before the heat season, early and peak lactation periods and mid and late at the start of the heat season, and 1-2 and [≥] 3 parity at the start of the heat season. Multiple regression analysis confirmed that RT was affected by low MY, early to peak and 1-2 parity cows, low MY, mid to late and 1-2 parity cows, and low MY, early to peak and [≥] 3 parity cows (coefficients = -37.039, -25.353, -44.805 respectively, P < 0.05). When the cows were classified by MY before the heat season, the RT and MY of the high-MY group remained higher than the low-MY group until before the severe heat (THI [≥] 84 continued). However, when cows were classified by RT before the heat season, there was no difference in MY between the low-RT (< 485 min/d) and the high-RT group (> 485 min/d). In conclusions, MY is a factor affecting RT up to moderate heat but RT is not a sufficient condition affecting MY.

category: zoology


title: Critical periods in which dopamine exerts control over behavioral responding during Pavlovian learning

abstract:
RationaleLearning the association between rewards and predictive cues is critical for appetitive behavioral responding. The mesolimbic dopamine system is thought to play an integral role in establishing these cue-reward associations. The dopamine response to cues can signal differences in reward value, though this emerges only after significant training. This suggests that the dopamine system may differentially regulate behavioral responding depending on the phase of training.

ObjectivesThe purpose of this study was to determine whether antagonizing dopamine receptors elicited different effects on behavior depending on the phase of training or the type of Pavlovian task.

MethodsSeparate groups of male rats were trained on Pavlovian tasks in which distinct audio cues signaled either differences in reward size or differences in reward rate. The dopamine receptor antagonist flupenthixol was systemically administered prior to either the first ten sessions of training (acquisition phase) or the second ten sessions of training (expression phase) and we monitored the effect of these manipulations for an additional ten training sessions.

ResultsWe identified acute effects of dopamine receptor antagonism on conditioned responding, the latency to respond, and post-reward head entries in both Pavlovian tasks. Interestingly, dopamine receptor antagonism during the expression phase produced persistent deficits in behavioral responding only in rats trained on the reward size Pavlovian task.

ConclusionsTogether, our results illustrate that dopamines control over behavior in Pavlovian tasks depends upon ones prior training experience and the information signaled by the cues.


title: Critical periods when dopamine controls behavioral responding during Pavlovian learning


title: Dendrites use mechanosensitive channels to proofread ligand-mediated guidance during morphogenesis.

abstract: Ligand-receptor interactions guide axon navigation and dendrite arborization. Mechanical forces also influence guidance choices. However, the nature of such mechanical stimulations, the mechanosensors identity and how they interact with guidance receptors are unknown. Here, we demonstrate that mechanosensitive DEG/ENaC channels are required for dendritic arbor morphogenesis in Caenorhabditis elegans. Inhibition of DEG/ENaC channels causes reduced dendritic outgrowth and branching in vivo, a phenotype that is alleviated by overexpression of the mechanosensitive channels PEZO-1/Piezo or YVC1/TrpY1. DEG/ENaCs trigger local Ca2+ transients in growing dendritic filopodia via activation of L-type voltage-gated Ca2+ channels. Anchoring of filopodia by guidance ligand/receptor is required for the mechanical activation of DEG/ENaC channels. Therefore, mechanosensitive channels serve as a checkpoint for appropriate chemoaffinity by activating Ca2+ transients required for neurite growth.


title: Hominoid-specific sulcal variability is related to face perception ability

abstract: Human perception requires complex cortical networks that function at neuroanatomical scales of microns and temporal scales of milliseconds. Despite this complexity, what if just one morphological feature of the brain could predict perceptual ability? Here, we tested this hypothesis with pre-registered analyses of neuroanatomy and face perception in neurotypical controls (NTs) and individuals with developmental prosopagnosia (DPs). Results show that the length of the mid-fusiform sulcus (MFS), a hominoid-specific tertiary sulcus in ventral temporal cortex (VTC), was shorter in DPs than NTs. Furthermore, individual differences in MFS length in the right, but not left, hemisphere predicted individual differences in face perception. These results support theories linking brain structure and function to perception, as well as indicate that one feature - variability in MFS length - can predict face perception. Finally, these findings add to growing evidence supporting a role of morphological variability of late developing, tertiary sulci and individual differences in cognition.


title: The ITPRIPL1- CD3ϵ axis: a novel immune checkpoint controlling T cells activation

abstract: The immune system is critical to fighting infections and disease. The molecular recognition of harmful entities takes place when antigen-presenting cells (APC) harboring major histocompatibility complex (MHC) molecules bound to peptides derived from harmful antigens (ligand) dock on specific T cell receptor (TCR)-CD3 complex (receptor) at the surface of CD8+ T cells. The discovery of a general immune checkpoint mechanism to avoid the harmful impact of T cell hyperactivation provoked a paradigm shift. The clinical relevance of this mechanism is highlighted by the fact that PD-1 and PD-L1 inhibitors are very effective at boosting immune reactions. Still, immune evasion frequently happens. The observation that some PD-1/PD-L1 negative tumors have a poor immune response opens the door to identifying a novel immune checkpoint mechanism. Here, we discovered that ITPRIPL1, a gene with unknown function, impairs T cell activation. Surprisingly, we found that CD3{varepsilon} is the direct receptor of ITPRIPL1. This novel immune checkpoint was validated as a drug target using ITPRIPL1 KO mice and monoclonal antibodies. Thus, targeting the ITPRIPL1-CD3e axis, especially in PD-1 - PDL-1 negative patients, is a promising therapeutic strategy to reduce immune evasion.


title: Decoupling diet from microbiome dynamics results in model mis-specification that implicitly annuls potential associations between the microbiome and disease phenotypes-ruling out any role of the microbiome in autism (Yap et al. 2021) likely a premature conclusion

abstract: Recently, and in a tour de force effort, Yap et al. performed a comprehensive association analysis of factors such as demographics, psychometrics, diet, stool metagenomics, stool consistency, and genome-wide SNP genotypes with autism in 247 Australian children. Surprisingly, the authors suggest their data show a strong correlation between diet and autism spectrum disorder (ASD) but only negligible, if any, ASD-specific microbiome signals. While the first conclusion comes as no surprise, we were rather puzzled by the second conclusion given the growing evidence of strong associations between the microbiome and ASD phenotype and the wide consensus on a close connection between diet and microbiome composition and function. The causal model proposed by Yap et al. seemed to imply that diet and the microbiome were two independent variables. A careful review of the approach used by the authors confirmed our suspicions that the statistical models were mis-specified, i.e. they had a questionable biological assumption--the independence of diet and microbiome--embedded in them. We have run side-by-side simulations of the causal linear model proposed by Yap et al. and of an analogous model in which diet and the microbiome are treated as co-dependent variables. We show how the Yap et al. model can preemptively exclude any potential host-microbe interactions if the diet-microbiome independence assumption is violated. We believe large-scale efforts such as the one described by Yap et al. are essential to advance our understanding of the potential role of the microbiome in ASD and other diseases. But these are highly complex systems to analyze and thus ensuring that the statistical methods used are accurate is essential to avoid drawing any potentially misleading conclusions due to subtle causal assumptions propagated by the statistical models themselves.


title: Levels of p53 expression determine the competitive ability of embryonic stem cells during the onset of differentiation.

abstract: During development, the rate of tissue growth is determined by the relative balance of cell division and cell death. Cell competition is a fitness quality control mechanism that contributes to this balance by eliminating viable cells that are less-fit than their neighbours. What mutations confer cells with a competitive advantage or the dynamics of the interactions between winner and loser cells are not well understood. Here, we show that embryonic cells lacking the tumour suppressor p53 are super-competitors that eliminate their wild-type neighbours through the direct induction of apoptosis. This elimination is context dependant, as does not occur when cells are pluripotent and is triggered by the onset of differentiation. Furthermore, by combining mathematical modelling and cell-based assays we show that the elimination of wild-type cells is not through a competition for space or nutrients, but instead is mediated by short range interactions that are dependent on the local cell neighbourhood. This highlights the importance of the local cell neighbourhood and the competitive interactions within this neighbourhood for the regulation of proliferation during early embryonic development.

category: developmental biology


title: Model selection in occupancy models: inference versus prediction

abstract:
Occupancy models are a vital tool for applied ecologists studying the patterns and drivers of species occurrence, but their use requires a method for selecting between models with different sets of occupancy and detection covariates. The information-theoretic approach, which employs information criteria such as Akaikes Information Criterion (AIC) is arguably the most popular approach for model selection in ecology and is often used for selecting occupancy models. However, the information-theoretic approach risks selecting models which produce inaccurate parameter estimates, due to a phenomenon called collider bias. Using simulations, we investigated the consequences of collider bias (using an illustrative example called M-bias) in the occupancy and detection processes of an occupancy model, and explored the implications for model selection using AIC and a common alternative, the Schwarz Criterion (or Bayesian Information Criterion, BIC). We found that when M-bias was present in the occupancy process, AIC and BIC selected models which inaccurately estimated the effect of the focal occupancy covariate, while simultaneously producing more accurate predictions of the site-level occupancy probability. In contrast, M-bias in the detection process did not impact the focal estimate; all models made accurate inferences, while the site-level predictions of the AIC/BIC-best model were slightly more accurate. Our results demonstrate that information criteria can be used to select occupancy covariates if the sole purpose of the model is prediction, but must be treated with more caution if the purpose is to understand how environmental variables affect occupancy. By contrast, detection covariates can usually be selected using information criteria regardless of the models purpose. These findings illustrate the importance of distinguishing between the tasks of parameter inference and prediction in ecological modelling. Furthermore, our results underline concerns about the use of information criteria to compare different biological hypotheses in observational studies.

Open Research StatementCode to fully reproduce our simulations and analyses is available at: https://zenodo.org/badge/latestdoi/462801230

category: ecology


title: Spatio-temporal model and machine learning method reveal process of phenological shift under climate change of North Pacific spiny dogfish

abstract: Climate change has disrupted natural phenological patterns, including migration. Despite extensive studies of phenological shifts in migration by climate change and driving factors of migration, a few issues remain unresolved. In particular, little is known about the complex effects of driving factors on migration with interactions and nonlinearity, and partitioning of the effects of factors into spatial, temporal, and spatio-temporal effects. The Pacific spiny dogfish Squalus suckleyi (hereafter "spiny dogfish") is a coastal elasmobranchii that migrates southward for parturition and northward for feeding in the western North Pacific. Here, to elucidate the migration patterns as well as the driving factors under climate change, we first examined long-term changes in the timing and geographic location of migration by applying a spatio-temporal model to ca. 5-decade time series data for the presence/absence of spiny dogfish in the western North Pacific. We then evaluated the spatial, temporal, and spatio-temporal effects of driving factors (sea surface temperature [SST], depth, productivity, and magnetic fields) on seasonal occurrence patterns using a machine learning model. We found that the migration area did not change over ca. 5 decades, whereas the migration timing advanced by a month after 2000. The spatial effects of magnetic fields and depth were consistently large and the spatial and spatio-temporal effects of SST increased in the migration season, even though temporal effect of SST was always weak. These results suggest that the migration area of spiny dogfish was stable over time because their spatial distribution was determined by geographic features, whereas the migration timing advanced by tracking a suitable SST location which increased steeply after 2000. Therefore, temperature as well as other factors influence migration simultaneously under climate change and underline the importance of paying attention biotic/abiotic factors including temperature and process-based understanding to predict future impacts of climate change on phenological shifts.


title: Fine-scale survey of intertidal macroalgae reveals recent changes in a cold-water biogeographic stronghold

abstract:
Global warming has been causing severe impacts on marine ecosystems, a notorious one being shifts in the geographical ranges of species. The north-western coast of the Iberian Peninsula is an especially interesting zone to study distributional shifts as it has a strong latitudinal thermal gradient, is influenced by the Canary upwelling system (which partially cancels coastal warming) and holds some of the most diverse macroalgae communities in Europe. Notably, it is within this region that many cold-water species, common in northern Europe, have their southernmost distribution refuge. Recent studies hypothesize that the environmental conditions may be nonetheless changing and already threatening this biodiversity hotspot.

The main goal of this study was to carry out a fine-scale assessment of the distributional limits of several macroalgae in North-western Iberia, as well as identify possible population and range shifts using historical data (2001-2005) as reference. In addition, invasive species were also surveyed. We also assessed if the regions of (i) Galicia, (ii) Northern Portugal, and (iii) Central Portugal displayed distinctive characters regarding macroalgae composition and abundance.

We identified an increase in abundance of some invasive macroalgae as well as a decrease in the abundance of some cold-water species. In the most severe cases, cold-water species were extirpated along hundreds of km. The compounded effect of the decrease in the abundance of cold-water species and the increase in the abundance of invasive species is leading to the homogenization of macroalgae communities in north-western Iberia.


title: Experimental evidence that animal societies vary in size due to sex differences in cooperation

abstract: Cooperative breeding societies allow the costs of reproduction to be shared. However, as groups become larger, such benefits often decrease and competition increases. This is predicted to select for an optimal group size, yet variation in groups is a ubiquitous feature of cooperative breeding animals. Here we experimentally established groups (ngroups=96) of cooperative breeding ostriches, Struthio camelus, with different numbers of males and females and manipulated the potential for cooperation over incubation. There was a clear optimal group size for males. Their reproductive success was maximized in groups with four or more females and no other males, irrespective of cooperation over incubation. Conversely, female reproductive success was strongly dependent on the benefits of cooperating over incubation, being maximized in groups with either many males or many females. In intermediate sized groups, both male and female reproductive success was reduced by sexual conflict over the timing of mating and incubation. Our experiments show that sex differences in the opposing forces of cooperation and competition can explain why variation in cooperative groups is widespread.


title: Reconstructing the ancestral vertebrate brain using a lamprey neural cell type atlas

abstract: The vertebrate brain emerged more than [~]500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey - a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits - based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of the core cell type composition, tissue structures, and gene expression programs of the ancestral brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral vertebrate brain was likely devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors on the jawed vertebrate lineage. Our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.


title: Rapid adaptation and range expansion in response to agriculture over the last two centuries

abstract:
North America has seen a massive increase in cropland use since 1800, accompanied more recently by the intensification of agricultural practices. Through genome analysis of present-day and historical samples spanning environments over the last two centuries, we studied the impact of these changes in farming on the extent and tempo of evolution in the native common waterhemp (Amaranthus tuberculatus), a now pervasive agricultural weed. Modern agriculture has imposed strengths of selection rarely observed in the wild (0.027-0.10), with striking shifts in allele frequency trajectories since agricultural intensification in the 1960s. An evolutionary response to this extreme selection was facilitated by a concurrent human-mediated range shift. By reshaping genome-wide diversity and variation for fitness, agriculture has driven the success of this 21st-century weed.

One Sentence SummaryModern agriculture has dramatically shaped the evolution of a native plant into an agricultural weed through imposing strengths of selection rarely observed in the wild.


title: Hrq1/RECQL4 regulation is critical for preventing aberrant recombination during DNA intrastrand crosslink repair and is upregulated in breast cancer.

abstract: Human RECQL4 is a member of the RecQ family of DNA helicases and functions during DNA replication and repair. RECQL4 mutations are associated with developmental defects and cancer. Although RECQL4 mutations lead to disease, RECQL4 overexpression is also observed in cancer, including breast and prostate. Thus, tight regulation of RECQL4 protein levels is crucial for genome stability. Because mammalian RECQL4 is essential, how cells regulate RECQL4 protein levels is largely unknown. Utilizing budding yeast, we investigated the RECQL4 homolog, HRQ1, during DNA crosslink repair. We find that Hrq1 functions in the error-free template switching pathway to mediate DNA intrastrand crosslink repair. Although Hrq1 repairs cisplatin-induced lesions, it is paradoxically degraded by the proteasome following cisplatin treatment. By identifying the targeted lysine residues, we show that preventing Hrq1 degradation results in increased recombination and mutagenesis. Like yeast, human RECQL4 is similarly degraded upon exposure to crosslinking agents. Furthermore, over-expression of RECQL4 results in increased RAD51 foci, which is dependent on its helicase activity. Using bioinformatic analysis, we observe that RECQL4 overexpression correlates with increased recombination and mutations. Overall, our study uncovers a role for Hrq1/RECQL4 in DNA intrastrand crosslink repair and provides further insight how misregulation of RECQL4 can promote genomic instability, a cancer hallmark.


title: Estimating the overall fraction of phenotypic variance attributed to high-dimensional predictors measured with error

abstract: In prospective genomic studies (e.g., DNA methylation, metagenomics, and transcriptomics), it is crucial to estimate the overall fraction of phenotypic variance (OFPV) attributed to the high-dimensional genomic variables, a concept similar to heritability analyses in genome-wide association studies (GWAS). Unlike genetic variants in GWAS, these genomic variables are typically measured with error due to technical limitation and temporal instability. While the existing methods developed for GWAS can be used, ignoring measurement error may severely underestimate OFPV and mislead the design of future studies. Assuming that measurement error variances are distributed similarly between causal and noncausal variables, we show that the asymptotic attenuation factor equals to the average intraclass correlation coefficients of all genomic variables, which can be estimated based on a pilot study with repeated measurements. We illustrate the method by estimating the contribution of microbiome taxa to body mass index and multiple allergy traits in the American Gut Project. Finally, we show that measurement error does not cause meaningful bias when estimating the correlation of effect sizes for two traits.


title: Agency as Planning for Known Unknowns

abstract: The utility of a given experience, like interacting with a particular friend or tasting a particular food, fluctuates continually according to homeostatic and hedonic principles. Consequently, to maximize reward, an individual must be able to escape or attain outcomes as preferences change, by switching between actions. Recent work on human and artificial intelligence has defined such control in information theoretic terms. However, information theoretic measures do not necessarily correspond to an agents ability to escape or obtain sensory-specific states. In the current study, choice behavior was best described by a forward consideration of action values based on possible changes in outcome utilities. Consistent with previous work on agency and personality, individual differences in dimensional schizotypy were correlated with choice preferences in conditions with the greatest and lowest levels of instrumental control. The results contribute to a growing literature on the role of agency in goal-directed choice.

category: animal behavior and cognition


title: Nonlinear micro finite element models based on digital volume correlation measurements predict early microdamage in newly formed bone

abstract: Bone regeneration in critical-sized defects is a clinical challenge, with biomaterials under constant development aiming at enhancing the natural bone healing process. The delivery of bone morphogenetic proteins (BMPs) in appropriate carriers represents a promising strategy for bone defect treatment but optimisation of the spatial-temporal release is still needed for the regeneration of bone with biological, structural, and mechanical properties comparable to the native tissue. Nonlinear micro finite element ({micro}FE) models can address some of these challenges by providing a tool able to predict the biomechanical strength and microdamage onset in newly formed bone when subjected to physiological or supraphysiological loads. Yet, these models need to be validated against experimental data. In this study, experimental local displacements in newly formed bone induced by osteoinductive biomaterials subjected to in situ X-ray computed tomography compression in the apparent elastic regime and measured using digital volume correlation (DVC) were used to validate ({micro}FE) models. Displacement predictions from homogeneous linear {micro}FE models were highly correlated to DVC-measured local displacements, while tissue heterogeneity capturing mineralisation differences showed negligible effects. Nonlinear {micro}FE models improved the correlation and showed that tissue microdamage occurs at low apparent strains. Microdamage seemed to occur next to large cavities or in biomaterial-induced thin trabeculae, independent of the mineralisation. While localisation of plastic strain accumulation was similar, the amount of damage accumulated in these locations was slightly higher when including material heterogeneity. These results demonstrate the ability of the nonlinear {micro}FE model to capture local microdamage in newly formed bone tissue and can be exploited to improve the current understanding of healing bone and mechanical competence. This will ultimately aid the development of BMPs delivery systems for bone defect treatment able to regenerate bone with optimal biological, mechanical, and structural properties.


title: Generating publication ready visualizations for Single Cell transcriptomics using SCpubr

abstract: Single Cell transcriptomic analysis has become a widespread technology of choice when it comes to understanding the differences at a transcriptomic level in heterogeneous samples. As a consequence, a plethora of analysis tools have been published to tackle the different analysis steps from count matrix generation to downstream analysis. Many of them provide ways to generate visualizations of the data. While some design choices are made, it is a common practice to provide the user with visualizations as raw as possible so that they can be customized to the user needs. However, in many cases these final customization steps are either time consuming or demand a very specific set of skills. This problem is addressed by SCpubr, which sacrifices some of this initial freedom of choice in aesthetics to provide the user a more streamlined way of generating high quality Single Cell transcriptomic visualizations.


title: Automated Analysis of Neuronal Morphology through an Unsupervised Classification Model of Neurites

abstract: Brain function emerges from a highly complex network of specialized cells that are interlinked by billions of synapses. The synaptic connectivity between neurons is established between the elongated processes of their axons and dendrites or, together, neurites. To establish these billions of often far-reaching connections, cellular neurites have to grow in highly specialized, cell-type dependent patterns covering often mm distances and connecting with thousands of other neurons. The outgrowth and branching of neurites are tightly controlled during development and are a commonly used functional readout of imaging in the neurosciences. Manual analysis of neuronal morphology from microscopy images, however, is very time intensive and error prone. Especially fully automated segmentation and classification of all neurites remain unavailable in open-source software. Here we present a standalone, GUI-based software for batch-quantification of neuronal morphology in fluorescence micrographs with minimal requirements for user interaction. Neurons are segmented using a Hessian-based algorithm to detect thin neurite structures combined with intensity- and shape-based detection of the cell body. To measure the number of branches in a neuron accurately, rather than just determining branch points, neurites are classified into axon, dendrites and their branches of increasing order by their length using a geodesic distance transform of the cell skeleton. The software was benchmarked against a large, published dataset and reproduced the phenotype observed after manual annotation before. Our tool promises greatly accelerated and improved morphometric studies of neuronal morphology by allowing for consistent and automated analysis of large datasets.


title: Dark kinase annotation, mining and visualization using the Protein Kinase Ontology

abstract: The Protein Kinase Ontology (ProKinO) is an integrated knowledge graph that conceptualizes the complex relationships connecting protein kinase sequence, structure, function, and disease in a human and machine-readable format. Here we extend the scope of ProKinO as a discovery tool by including new classes and relationships capturing information on kinase ligand binding sites, expression patterns, and functional features, and demonstrate its application in uncovering new knowledge regarding understudied members of the protein kinase family. Specifically, through graph mining and aggregate SPARQL queries, we identify the p21-activated protein kinase 5 (PAK5) as one of the most frequently mutated dark kinase in human cancers with abnormal expression in multiple cancers, including an unappreciated role in acute myeloid leukemia. We identify recurrent oncogenic mutations in the PAK5 activation loop predicted to alter substrate binding and phosphorylation and identify common ligand/drug binding residues in PAK family kinases, highlighting the potential application of ProKinO in drug discovery. The updated ontology browser and a web component, ProtVista, which allows interactive mining of kinase sequence annotations in 3D structures and Alphafold models, provide a valuable resource for the signaling community. The updated ProKinO database is accessible at http://prokino.uga.edu/browser/.


title: Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels

abstract: Otopetrin (OTOP) channels are proton-selective ion channels conserved among vertebrates and invertebrates and with no structural similarity to other ion channels. There are three vertebrate OTOP channels (OTOP1, OTOP2, and OTOP3), of which one (OTOP1), functions as a sour taste receptor. Whether OTOP channels are gated by, as well as permeating, protons was not known. Here, by comparing functional properties of the three vertebrate proton channels with patch-clamp recording and cytosolic pH microfluorimetry, we provide evidence that each is gated by external protons. OTOP1 and OTOP3 are both activated by extracellular protons, with a sharp threshold of pHe <6.0 and 5.5 respectively, while OTOP2 is negatively gated by protons, and more conductive at alkaline extracellular pH (>pH 9.0). Strikingly, we found that we could change pH-sensitive gating of OTOP2 and OTOP3 channels by swapping extracellular linkers that connect transmembrane domains. Swaps of linkers within the N domain changed the relative conductance at alkaline pH, while swaps within the C domain tended to change the rates of OTOP3 current activation. We conclude that members of the OTOP channel family are proton-gated (acid-sensitive) proton channels and that the gating apparatus is distributed across multiple extracellular regions within both the N and C domains of the channels. In addition to the taste system, OTOP channels are found in the vestibular and digestive systems, where pH sensitivity may be tuned to specific functions.


title: Quantitative imaging reveals PI3Kδ inhibition reduces rhinovirus-induced damage of small airway epithelia in ex vivo cultured human precision cut lung slices from COPD patients

abstract:
Chronic obstructive pulmonary disease (COPD) is one of the major causes of disability and death worldwide and a significant risk factor for respiratory infections. Rhinoviral infections are the most common trigger of COPD exacerbations which lead to a worsening of disease symptoms, decline in lung function and increased mortality. The lack of suitable disease models to study the relevant cellular and molecular mechanism hinders the discovery of novel medicines that prevent disease progression in exacerbating COPD patients. We used quantitative multi-color imaging of COPD and control patient derived human precision-cut lung slices (hPCLS) to study the impact of rhinovirus infection on the structure and function of the small airway epithelium. Data analysis highlighted that COPD-derived hPCLS have a higher cellular density and basal cell hyperplasia, more unciliated airway surface areas with mucus overproduction, and shorter cilia length compared to control hPCLS. In response to rhinovirus 16 infection, COPD-derived hPCLS secreted higher amounts of pro-inflammatory cytokines and displayed decreased epithelial integrity and reduced airway ciliation. Finally, treatment with a selective PI3K{delta} inhibitor reduced secretion of rhinovirus-induced cytokines and ameliorated rhinovirus-induced damage to COPD small airway epithelia. Thus, these data demonstrate the potential of quantitative imaging to assess complex airway functions in a patient-derived lung tissue model system, and indicate that targeting PI3K{delta} might be a promising therapeutic opportunity to limit rhinovirus-induced airway damage in exacerbating COPD patients.

SummaryPI3K{delta} inhibition reduces rhinovirus-mediated damage of small airway epithelia from chronic obstructive pulmonary disease (COPD) patients

category: pathology


title: On the regulation of photosynthesis in pea leaves exposed to oscillating light.

abstract:
Plants grow in nature often in fluctuating irradiance. In the laboratory, the dynamics of photosynthesis is usually explored by instantaneously exposing dark-adapted plants to constant light and detecting the dark-to-light transient, which is only a poor approximation of natural phenomena. Aiming at a better approximation, we exposed pea leaves to oscillating light and measured, during oscillations, changes in function of photosystem I and II and of the proton-motive force at the thylakoid membrane. The dynamics depends on the oscillation period, leaving information about the regulatory networks. As demonstrated for selected period of the oscillation of 60 s, the regulations try to keep reactions centres of photosystems I and II open. A possible evaluation of obtained data is presented and involvement of particular processes in regulation of photosynthesis is discussed. The forced oscillations provide information-rich fingerprint of complex regulatory networks. Further progress in understanding the networks is expected from experiments involving chemical interventions, plant mutants, and by using mathematical modelling and the system identification and system control tools, as already applied in other parts of science.

HighlightMeasurement of photosynthetic signals during illumination of plants by light, whose intensity oscillates as sinus function provides information about regulation of photosynthesis in fluctuating light.

category: plant biology


title: Balancing activation and costimulation of CAR tunes signaling dynamics and enhances therapeutic potency

abstract:
BackgroundPrimary human T cells engineered with chimeric antigen receptors (CARs) ex vivo can be adoptively transferred to treat cancer. CD19-targeting CAR with CD28 costimulatory domain and CD3{zeta} activation domain have been approved by the US FDA for treating B cell malignancies.

MethodsHere we generated mutation of immunorecpetor tyrosine-based activation motifs (ITAMs) in CD3{zeta}, namely 1XX CAR, which altered the balance of activation and costimulation. Next we investigated whether 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their WT counterparts.

ResultsEven though 1XX CARs decreased cytotoxicity against tumor cells in vitro, they showed better anti-tumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells proliferated more in response to antigen stimulation in vitro, persisted longer in vivo and had higher percentage of central memory cells. As 1XX modification directly calibrates CAR activation potential, we utilized fluorescence resonance energy transfer (FRET)-based biosensor to monitor signaling dynamics downstream of CARs. Decreased ITAM numbers in 1XX resulted in similar ZAP70 activation, while 1XX induced higher Ca2+ elevation and faster Erk activation than WT CAR, which may contribute to the better therapeutic potency of 1XX.

ConclusionsOur results established the surpiosity of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical application of 1XX CARs against solid tumors.


title: Alternative splicing downstream of EMT enhances phenotypic plasticity and malignant behavior in colon cancer

abstract:
Phenotypic plasticity allows carcinoma cells to transiently acquire the quasi-mesenchymal features necessary to detach from the primary mass and proceed along the invasion-metastasis cascade. A broad spectrum of epigenetic mechanisms is likely to cause the epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) transitions necessary to allow local dissemination and distant metastasis. Here, we report on the role played by alternative splicing (AS) in eliciting phenotypic plasticity in colon cancer.

By taking advantage of the identification of a subpopulation of quasi-mesenchymal and highly metastatic EpCAMlo colon cancer cells, we here show that the differential expression of ESRP1 and other RNA-binding proteins (RBPs) downstream of the EMT master regulator ZEB1, alters the AS pattern of a broad spectrum of targets including CD44 and NUMB, thus resulting in the generation of specific isoforms functionally associated with increased invasion and metastasis. Additional functional and validation studies indicate that both the newly identified RBPs and the CD44s and NUMB2/4 splicing isoforms promote local invasion and distant metastasis and are associated with poor survival.

The systematic elucidation of the spectrum of EMT-related RBPs and AS targets in colon cancer and in other malignancies, apart from the insights in the mechanisms underlying phenotypic plasticity, will lead to the identification of novel and tumor-specific therapeutic targets.

category: cancer biology


title: Numerical discrimination in Drosophila melanogaster

abstract: Sensitivity to numbers is a crucial and evolutionarily conserved cognitive ability. The lack of experimental models amenable to systematic genetic and neural manipulation has precluded discovering circuits required for numerical cognition. Here, we demonstrate that in a two-choice task Drosophila fruit flies spontaneously prefer sets containing more objects. This preference is determined by the ratio between the two numerical quantities tested, a characteristic signature of numerical cognition across species. Individual flies maintained their numerical choice over consecutive days. Using a numerical visual conditioning paradigm, we found that flies are capable of associating sucrose with numerical quantities and can be trained to reverse their spontaneous preference for large quantities. Finally, we show that silencing LC11 neurons reduces the preference for more objects, thus identifying a neuronal substrate for numerical cognition in invertebrates. This discovery paves the way for the systematic analysis of the behavioral and neural mechanisms underlying sensitivity to numerosity.