title: Transcriptome analysis of alcohol dependence and stress interactions in the nucleus of the solitary tract

abstract: Stress exposure contributes to the development of drug and alcohol use disorders. In animal models, stress exacerbates escalations in alcohol consumption in alcohol-dependent animals. The nucleus of the solitary tract (NTS) is a critical brainstem region for integrating and relaying peripheral signals to regulate stress responses. To define the molecular adaptions within this brain region that may contribute to stress-induced alcohol drinking, we exposed animals to chronic intermittent bouts of ethanol vapor (CIE), forced swim stress (FSS), or both (CIE + FSS) and then transcriptionally profiled the NTS at three different timepoints after the last vapor exposure (0-hr, 72-hr, and 186-hr). We identified interferon (IFN) signaling as a critical gene network correlated with alcohol consumption levels. Using a likelihood ratio test, we identified genes that were differentially expressed across time and between groups. Clustering analysis of these genes to identify unique expression patterns identified a subset of genes that fail to normalize in the CIE + FSS group, but not the others. These genes were enriched for cell-to-cell interaction and cellular movement pointing to long-term structural and functional changes in this brain region caused by the unique interaction of alcohol dependence and stress. Specific genes of interest identified in this group include Aqp4, Il16, Reln, Grm4, Gabrd, and Gabra6. We also compared gene expression changes in the NTS to the PFC and found a significant overlap of genes between the two brain regions. Overlapping NTS/PFC genes in the CIE + FSS group were enriched for type I IFN signaling. Finally, we tested the hypothesis that activation of type I IFN signaling increases alcohol consumption based on the three lines of evidence identifying type I IFN signaling as critical for escalations in alcohol intake. Mice treated with recombinant IFN{beta} showed significantly elevated levels of alcohol intake in a two-bottle choice procedure compared to saline-treated controls. Overall, these results define the transcriptomic changes across time in the NTS that may be critical to the development of stress-induced increases in alcohol consumption and alcohol dependence.

category: molecular biology


title: Arginine 65 methylation of Neurogenin 3 by PRMT1 is a prerequisite for development of hESCs into pancreatic endocrine cells

abstract: In the developing pancreas, Neurogenin 3 (NGN3) is a key transcription factor in the cell fate determination of endocrine progenitors (EPs). Although the activation and stability of NGN3 are regulated by phosphorylation, the role of arginine methylation of NGN3 is poorly understood. Here, we report arginine 65 methylation of NGN3 is absolutely required for the pancreatic lineage development of human embryonic stem cells (hESCs) in vitro. First, we found inducible protein arginine methyltransferase 1 (PRMT1)-knockout (P-iKO) hESCs did not differentiate from EPs to endocrine cells (ECs) in the presence of doxycycline. Loss of PRMT1 caused an accumulation of NGN3 in the cytoplasm of EPs and blocked NGN3s transcriptional activity in PRMT1-KO EPs. We also found that PRMT1 specifically methylates NGN3 arginine 65, and this modification is a prerequisite for ubiquitin-mediated NGN3 degradation. Our findings indicate arginine 65 methylation of NGN3 is a key molecular switch in hESCs in vitro permitting the differentiation into pancreatic endocrine lineages.

category: developmental biology


title: Evaluating the potential benefits and pitfalls of combining protein and expression quantitative trait loci in evidencing drug targets

abstract:
Molecular quantitative trait loci (molQTL), which can provide functional evidence on the mechanisms underlying phenotype-genotype associations, are increasingly used in drug target validation and safety assessment. In particular, protein abundance QTLs (pQTLs) and gene expression QTLs (eQTLs) are the most commonly used for this purpose. However, questions remain on how to best consolidate results from pQTLs and eQTLs for target validation.

In this study, we combined blood cell-derived eQTLs and plasma-derived pQTLs to form QTL pairs representing each gene and its product. We performed a series of enrichment analyses to identify features of QTL pairs that provide consistent evidence for drug targets based on the concordance of the direction of effect of the pQTL and eQTL. We repeated these analyses using eQTLs derived in 49 tissues.

We found that 25-30% of blood-cell derived QTL pairs have discordant effects. The difference in tissues of origin for molecular markers contributes to, but is not likely a major source of, this observed discordance. Finally, druggable genes were as likely to have discordant QTL pairs as concordant.

Our analyses suggest combining and consolidating evidence from pQTLs and eQTLs for drug target validation is crucial and should be done whenever possible, as many potential drug targets show discordance between the two molecular phenotypes that could be misleading if only one is considered. We also encourage investigating QTL tissue-specificity in target validation applications to help identify reasons for discordance and emphasise that concordance and discordance of QTL pairs across tissues are both informative in target validation.

category: genetics


title: Structure and functional mapping of the KRAB-KAP1 repressor complex

abstract: Transposable elements (TEs) are a genetic reservoir from which new genes and regulatory elements can emerge. Expression of TEs can be pathogenic, however, and is tightly regulated. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) recruit co-repressor KRAB-associated protein 1 (KAP1/TRIM28) to regulate many TEs but how KRAB-ZFPs and KAP1 interact remains unclear. Here, we report the crystal structure of the KAP1 tripartite motif (TRIM) in complex with the KRAB domain from a human KRAB-ZFP, ZNF93. Structure-guided mutations in the KAP1-KRAB binding interface abolished repressive activity in an epigenetic TE silencing assay. Deposition of H3K9me3 over thousands of loci is lost genome-wide in cells expressing a KAP1 variant with mutations that abolish KRAB binding. Our work identifies and functionally validates the KRAB-KAP1 molecular interface, which forms the nexus of a transcriptional control axis vital to vertebrates and underpins programmable gene repression by CRISPRi.

category: biochemistry


title: Eight week dietary inclusion of nitrite-containing frankfurter exacerbates colorectal cancer pathology, increases oxidative stress, alters metabolism and causes gut dybiosis in APCmin mice

abstract: Colorectal cancer (CRC) is the second most prevelant malignancy in Europe and diet is an important modifiable risk factor. Processed meat consumption, including meats with preservative salts such as sodium nitrite, have been implicated in CRC pathogenesis. This study investigated how the CRC pathology and metabolic status of adenomatous polyposis coli (APC) multiple intestinal neoplasia (min) mice was perturbed following 8 weeks of pork meat consumption. Dietary inclusions (15%) of either nitrite-free pork, nitrite-free sausage or nitrite-containing sausage (frankfurter) were compared against a parallel control group (100% chow). Comprehensive studies investigated: gastrointestinal tract histology (tumours, aberant crypt foci (ACF) and mucin deplin foci (MDF), lipid peroxidation (urine and serum), faecal microbiota and serum metabolomics (599 metabolites). After 8 weeks mice consuming the frankfurter diet had 53% more (P=0.014) gastrointestinal tumours than control, although ACF and MDF did not differ. Urine and serum lipid peroxidation markers were 59% (P=0.001) and 108% (P=0.001) higher, respectively in the frankfurter group. Gut dysbiosis was evident in these mice with comparably fewer Bacteriodes and more Fermicutes. Fasting serum levels of trimethylamine N-oxide (TMAO) and numerous triglycerides were elevated. Various serum phosphotidylcholine species were decreased. These results demonstrate that nitrite-containing sausages may exaccerbate the development of CRC pathology in APCMin mice to a greater extent than nitrite-free sausages, and this is associated with greater lipid peroxidation, wide-ranging metabolic alternation and gut dysbiosis.


title: Structure of a Rad52 homolog from bacteriophage in complex with a novel duplex intermediate of DNA annealing

abstract: Human Rad52 protein binds to ssDNA and promotes the annealing of complementary strands. This activity is central to multiple DNA repair pathways and Rad52 is a target for cancer therapeutics. Previous crystal structures of the DNA binding domain of Rad52 revealed an 11-mer ring that binds to ssDNA in an extended conformation with the bases exposed for homology recognition. While this complex is likely involved in the early stages of annealing, there is no structure of Rad52 with two strands of DNA bound simultaneously, and its mechanism of annealing is poorly understood. To approach this problem, we have turned to the RecT/Red{beta} family of annealing proteins from bacteriophage, which are distant homologs of Rad52 that form stable complexes with a duplex intermediate of annealing. We have used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 [A] structure of a RecT homolog from a prophage of Listeria innocua (LiRecT) in complex with two complementary 83-mer oligonucleotides that were added to the protein sequentially. The structure reveals a left-handed helical filament of the protein bound to a novel conformation of DNA duplex that is highly extended and under-wound. The duplex is bound at a stoichiometry of 5 bp/monomer to a deep, narrow, positively-charged groove that runs along the outer surface of the filament. Data from native mass spectrometry confirm that the filament complex seen by cryo-EM also exists in solution. Collectively, these data provide new insights into the mechanism of annealing by LiRecT and by homologous proteins including human Rad52.


title: Groundwater metabolome responds to recharge in fractured sedimentary strata

abstract: Understanding the sources, structure and fate of dissolved organic matter (DOM) in groundwater is paramount for protection and sustainable use of this vital resource. On its passage through the Critical Zone, DOM is subject to biogeochemical conversions. Therefore, it carries valuable cross-habitat information for monitoring and predicting the stability of groundwater ecosystem services and assessing these ecosystems response to fluctuations caused by external impacts such as climatic extremes. Challenges arise from insufficient knowledge on groundwater metabolite composition and dynamics due to a lack of consistent analytical approaches for long-term monitoring. Our study establishes groundwater metabolomics to decipher the complex biogeochemical transport and conversion of DOM. We explore fractured sedimentary bedrock along a hillslope recharge area by a 5-year untargeted metabolomics monitoring of oxic perched and anoxic phreatic groundwater. A summer with extremely high temperatures and low precipitation was included in the monitoring. Water was accessed by a sampling well-transect and regularly collected for liquid chromatography-mass spectrometry (LC-MS) investigation. Dimension reduction of the resulting complex data set by principal component analysis revealed that metabolome dissimilarities between distant wells coincide with transient cross-stratal flow indicated by groundwater levels. Time series of the groundwater metabolome data provides detailed insights into subsurface responses to recharge dynamics. We demonstrate that dissimilarity variability between groundwater bodies with contrasting aquifer properties coincides with recharge dynamics. This includes groundwater high- and lowstands as well as recharge and recession phases. Our monitoring approach allows to survey groundwater ecosystems even under extreme conditions. The metabolome was otherwise highly variable lacking seasonal patterns and did not segregate by geographic location of sampling wells, thus ruling out vegetation or (agricultural) land use as primary driving factor. Patterns that emerge from metabolomics monitoring give insight into subsurface ecosystem functioning and water quality evolution, essential for sustainable groundwater use and climate change-adapted management.


title: Visualizing the Transiently Populated Closed-State of Human HSP90 ATP Binding Domain

abstract: HSP90 are abundant molecular chaperones, assisting the folding of several hundred client proteins, including substrates involved in tumor growth or neurodegenerative diseases. A complex set of large ATP-driven structural changes occurs during HSP90 functional cycle. However, the existence of such structural rearrangements in apo HSP90 has remained unclear. Here, we identified a metastable excited state in the isolated HSP90 ATP binding domain. We used solution NMR and mutagenesis to characterize structures of both ground and excited states. We demonstrated that in solution the HSP90 ATP binding domain transiently samples a functionally relevant ATP-lid closed state, distant by more than 30 [A] from the ground state. NMR relaxation and molecular dynamics were combined to characterize the energy landscape corresponding to the transition between these interconverting states. The precise description of the dynamics and structures sampled by human HSP90 ATP binding domain is a paramount piece of information for the future design of new therapeutic ligands.

category: biophysics


title: A microfluidic platform to investigate the role of mechanical constraints on tissue reorganization

abstract: Mechanical constraints have a high impact on development processes, and there is a need for new tools to investigate the role of mechanosensitive pathways in tissue reorganization during development. We present here experiments where embryonic cell aggregates are aspired through constrictions in microfluidic channels, generating heterogeneous flows and cell deformations that can be imaged using biphoton microscopy. This tool provides a way to measure in situ local viscoelastic properties of 3D tissues and connect them to intercellular and extracellular events such as cell shape changes and cell rear-rangements. Perspectives include applications on organoids to investigate and quantify rheological properties of tissues, and to understand how constraints affect development.


title: Allele-specific activation, enzyme kinetics, and inhibitor sensitivities of EGFR exon 19 deletion mutations in lung cancer

abstract:
Oncogenic mutations within the epidermal growth factor receptor (EGFR) are found in 15-30% of all non-small cell lung carcinomas. The term exon 19 deletion (ex19del) is collectively used to refer to more than 20 distinct genomic alterations within exon 19 that comprise the most common EGFR mutation subtype in lung cancer. Despite this heterogeneity, clinical treatment decisions are made irrespective of which EGFR ex19del variant is present within the tumor, and there is a paucity of information regarding how individual ex19del variants influence protein structure and function. Herein, we identify allele-specific functional differences among ex19del variants attributable to recurring sequence and structure motifs. We build all-atom structural models of 60 ex19del variants identified in patients and combine ~400 s of molecular dynamics simulations with biochemical and biophysical experiments to analyze three ex19del mutations (E746_A750, E746_S752>V, and L747_A750>P). We demonstrate that sequence variation in ex19del alters oncogenic cell growth, dimerization propensity, and tyrosine kinase inhibitor (TKI) sensitivity. We show that in contrast to E746_A750 and E746_S752>V, the L747_A750>P variant forms highly active ligand-independent dimers. E746_S752>V displays the least TKI sensitivity among the variants tested, which enzyme kinetic analysis and TKI inhibition experiments suggest is due to increased ATP Km relative to the common E746_A750 variant. Through these analyses, we propose an expanded framework for interpreting ex19del variants and new considerations for therapeutic intervention.

SignificanceEGFR mutations are detected in approximately 30% of all lung adenocarcinomas, and the most common EGFR mutation occurring in ~50% of patients is termed "exon 19 deletion" (ex19del). Despite the existence of dozens of different genomic variants comprising what is generically referred to clinically as ex19del, clinicians currently do not distinguish between ex19del variants in considering treatment options, and the differences between ex19del variants are largely unstudied in the broader scientific community. Herein, we describe functional differences between distinct EGFR ex19del variants attributable to the structural features of each variant. These findings suggest a possible explanation for observed differences in patient outcomes stratified by ex19del subtype and reinforce the need for allele-specific considerations in clinical treatment decision making.

category: cancer biology


title: Multivalent, Bispecific ????B7-H3-????CD3 Chemically Self-Assembled Nanorings Direct Potent T-cell Responses Against Medulloblastoma

abstract:
Few therapeutic options have been made available for treating central nervous system tumors, especially upon recurrence. Recurrent medulloblastoma is uniformly lethal with no approved therapies. Recent preclinical studies have shown promising results for eradicating various solid tumors by targeting the overexpressed immune checkpoint molecule, B7-H3. However, due to several therapy-related toxicities and reports of tumor escape, the full potential of targeting this pan-cancer antigen has yet to be realized. Here, we designed and characterized bispecific chemically self-assembling nanorings (CSANs) that target the T cell receptor, CD3{varepsilon}, and tumor associated antigen, B7-H3, derived from the humanized 8H9 single chain variable fragment (scFv). We show that the B7-H3-CD3 CSANs increase T cell infiltration and facilitate selective cytotoxicity of B7-H3+ medulloblastoma spheroids and that activity is independent of target cell MHC class I expression. Importantly, non-specific T cell activation against the ONS 2303 medulloblastoma cell line can be reduced by tuning the valency of the CD3 targeted monomer in the oligomerized CSAN. Intraperitoneal injections of B7-H3-CD3 bispecific CSANs were found to effectively cross the blood-tumor barrier into the brain and elicit significant anti-tumor T cell activity intracranially as well as systemically in an orthotopic medulloblastoma model. Moreover, following treatment with B7-H3-CD3 CSANs, intratumoral CD4+ and CD8+ T cells were found to primarily have a central memory phenotype that displayed significant levels of characteristic activation markers. Collectively, these results demonstrate the ability of our multi-valent, bispecific CSANs to direct potent anti-tumor T cell responses and indicate its potential utility as an alternative or complementary therapy for immune cell targeting of B7-H3+ brain tumors.

SignificanceThis study presents B7-H3-CD3 bispecific CSANs as a promising, new immunotherapeutic option for patients with established B7-H3+ medulloblastomas.


title: Stress-tolerant, recyclable, and autonomously renewable biocatalyst platform enabled by engineered bacterial spores

abstract:
Here, we describe a stress-tolerant, recyclable, and autonomously renewable biocatalyst platform based on T7 RNA polymerase-enabled high-density protein display on bacterial spores (TIED). TIED uses high-level T7 RNA polymerase-driven expression of recombinant proteins specifically in sporulating cells to allow spontaneous assembly of recombinant fusion proteins on B. subtilis spore surface. TIED enables a high loading density in the range of 106-107 recombinant enzymes per spore, robust catalytic activities of displayed enzymes comparable to the respective free enzymes, and enhanced kinetic stability of displayed enzymes in methanol and elevated temperatures. Further, we demonstrate TIED-enzymes to be not only recyclable, but fully renewable after loss of activity through induction of germination and sporulation, enabling perpetual reuse of these immobilized biocatalysts.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/484680v1_ufig1.gif" ALT="Figure 1">
View larger version (46K):
org.highwire.dtl.DTLVardef@6f50d0org.highwire.dtl.DTLVardef@22a9c3org.highwire.dtl.DTLVardef@1b74e9corg.highwire.dtl.DTLVardef@18a11c8_HPS_FORMAT_FIGEXP  M_FIG C_FIG Schematic illustration of the T7 RNA polymerase-enabled high-density protein display (TIED) on bacterial spores and its unique features as a biocatalyst platform.

category: synthetic biology


title: Lineage tracing via associative chromosome/plasmid barcoding with siBar

abstract: We describe a new method, siBar, which enables simultaneous tracking of plasmids and chromosomal lineages within a bacterial host. siBar involves integration of a linearized plasmid construct carrying a unique combination of two molecular barcodes. Upon recircularization one barcode remains integrated into the host chromosome, while the other remains on the plasmid allowing direct observation of expansion and contraction of adaptive lineages. We also performed a pilot evolution experiment that allowed us to assess the barcode complexity and establish an analytical framework for the analysis of siBar data.

category: microbiology


title: The faecal microbiome of the wild European badger Meles meles; a comparison against other wild omnivorous mammals from across the globe

abstract: Here we investigate the faecal microbiome of wild European badgers Meles meles using samples collected at post-mortem as part of the All Wales Badger Found Dead study based on sequencing the V3-V4 region of the 16S rRNA gene. This is the first published characterisation of the badger microbiome. We initially undertook a sex-matched age comparison between the adult and cub microbiomes. Analysis used the QIIME 2 pipeline utilising DADA2 and the Silva database for taxonomy assignment. Fusobacteria appeared to be more abundant in the microbiomes of the cubs than the adults although no significant difference was seen in alpha or beta diversity between the adult and cub badger microbiomes. Comparisons were also made against other wild, omnivorous, mammals faecal microbiomes using publicly available data. Significant differences were seen in both alpha and beta diversity between the microbiomes from different species. As a wildlife species of interest to the disease bovine tuberculosis, knowledge of the faecal microbiome could assist in identification of infected badgers. Our work here suggests that if comparisons were made between the faeces of bTB infected and non-infected badgers, its possible age may not have a significant impact on the microbiome.


title: New features of micro-RNA regulation of mRNA translation and stability revealed by expression of targeted or not targeted reporter genes

abstract:
The existence of translation regulation by RNA-induced silencing complexes (RISCs) composed from Argonaute proteins and micro-RNAs is well established, however the mechanisms underlying specific cellular and miRNA effects and the way in which specific complexes arise are not completely clear.

Here we describe experiments with Renilla and Firefly luciferase reporter genes transfected on a PsiCheck2 plasmid into human cancer HCT116 or Me45 cells where only the Renilla gene contained or not sequences targeted by micro RNAs (miRNAs) in the 3UTR. The effects of targeting were miRNA-specific; miRNA-21 caused strong inhibition of translation whereas miRNA-24 or Let-7 caused no change or an increase in global reporter Renilla luciferase synthesis, and the mRNA-protein complexes formed by reporter transcripts in both cell types differed as shown by sucrose gradient sedimentation. In both cell types the presence of miRNA targets on Renilla transcripts affected expression of the co-transfected non-targeted Firefly luciferase, and Renilla and Firefly transcripts were found in the same sucrose gradient fractions. We also observed that specific anti-miRNA oligoribonucleotides influenced expression of the Firefly as well as of the Renilla gene, suggesting modulation of non-targeted transcript expression by miRNAs. Our results indicate the existence of interactions between miRNA-regulated and -unregulated transcripts and suggest that the use of the latter as a normalizers in experiments may be biased. We also discuss some hypothetical mechanisms which could explain the observed miRNA-induced effects.


title: Identification of Aspergillus niger aquaporins involved in hydrogen peroxide signaling

abstract:
Aspergillus niger is a robust microbial cell factory for organic acids production. However, the regulation of many industrially important pathways is still poorly understood. Recently the regulation of glucose oxidase (GOx) expression system involved in the biosynthesis of gluconic acid has been uncovered. Hydrogen peroxide, a by-product of the enzymatic conversion of glucose to gluconate, has a pivotal role as a signaling molecule in the induction of this system. In this study, facilitated diffusion of hydrogen peroxide via aquaporin water channels (AQPs) was studied. AQPs are transmembrane proteins of the major intrinsic proteins (MIPs) superfamily. In addition to water and glycerol, they may also transport small solutes such as hydrogen peroxide. Accordingly, the genome sequence of A. niger N402 was screened for putative AQPs. Seven were found which could be classified in three main groups. Their ability to facilitate diffusion of hydrogen peroxide was identified using yeast phenotypic growth assays and by studying AQP gene knock-outs in A. niger. Complementing from both yeast and A. niger experiments, the X-intrinsic protein AQPF plays apparent roles in facilitating hydrogen peroxide transport across cellular membrane.

IMPORTANCEAquaporins (AQP) are integral membrane proteins that facilitate transfer of water, small solutes and hydrogen peroxide across the plasma membrane. As they have conserved structural features, it is not difficult to detect their presence in fungal genomes. Their specific roles in fungi are however less understood. Previously we have elucidated the role of the hydrogen peroxide as a second messenger in the induction and regulation of the expression of glucose oxidase involved in the extracellular conversion of glucose in gluconate (1). Here we identified the involvement of AQPF, a member of the X-intrinsic protein class in facilitating transport of the hydrogen peroxide signal across the cell membrane.


title: The Arabidopsis Hop1 homolog ASY1 mediates cross-over assurance and interference

abstract:
The chromosome axis plays a crucial role in meiotic recombination. Here, we study the function of ASY1, the Arabidopsis homolog of the yeast chromosome axis associated component Hop1. Specifically, we characterized cross-over (CO) distribution in female and male meiosis by deep sequencing of the progeny of an allelic series of asy1 mutants. Combining data from nearly 1000 individual plants, we find that reduced ASY1 activity leads to genomic instability and sometimes drastic genomic rearrangements. We further observed that COs are less frequent and appear in more distal chromosomal regions in plants with no or reduced ASY1 activity, consistent with previous analyses. However, our sequencing approach revealed that the reduction in CO number is not as dramatic as suggested by cytological analyses. Analysis of double mutants of asy1 with mutants with three other CO factors, MUS81, MSH4 and MSH5 as well as the determination of foci number of the CO regulator MLH1 demonstrates that the majority of the COs in asy1, similar to the situation in the wildtype, largely belong to the class I, which are subject to interference. However, these COs are redistributed in asy1 mutants and typically appear much closer than in the wildtype. Hence, ASY1 plays a key role in CO interference that spaces COs along a chromosome. Conversely, since a large proportion of chromosomes do not receive any CO, we conclude that CO assurance, the process that ensures the obligatory assignment of one CO per chromosome, is also affected in asy1 mutants.

Significant statementThe regulation of the number and placement of cross-overs (COs) during meiosis is critical to ensure meiotic fidelity and promote new genetic combinations. Here, we investigated the function of one of the proteins of the chromosome axis, which plays a key role in CO formation: ASY1. Our results show that COs in asy1 mutants are positioned closer to each other than in the wildtype and that, despite a roughly similar number of COs, not every chromosome receives a CO. With this, our results shed light on the mechanisms regulating two important but still poorly understood aspects of meiosis: CO assurance, which safeguards at least one CO per chromosome pair, and CO interference, which prevents two COs from occurring close to each other.

category: cell biology


title: The DeepFaune initiative: a collaborative effort towards the automatic identification of the French fauna in camera-trap images

abstract:
Camera-traps have revolutionized the way ecologists monitor biodiversity and population abundances. Their full potential is however only realized when the hundreds of thousands of images collected can be rapidly classified with minimal human intervention. Machine learning approaches, and in particular deep learning methods, have allowed extraordinary progress towards this end. Trained classification models remain rare however, and for instance are only emerging for the European fauna. This can be explained by the technical expertise they require but also by the limited availability of large datasets of annotated pictures, which are key to obtaining successful recognition models.

In this context, we set-up the DeepFaune initiative (https://deepfaune.cnrs.fr), a large-scale collaboration between dozens of partners involved in research, conservation and management of wildlife in France. The aim of DeepFaune is to aggregate individual datasets of annotated pictures to train species classification models based on convolutional neural networks, an established deeplearning approach.

Here we report on our first milestone, a two-step pipeline built upon the MegaDetector algorithm for detection (discarding empty pictures and cropping the animal) and a classification model for 18 species or higher-level taxa as well as people and vehicles. The classification model achieved 92% validation accuracy and showed > 90% sensitivity and specificity for many classes. Most importantly, these performances were generally conserved when tested on an independent out-of-sample dataset. In addition, we developed a cross-platform graphical-user-interface that allows running the pipeline on images stored locally on a personal computer.

In conclusion, the DeepFaune initiative provides a freely available (for non-commercial purposes) toolbox with high performance to classify the French fauna in camera-trap images.

category: ecology


title: clampFISH 2.0 enables rapid, scalable amplified RNA detection in situ

abstract: RNA labeling in situ has enormous potential to visualize transcripts and quantify their levels in single cells, but it remains challenging to produce high levels of signal while also enabling multiplexed detection of multiple RNA species simultaneously. Here, we describe clampFISH 2.0, a method that uses an inverted padlock design to efficiently detect and exponentially amplify signals from many RNA species at once, while also reducing time and cost compared to the prior clampFISH method. We leverage the increased throughput afforded by multiplexed signal amplification and sequential detection by demonstrating the ability to detect 10 different RNA species in over 1 million cells. We also show that clampFISH 2.0 works in tissue sections. We expect the advantages offered by clampFISH 2.0 will enable many applications in spatial transcriptomics.

category: bioengineering


title: Rational design of Cas9 ribonucleoprotein with a "gRNA-shRNA" for multidimensional genome manipulation and enhanced homology-directed repair

abstract: Gene perturbation approaches have evolved as powerful tools for understanding the function of genes and curing inherited diseases. Here, we develop a method that combines the merits of RNAi and CRISPR technology by rational design of Cas9 ribonucleoprotein (RNP) with a "gRNA-shRNA" component. The RNP, termed Cas9-RNAi, has a gRNA containing a 3 extension that can be processed to a functional siRNA via dorsha/dicer enzyme mediated cleavage within cells. We prepared the Cas9-RNAi RNPs by streamline co-expression of Cas9 enzymes and the "gRNA-shRNA" ribonucleotides in Escherichia coli strain HT115(DE)3. Transferring the Cas9-RNAi RNPs into mammalian cells achieves multidimensional genome manipulation, e.g., simultaneously knock out and knock down the target genes. Moreover, by introduction of a shRNA against the gene of human DNA ligase 4 (LIG4), significantly improved homology-directed repair was attained. Together, we develop a simple-to-use CRISPR RNP tool that has great potentials in precise genome editing, gene function analysis and gene therapy.


title: TidyMass: An Object-oriented Reproducible Analysis Framework for LC-MS Data

abstract: Reproducibility and transparency have been longstanding but significant problems for the metabolomics field. Here, we present the tidyMass project (https://www.tidymass.org/), a comprehensive computational framework that can achieve the shareable and reproducible workflow needs of data processing and analysis for LC-MS-based untargeted metabolomics. TidyMass was designed based on the following strategies to address the limitations of current tools: 1) Cross-platform utility. TidyMass can be installed on all platforms; 2) Uniformity, shareability, traceability, and reproducibility. A uniform data format has been developed, specifically designed to store and manage processed metabolomics data and processing parameters, making it possible to trace the prior analysis steps and parameters; 3) Flexibility and extensibility. The modular architecture makes tidyMass a highly flexible and extensible tool, so other users can improve it and integrate it with their own pipeline easily.

category: bioinformatics


title: Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

abstract: The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including keto-isovalerate submodule for converting pyruvate to keto-isovalerate, ketoacid elongation submodule for producing longer carbon-chain keto-acids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23-31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. The engineered strains produced isoamyl acetate at a titer of 8.8 g/L (> 0.25 g/L toxicity limit), a yield of 0.17 g/g (47% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.


title: Structure of dynein-dynactin on microtubules shows tandem recruitment of cargo adaptors

abstract: Cytoplasmic dynein is a microtubule motor that is activated by its cofactor dynactin and a coiled-coil cargo adaptor. There is currently limited structural information on how the resulting complex interacts with microtubules and how adaptors are recruited. Here, we develop a cryo-EM processing pipeline to solve the high-resolution structure of dynein-dynactin and the adaptor BICDR1 bound to microtubules. This reveals the asymmetric interactions between neighbouring dynein motor domains and how it relates to their motile behaviour. We find unexpectedly that two adaptors occupy the complex. Both adaptors make similar interactions with the dyneins but diverge in their contacts with each other and dynactin. Our structure has implications for the stability and stoichiometry of motor recruitment by cargos.


title: A correlational study between microstructural, macrostructural and functional age-related changes in the human visual cortex

abstract: Age-related changes in the human brain can be investigated from either structural or functional perspectives. Analysis of structural and functional age-related changes throughout the lifespan may help to understand the normal brain development process and monitor the structural and functional pathology of the brain. This study, combining dedicated electroencephalography (EEG) and magnetic resonance imaging (MRI) approaches in adults (20-78 years), highlights the complex relationship between micro/macrostructural properties and the functional responses to visual stimuli. Here, we aimed to relate age-related changes of the latency of visual evoked potentials (VEPs) to micro/macrostructural indexes and find any correlation between micro/macrostructural features, as well. We studied age-related structural changes in the brain, by using the MRI and diffusion-weighted imaging (DWI) as preferred imaging methods for extracting brain macrostructural parameters such as the cortical thickness, surface area, folding and curvature index, gray matter volume, and microstructural parameters such as mean diffusivity (MD), radial diffusivity (RD) and axial diffusivity (AD). All the mentioned features were significantly correlated with age in V1 and V2 regions of the visual cortex. Furthermore, we highlighted, negative correlations between structural features extracted from T1-weighted images and DWI. The latency and amplitude of the three dominants peaks (C1, P1, N1) of the VEP were considered as the brain functional features to be examined for correlation with age and structural features of the corresponding age. We observed significant correlations between mean C1 latency and GM volume averaged in V1 and V2. In hierarchical models, the structural index did not contributed to significant additional variance in the C1 latency after accounting for the variance associated with age. However, the age explained significant additional variance in the model after accounting for the variance associated with the structural feature.


title: Effects of different environmental intervention durations on the intestinal mucosal barrier and the brain-gut axis in rats with colorectal cancer

abstract:
ObjectivesEnriched environment (EE) is a promising strategy to protect the intestinal mucosal barrier and regulate brain-gut peptide expression. However, the optimal enriched environment intervention duration is unknown. Here, different EE intervention durations were applied to assess the optimal intervention duration in rats with colorectal cancer.

MethodsWe used a rat model of 1,2-dimethylhydrazine-induced colorectal cancer. Rats were housed in an EE for 0, 2, 4 or 8 weeks. The intestinal mucosa and serum TNF-, IL-6, IL-10, DAO, ATP, CRF, and occludin levels and bacterial translocation (BT) were measured, and the intestinal mucosa morphology was evaluated.

ResultsEight-week EE intervention was more beneficial to the intestinal mucosal mechanical barrier than 2-week or 4-week intervention (P<0.05). There was a significant different between the 4-week and 8-week groups on BT (P=0.049). However, which intervention duration had the greatest advantages in intestinal mucosa and serum inflammatory factor regulation was not determined. There were no significant differences in the effects of different EE intervention durations on BT or brain intestinal peptide levels among the other groups (P>0.05).

ConclusionsThe effect of an 8-week environmental intervention duration on the intestinal mucosal barrier was better than that of 2-week and 4-week durations overall, but the effect of different environmental intervention durations on brain-gut peptide levels was not obvious. In the future, we can further explore the molecular biological mechanism of the effect of different EE intervention durations on the intestinal mucosal barrier and analyze the effect of an EE on other brain-gut peptides.


title: Fgf8 dosage regulates jaw shape and symmetry through pharyngeal-cardiac tissue relationships

abstract:
BackgroundAsymmetries in craniofacial anomalies are commonly observed. With respect to the facial skeleton, the left side is more commonly and/or severely affected than the right. Such asymmetries complicate treatment options. Mechanisms underlying variation in disease severity between individuals as well as within individuals (asymmetries) are still relatively unknown.

ResultsDevelopmental reductions in Fibroblast growth factor 8 (Fgf8) have a dosage dependent effect on jaw size, shape, and symmetry. Further, Fgf8 mutants have directionally asymmetric jaws with the left side being more affected than the right. Defects in lower jaw development begin with an early disruption to Meckels cartilage, which is discontinuous and appears as two separate condensations in Fgf8 mutants. All skeletal elements associated with the proximal condensation are dysmorphic in the mutants, which is exemplified by a malformed and mis-oriented malleus. At later stages, Fgf8 mutants exhibit syngnathia, which falls into 2 broad categories: bony fusion of the maxillary and mandibular alveolar ridges and zygomatico-mandibular fusion. All of these morphological defects exhibit both inter- and intra-individual variation.

ConclusionsWe hypothesize that these asymmetries are linked to asymmetries in heart development resulting in higher levels of Fgf8 on the right side of the face during development, which may buffer the right side to mild developmental perturbations. This mutant mouse is a good model for future investigations of mechanisms underlying human syngnathia and facial asymmetry.


title: Single-cell RNA sequencing of conditional 	Pten knockout neurons in mice

abstract: PTEN is a well-known tumor suppressor whose mutations are also strongly associated with Autism Spectrum Disorder (ASD). The activity and function of PTEN in neurons have been studied extensively in various settings, whereas animal models offer the best opportunity to experimentally modify and test the role of PTEN in neuronal development in vivo. On the molecular level, PTENs importance in the mTOR pathways suppression is well-known but many other interactions have been suggested and yet others likely remain undiscovered. Therefore, to systematically explore the regulatory landscape downstream of PTEN on the genetic level, we have set out to establish a single-cell RNA sequencing (scRNA-seq) workflow to measure gene expression changes as a result of knocking out the Pten gene in vivo. We were able to collect brain tissue from four conditionally knocked out mouse samples and dissociate them into single live cells, which were subsequently flow-sorted and a scRNA-seq library was prepared. All of these steps were completed in a single day, yielding sequencing data from hundreds of cells suitable for cell clustering, cell type identification, and differential expression measurements. Having demonstrated its feasibility, this approach promises to aid hypothesis generation and discovery in mapping the function of Pten as well other genes whose regulatory networks remain to be fully defined.

category: neuroscience


title: Distinct cortical responses to Auditory Statistics: pre-attentive discriminations based on local and global representations

abstract:
The auditory system relies on local and global representations, depending on the amount of entering temporal details. When local information exceeds system constraints, acoustic details are summarized into a set of average statistics, and global structure emerges. Whether computations subtending local and global representations of sounds can be distinguished at the cortical level is unknown. A computational auditory model was employed to extract auditory statistics from natural sound textures, such as fire, wind, and rain, and generate synthetic exemplars in which we had control of local and global properties. Participants were passively exposed to auditory streams with triplets of sounds including two standards and an oddball while the EEG was recorded. In Local Discrimination (exp 1) the oddball sound varied from standards only for its local features; in Global Discrimination (exp 2), it varied for its global structure. Different sound durations were employed to manipulate the amount of acoustic information and statistical variability between standards and oddballs. We expected short and long sounds to engage local (exp 1) or global (exp 2) representations. As predicted, neural responses associated with local discriminations exceeded global ones for short sounds, whereas the opposite pattern emerged for longer sounds. Electrical source estimates revealed that local discriminations mainly engaged auditory regions of the right hemisphere while global discriminations of the left hemisphere. These findings revealed distinct neural correlates of acoustic discriminations guided by the temporal scale at which acoustic variability occurs and thus by local or global structures. Ultimately results demonstrated that the auditory system developed a functional architecture to automatically detect changes occurring in the environment based on these two modes of sound representation.

SIGNIFICANT STATEMENTPrior to this study, it was not known whether it was possible to directly measure auditory cortical representations of sound discriminations depending on local or global acoustic properties. Results unveiled the existence of a mechanisms which pre-attentively determines at which timescale (local or global) a change in a stream occurred based on statistical variability between sound excerpts. By performing source reconstruction, we additionally revealed that the response to local change detection pertains to different cortical structures as compared to global one, displaying also hemispheric asymmetries. These findings expand our knowledge on fundamental mechanisms characterizing auditory system functioning.


title: Mesoaccumbal glutamate neurons drive reward via glutamate release, but aversion via dopamine co-release

abstract: Ventral tegmental area (VTA) projections to the nucleus accumbens medial shell (NAc) drive reward-related motivation. Although dopamine neurons are predominant, a substantial glutamatergic projection is also present, and a subset of these populations can release both dopamine and glutamate. Optogenetic stimulation of VTA glutamate neurons supports self-stimulation, but can also induce place avoidance, even in the same assay. Here, we parsed the selective contribution of glutamate or dopamine co-release from VTA glutamate neurons to reinforcement and avoidance. We expressed Channelrhodopsin (ChR2) in VTA glutamate neurons, in combination with CRISPR/Cas9 to disrupt either the gene encoding vesicular glutamate transporter 2 (VGLUT2) or Tyrosine hydroxylase (Th). Selective disruption of VGLUT2 abolished optogenetic self-stimulation, but left real-time place avoidance intact, while CRISPR/Cas9 deletion of Th preserved optogenetic self-stimulation but abolished place avoidance. Our results demonstrate that glutamate release from VTA glutamate neurons is positively reinforcing, but that dopamine release from these same neurons can induce avoidance behavior.


title: A brain-wide analysis maps structural evolution to distinct anatomical modules

abstract: Brain anatomy is highly variable and it is widely accepted that anatomical variation impacts brain function and ultimately behavior. The structural complexity of the brain, including differences in volume and shape, presents an enormous barrier to define how variability underlies differences in function. In this study, we sought to investigate the evolution of brain anatomy in relation to brain region volume and shape across the brain of a single species with variable genetic and anatomical morphs. We generated a high-resolution brain atlas for the blind Mexican cavefish and coupled the atlas with automated computational tools to directly assess brain region shape and volume variability across all populations. We measured the volume and shape of every neuroanatomical region of the brain and assess correlations between anatomical regions in surface, cavefish and surface to cave F2 hybrids, whose phenotypes span the range of surface to cave. We find that dorsal regions of the brain are contracted in cavefish, while ventral regions have expanded. Interestingly, in hybrid fish the volume and shape of dorsal regions are inversely proportional to ventral regions. This trend is true for both volume and shape, suggesting that these two parameters share developmental mechanisms necessary for remodeling the entire brain. Given the high conservation of brain anatomy and function among vertebrate species, we expect these data to studies reveal generalized principles of brain evolution and show that Astyanax provides a system for functionally determining basic principles of brain evolution by utilizing the independent genetic diversity of different morphs, to test how genes influence early patterning events to drive brain-wide anatomical evolution.


title: Surface motility favors co-dependent interaction between Pseudomonas aeruginosa and Burkholderia cenocepacia

abstract:
Interactions between different bacterial species shape bacterial communities and their environments. The opportunistic pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia both can colonize the lungs of individuals affected by cystic fibrosis. Using the social surface behavior called swarming motility as a study model of interactions, we noticed intricate interactions between B. cenocepacia K56-2 and P. aeruginosa PA14. While strain K56-2 does not swarm under P. aeruginosa favorable swarming conditions, co-inoculation with a non-motile PA14 flagellum-less {Delta}fliC mutant restored spreading for both strains. We show that P. aeruginosa provides the wetting agent rhamnolipids allowing K56-2 to perform swarming motility, while aflagellated PA14 seems able to <<hitchhike>> along with K56-2 cells in the swarming colony.

ImportancePseudomonas aeruginosa and Burkholderia cenocepacia are important opportunistic pathogens often found together in the airways of persons with cystic fibrosis. Laboratory co-culture of both species often ends with one taking over the other. We used a surface motility assay to study the social interactions between population of these bacterial species. Under our conditions, B. cenocepacia cannot swarm without supplementation of the wetting agent produced by P. aeruginosa. In a mixed colony of both species, an aflagellated mutant of P. aeruginosa provides the necessary wetting agent to B. cenocepacia, allowing both bacteria to swarm and colonize a surface. We highlight this peculiar interaction where both bacteria set aside their antagonistic tendencies to cooperate.


title: Genomics epidemiology analysis reveals hidden signatures of drug resistance in Mycobacterium tuberculosis

abstract:
Mycobacterium tuberculosis (Mtb) causes the majority of reported cases of human tuberculosis (TB), one of the deadliest infectious diseases worldwide. New diagnostic tools and approaches to detect drug-resistance must be introduced by 2025 to achieve the End-TB Strategy goals set for 2030 by the WHO. Genomic epidemiology of TB has allowed the expansion of catalogs listing genetic signatures of Mtb drug-resistance. However, very few Mtb strains from Latin America have participate in previous genomic epidemiologic efforts. Here we present the first functional genomic epidemiology study of drug-resistant Mtb strains in Mexico, incorporating the genomic characterization of 133 genomes, including 53 newly sequenced isolates, to provide a comprehensive phylogeographic analysis of drug resistant Mtb in Mexico. The study evidences the prevalence of Euro-American Lineage L4 (96.2%), featuring a uniform distribution of the sublineages X-type (33.08%), LAM (22.56%), and Haarlem (21.05%). Our results demonstrate low levels of agreement with traditional drug sensitivity tests (DST), raising concerns for drug-resistant isolates lacking any previously reported genetic signatures of resistance. Finally, we propose a novel functional networking tool (FuN-TB) to explore metabolic and cellular signatures of drug resistance. Applying functional genomics approaches to Latin American Mtb genomes will provide new drug-resistance screening targets that can contribute to bed side decision-making and advise local public policy.

Abstract importanceWe presented the first phylogeographic analysis of Mycobacterium tuberculosis (Mtb) of Mexico. Our analysis integrates 133 genome sequences and is focused on the identification of genetic signatures associated to drug-resistance. The results show the geographic distribution of sublineages and drug-resistance phenotypic classes. Additionally, we propose a novel functional networking tool (FuN-TB) to explore metabolic and cellular signatures of drug resistance associated. We show for the first time that Mtb isolates from Mexico encode for region-specific genetic signarures of antimicrobial resistance. Applying functional genomics approaches to Latin American Mtb genomes will provide new drug-resistance screening targets that can contribute to bed side decision-making and advise local public policy.

category: genomics


title: Brain-wide screen of prelimbic cortex inputs reveals a functional shift during early fear memory consolidation

abstract: Memory formation and storage rely on multiple interconnected brain areas, the contribution of which varies during memory consolidation. The medial prefrontal cortex, in particular the prelimbic cortex (PL), was traditionally found to be involved in remote memory storage, but recent evidence points towards its implication in early consolidation as well. Nevertheless, the inputs to the PL governing these dynamics remain unknown. Here, we first performed a brain-wide, rabies-based retrograde tracing screen of PL engram cells activated during contextual fear memory formation to identify relevant PL input regions. Next, we assessed the specific activity pattern of these inputs across different phases of memory consolidation, from fear memory encoding to recent and remote memory recall. Using projection-specific chemogenetic inhibition, we then tested their functional role in memory consolidation, which revealed a hitherto unknown contribution of claustrum to PL inputs at encoding, and of insular cortex to PL inputs at recent memory recall. Both of these inputs further impacted how PL engram cells were reactivated at memory recall, testifying to their relevance for establishing a memory trace in the PL. Collectively, this data identify a spatiotemporal shift in PL inputs important for early memory consolidation, and thereby help to refine the working model of memory formation.


title: Circulating miR-30b-5p is up regulated in Cavalier King Charles Spaniels affected by early myxomatous mitral valve disease

abstract: There is a growing interest in developing new molecular markers of heart disease in young Cavalier King Charles Spaniels affected by myxomatous mitral valve disease. The aim of the study was to measure the abundance of 3 circulating microRNAs and their application as potential biomarkers in the plasma of Cavalier King Charles Spaniels with early asymptomatic myxomatous mitral valve disease. 33 dogs affected by the disease in American College of Veterinary Internal Medicine (ACVIM) stage B1 were divided in three groups (11 younger than 3 years, 11 older than 3 years and younger than 7 years, and 11 older than 7 years), and 11 healthy (ACVIM stage A) Cavalier King Charles Spaniels were included as the control group. This is a prospective cross-sectional study. The abundance of three circulating microRNAs (miR-1-3p, miR30b-5p, and miR-128-3p) was measured by quantitative real-time PCR using TaqMan(R) probes. Diagnostic performance was evaluated by calculating the area under the receiver operating curve (AUC). miR-30b-5p was significantly higher in ACVIM B1 dogs compared to ACVIM A subjects, and the area under the receiver operating curve was 0.79. According to the age of dogs, the abundance of miR-30b-5p was statistically significantly higher in group B1<3y (2.3 folds, P = 0.034), B1 3-7y (2.2 folds, P = 0.028), and B1>7y (2.7 folds, P = 0.018) than in group A. The area under the receiver operating curves were fair in discriminating between group B1<3y and group A (AUC 0.780), between B1 3-7y and A (AUC 0.78), and good in discriminating between group B1>7y and A (AUC 0.822). miR-30b-5p changed in the plasma of dogs at the asymptomatic stage of disease, particularly at a young age.


title: Genomic analyses identify key molecules and significant signaling pathways in Sorafenib treated hepatocellular cancer cells

abstract: Sorafenib (Sfb) is a multikinase inhibitor that is significantly effective in killing liver cancer. However, the potential signaling pathways and mechanisms remain unknown. Here, we aim to identify the functional molecules and signaling pathways by analyzing the RNA-seq data. The GSE186280 was produced by the Illumina HiSeq 2000 (Homo sapiens). The KEGG and GO analyses indicated the MAPK signaling pathway and transcriptional misregulation in cancer are the major signaling pathways during the treatment of Sfb in liver cancer. Moreover, we identified ten key molecules including TP53, MYC, ACTB, CREBBP, HSP90AA1, JUN, UBB, SIRT1, CCNA2, RBX1. Therefore, this study provides novel knowledge of Sfb treated liver cancer.


title: Tempo: an unsupervised Bayesian algorithm for circadian phase inference in single-cell transcriptomics

abstract: The circadian clock is a 24-hour cellular timekeeping mechanism that temporally regulates human physiology. Answering several fundamental questions in circadian biology will require joint measures of single-cell circadian phases and transcriptomes. However, no widespread experimental approaches exist for this purpose. While computational approaches exist to infer cell phase directly from single-cell RNA-sequencing (scRNA-seq) data, existing methods yield poor circadian phase estimates, and do not quantify estimation uncertainty, which is essential for interpretation of results from highly sparse scRNA-seq data. To address these unmet needs, we developed Tempo, a Bayesian variational inference approach that incorporates domain knowledge of the clock and quantifies phase estimation uncertainty. Through simulations and analyses of real data, we demonstrate that Tempo yields more accurate estimates of circadian phase than existing methods and provides well-calibrated uncertainty quantifications. We further demonstrate that these properties generalize to the cell cycle. Tempo will facilitate large-scale studies of single-cell circadian transcription.


title: The Trichloroethylene Metabolite S-(1,2-dichlorovinyl)-l-cysteine Inhibits Lipopolysaccharide-induced Transcriptomic Inflammation Pathways and Cytokine Secretion in a Macrophage Cell Model

abstract: Previous studies have shown that the trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC) inhibits cytokine secretion in pathogen stimulated fetal membrane tissue but little is known about the mechanism for these effects, including which cell types or transcriptomic pathways are impacted. Macrophages play a critical role in the fetal membrane innate immune response during infection. We tested the hypothesis that DCVC inhibits lipopolysaccharide (LPS) stimulated inflammation pathways in differentiated (macrophage-like) THP-1 cells. THP-1 cells were differentiated with phorbol 12-myristate 13-acetone for 24 hours and subsequently treated with 1, 5, or 10 {micro}M DCVC for 24 hours. After an additional 4 hour incubation with lipopolysaccharide (LPS), we collected RNA and cell media. We performed transcriptomic analysis using RNA sequencing analysis for 5{micro}M DCVC treatments and quantified cytokine release (IL-1{beta}, IL-6, and TNF-) into cell media for 1, 5 and 10 {micro}M DCVC treatments. RNAseq analysis revealed 1,399 differentially expressed genes (FDR<0.05 and log2fold change magnitude>2.5) in the cells co-treated with DCVC and LPS compared to LPS alone. For example, TNF was 9-fold downregulated with the addition of DCVC. Major pathways downregulated (adjusted p-value<0.05) in DCVC+LPS treatments versus LPS-only treatments, included: "acute inflammatory response", "production of molecular mediator of immune response" and "phagocytosis". LPS increased IL-1{beta}, IL-6, and TNF- levels in culture media (p<0.001), but this effect which was inhibited by co-treatment with DCVC (p<0.001 for LPS vs. LPS+DCVC treatments). Our results demonstrate that DCVC suppresses inflammatory responses in macrophages.

category: pharmacology and toxicology


title: Feature-based attention multiplicatively scales the fMRI-BOLD contrast-response function

abstract:
Functional MRI (fMRI) plays a key role in the study of attention. However, there remains a puzzling discrepancy between attention effects measured with fMRI and with electrophysiological methods. While electrophysiological studies find that attention increases sensory gain, amplifying stimulus-evoked neural responses by multiplicatively scaling the contrast-response function (CRF), fMRI appears to be insensitive to these multiplicative effects. Instead, fMRI studies typically find that attention produces an additive baseline shift in the blood-oxygen-level-dependent (BOLD) signal. These findings suggest that attentional effects measured with fMRI reflect top-down inputs to visual cortex, rather than the modulation of sensory gain. If true, this drastically limits what fMRI can tell us about how attention improves sensory coding. Here, we re-examined whether fMRI is sensitive to multiplicative effects of attention using a feature-based attention paradigm designed to preclude any possible additive effects. We measured BOLD activity evoked by a probe stimulus in one visual hemifield while participants (6 male, 6 female) attended to the probe orientation (attended condition), or to an orthogonal orientation (unattended condition), in the other hemifield. To measure CRFs in visual areas V1-V3, we parametrically varied the contrast of the probe stimulus. In all three areas, feature-based attention increased contrast gain, improving sensitivity by shifting CRFs towards lower contrasts. For a subset of visual eccentricities, we also found an increase in response gain, an increase in the responsivity of the CRF. These results provide clear evidence that the fMRI-BOLD signal is sensitive to multiplicative effects of attention.

Significance StatementFunctional MRI (fMRI) plays a central role in the study of attention because it allows researchers to precisely and non-invasively characterize the effects of attention throughout the brain. Electrophysiological studies have shown that attention increases sensory gain, amplifying stimulus-evoked neural responses. However, a growing body of work suggests that the BOLD signal that is measured with fMRI is not sensitive to these multiplicative effects of attention, calling into question what we can learn from fMRI about how attention improves sensory codes. Here, using a feature-based attention paradigm, we provide evidence that the BOLD signal can pick up multiplicative effects of attention.


title: NFAT5 induction by the tumor microenvironment enforces CD8 T cell exhaustion

abstract: Persistent exposure to antigen during chronic infection or cancer renders T cells dysfunctional. The molecular mechanisms regulating this state of exhaustion are thought to be common in infection and cancer, despite obvious differences in their microenvironments. We discovered that NFAT5, an NFAT family member lacking an AP-1 docking site, is highly expressed in exhausted T cells from murine and human tumors and is a central player in tumor-induced exhaustion. While NFAT5 overexpression reduced tumor control, NFAT5 deletion improved tumor control by promoting the accumulation of tumor-specific CD8+ T cells that expressed less TOX and PD-1 and produced more cytokines particularly among precursor exhausted cells. Conversely, NFAT5 had no effect on chronic infection-induced T cell exhaustion. Mechanistically we found that TCR triggering induced NFAT5 expression and that hyperosmolarity stimulated transcriptional activity of NFAT5. We propose that NFAT5 takes over NFAT1/2 to promote exhaustion specifically in tumor-infiltrating CD8+ T cells.

category: immunology


title: Are trapping data still suited for home range estimation? An analysis with various estimators, asymptotic models and data ordering procedures

abstract: Understanding the size of animals home ranges is vital for studies in ecology and conservation. Trapping datasets are an important source of information when targeting the biodiversity of an area, inconspicuous species, or high numbers of individuals in contrast to more expensive telemetry-based methods such as radio- or GPS-collaring. Currently, studies relying on trapping lack an evaluation of the performance of existing home range estimation procedures comparable to those developed for telemetry. Using animal movement simulations, we evaluate three variables reflecting the trade-offs faced by ecologists when designing a trapping study, 1) the number of observations obtained per individual, 2) the trap density and 3) the proportion of the home range area falling inside of the trapping grid. We compare the performance of five estimators on these conditions, four commonly used (AKDE, KDE, MCP, LoCoH) and a possible alternative for situations with low trap density or high number of observations (bicubic interpolation). We further test suggested benefits of using asymptotic models (Michaelis-Menten and monomolecular) to assess the total home range area when information obtained per individual is scarce, as this situation might be common in trapping datasets. In addition, we propose sorting the observations based on the distance between locations to improve the performance of asymptotic models estimates. Using the results of the different procedures we constructed a generalized additive model (GAM) that allows predicting the bias in home range size under the different scenarios investigated. Our results show that the proportion of the area covered by the trapping grid and the number of observations were the most important factors predicting the accuracy and reliability of the estimates. The use of asymptotic models helped obtaining an accurate estimation at lower sample sizes and this effect was further improved by distance-ordering. The autocorrelation informed KDE was the estimator performing best under most conditions evaluated. Nevertheless, bicubic interpolation can be an alternative under common trapping conditions with low density of traps and low area covered. We provide the current results to the constructed GAM as a prospective tool for ecologists planning a new study or with already collected datasets that aim at assessing the potential biases in their estimates. Reliable and accurate home range estimates using trapping data can optimize monetary costs of home range studies, potentially enlarging the span of species, researchers and questions studied in ecology and conservation.


title: The use of pigs vocalisation structure to assess the quality of human-pig relationship

abstract: In domestic species, studying human-animal interactions and their consequences on the establishment of a positive Human-Animal Relationship (HAR) would have applications for both improving animal welfare. Objectify the quality of a HAR requires information on several aspects of the animal biology and emotional states (spatial behaviour, physiological and cognitive states). Growing evidence show that acoustic features of animal vocalisations may be a indicators of emotional states. Here, we tested the hypothesis that the quality of vocal expression may indicate the quality of HAR. Thirty piglets were tamed thanks to regular interactions with an experimenter talking and physically interacting with them, three times a day from weaning; while 30 other piglets received only contact necessary for their good breeding. Two weeks later, we recorded behaviours and vocalisations produced in presence of the static experimenter for five minutes. The test was repeated two weeks later, after a period of conditioning using human presence and contacts as a reward for all piglets, supposed to lead to a positive human-piglet relationship for all piglets. As expected, taming led to an attraction toward the experimenter, and, after the conditioning, untamed piglets expressed a similar level of attraction than previously tamed piglets. Tamed piglets generally produced shorter grunts than untamed piglets. However untamed piglets expressed more flexibility in call structure when vocalising close to a human, with a decrease of grunt duration and an increase of pitch, frequency range and noisiness in their grunt. This differential effect of proximity in tamed and untamed piglets was attenuated after the conditioning during a standard reunion with a static human but remained over time when the human was providing additional positive contacts. Results suggest that first, changes in vocal structure are consistent with indicators of positive states in the presence of a human. Second, increasing familiarity and proximity between a human and a piglet may induce changes in the acoustic structure of their grunts. Third, a carrying human trigger more changes in vocalisation structure than by their presence only. We show that vocalisation structure may allow to assess the quality of human-pig relationship.

category: animal behavior and cognition


title: Cavitation Induced Fracture of Intact Brain Tissue

abstract:
Nonpenetrating traumatic brain injuries (TBI) are linked to cavitation. The structural organization of the brain makes it particularly susceptible to tears and fractures from these cavitation events, but limitations in existing characterization methods make it difficult to understand the relationship between fracture and cavitation in this tissue. More broadly, fracture energy is an important, yet often overlooked, mechanical property of all soft tissues. We combined needle-induced cavitation (NIC) with hydraulic fracture models to induce and quantify fracture in intact brains at precise locations. We report here the first measurements of the fracture energy of intact brain tissue that range from 1.5 to 8.9 J/m2, depending on the location in the brain and the model applied. We observed that fracture consistently occurs along interfaces between regions of brain tissue. These fractures along interfaces allow cavitation-related damage to propagate several millimeters away from the initial injury site. Quantifying the forces necessary to fracture brain and other soft tissues is critical for understanding how impact and blast waves damage tissue in vivo and has implications for the design of protective gear and tissue engineering.

SignificanceMild injuries associated with concussion and blast waves cause tearing of brain tissue, which leads to traumatic brain injury (TBI). TBI is a leading cause of death and disability among children and young adults in the U.S., with 1.5 million Americans reporting a TBI each year. We introduce a novel approach to visualize these tears in intact brain tissue, and report the energies associated with brain fracture. Quantifying the fracture energy of brain, as we have done here, is critical to understand the forces from injury that lead to TBI.


title: A systematic comparison of current bioinformatic tools for glycoproteomics data

abstract:
Glycosylation is one of the most common post-translational modifications and generates an enormous amount of proteomic diversity; changes in glycosylation are associated with nearly all disease states. Intact glycoproteomics seeks to determine the site-localization and composition of glycans along a protein backbone via mass spectrometry. Following data acquisition, raw files are analyzed using search algorithms to define peptide sequence, glycan composition, and site localization. Glycoproteomics is rapidly expanding, creating the pressing need to establish bioinformatic community standards. Recently, several new search algorithms were released, many of which vary in terms of search strategy, localization system, score cutoffs, and glycan databases, thus warranting a comprehensive comparison of these new programs along with existing programs. Here, we analyzed three common samples: an enriched cell lysate, a mixture of 6 glycoproteins, and a mucin-domain glycoprotein. All raw files were searched with comparable parameters among software and the results were extensively manually validated to compare accuracy and completion of the output. Our results highlight the continued need for manual validation of glycopeptide spectral matches, especially for O-glycopeptides. Despite this, O-Pair outperformed all other programs in correct identification of O-glycopeptides and its localization system proved to be useful. On the other hand, Byonic and pGlyco performed best for N-glycoproteomics; the former was best for proteome-wide searches, but the latter identified more N-glycosites in less complex samples. Overall, we summarize the strengths, weaknesses, and potential improvements for these search algorithms.

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title: Unsupervised discovery of tissue architecture in multiplexed imaging

abstract: Multiplexed imaging and spatial transcriptomics enable highly resolved spatial characterization of cellular phenotypes, but still largely depend on laborious manual annotation to understand higher-order patterns of tissue organization. As a result, higher-order patterns of tissue organization are poorly understood and not systematically connected to disease pathology or clinical outcomes. To address this gap, we developed UTAG, a novel method to identify and quantify microanatomical tissue structures in multiplexed images without human intervention. Our method combines information on cellular phenotypes with the physical proximity of cells to accurately identify organ-specific microanatomical domains in healthy and diseased tissue. We apply our method to various types of images across physiological and disease states to show that it can consistently detect higher level architectures in human organs, quantify structural differences between healthy and diseased tissue, and reveal tissue organization patterns with relevance to clinical outcomes in cancer patients.


title: Adversarial attacks and adversarial robustness in computational pathology

abstract:
Artificial Intelligence (AI) can support diagnostic workflows in oncology by aiding diagnosis and providing biomarkers. AI applications are therefore expected to evolve from academic prototypes to commercial products in the coming years. However, AI applications are vulnerable to adversarial attacks, such as malicious interference with test data aiming to cause misclassifications. Therefore, it is essential for the use of AI-based diagnostic devices to secure them against such attacks before widespread use. Unfortunately, no resistant systems exist in computational pathology so far.

To address this problem, we investigate the susceptibility of convolutional neural networks (CNNs) to multiple types of white- and black-box attacks. We demonstrate that both attacks can easily confuse CNNs in clinically relevant pathology tasks and impair classification performance. Classical adversarially robust training and dual batch normalization (DBN) are possible mitigation strategies but require precise knowledge of the type of attack used in the inference.

We demonstrate that vision transformers (ViTs) perform equally well compared to CNNs at baseline and are orders of magnitude more robust to different types of white-box and black-box attacks. At a mechanistic level, we show that this is associated with a more robust latent representation of clinically relevant categories in ViTs compared to CNNs.

Our results are in line with previous theoretical studies. We show that ViTs are robust learners in computational pathology. This implies that large-scale rollout of AI models in computational pathology should rely on ViTs rather than CNN-based classifiers to provide inherent protection against adversaries.


title: metaPathwayMap: A tool to predict metabolic pathway neighborhoods from structural classes of untargeted metabolomics peaks

abstract:
SummaryThousands of peaks detected via untargeted tandem liquid chromatography mass spectrometry (LC-MS/MS) of natural extracts typically go unannotated, limiting our understanding of the metabolic pathways perturbed under different conditions. Current tools for predicting metabolic pathways from untargeted metabolomics data either require prior compound identification or are more focused on specific model species. metaPathwayMap makes use of recent advances in computational metabolomics to map peaks detected in untargeted LC-MS/MS experiments to MetaCyc pathway representations using their structural class predictions. This approach enables better insights into metabolomes of model and non-model species.

Availability and ImplementationRequired Python scripts can be downloaded from the moghelab/metaPathwayMap GitHub repository and implemented on a Unix machine. This tool is also available for use through the SolCyc website (https://metapathwaymap.solgenomics.net) and via DockerHub (srs57/metapathwaymap).

Contactgdm67@cornell.edu

Supplementary InformationAdditional information is provided in Supplementary Methods, Supplementary Files 1-3 and on GitHub (https://github.com/moghelab/metaPathwayMap)


title: Dynamic play between human N-α-acetyltransferase D and H4-mutant histones: Molecular dynamics study

abstract:
N-terminal acetyltransferases (NATs) are overexpressed in various cancers. Specifically in lung cancer, human N--acetyltransferase D (hNatD) is upregulated and prevents the histone H4 N-terminal serine phosphorylation, leading to the epithelial-to-mesenchymal transition (EMT) of cancer cells. hNatD facilitates histone H4 N--terminal serine acetylation and halts the CK2-mediated serine phosphorylation. In the present study, we report the effects of four N-terminal mutant (S1C, R3C, G4D and G4S) histone H4 peptides on their bindings with hNatD by employing a molecular dynamics simulation. We also used graph theory-based analyses to understand residue correlation and communication in hNatD under the influence of WT and MT H4 peptides. Results show that S1C, R3C and G4S mutant peptides have significant stability at the catalytic site of hNatD. However, S1C, G4D and G4S peptides disrupt hNatD structure. Additionally, intramolecular hydrogen bond analysis reveals greater stability of hNatD in complex with R3C peptide. Further, intermolecular hydrogen bond analysis of acetyl-CoA with hNatD and its RMSD analysis in five complexes indicate that cofactor has greater stability in WT and R3C complexes. Our findings support previously reported experimental study on impacts of H4 mutations on its hNatD-mediated acetylation catalytic efficiency. The betweenness centrality (BC) analysis further gives insight into the hNatD residue communication dynamics that can be exploited to target hNatD using existed or novel drug candidates therapeutically.

SECONDARY ABSTRACTMany N-terminal acetyltransferases (NATs) enzymes play important role in post-translational modification of histone tails. Research showed that these enzymes have been reported upregulated in many cancers. NatD is known to acetylate H4/H2A at the N-terminal. During lung cancer, this enzyme competes with the protein kinase CK2 and block the phosphorylation of H4 and, acetylates. Also, we observed that H4 has various mutations at the N-terminal and we considered only four mutations (S1C, R3C, G4D and G4S) to study the impacts of these mutations on H4 binding with NatD using MD simulation. Our results show that R3C stabilizes the NatD whereas remaining mutations destabilize the NatD. Thus, mutations have significant impacts on NatD structure. Our finding supports previous analysis also.

SIGNIFICANCEOur main objective in this study was to understand the structural and dynamics of hNatD under the influence of WT and MT H4 histones bindings. Previous experimental study reported that mutations on H4 N-terminus reduce the catalytic efficiency of N-Terminal acetylation. But here, we performed molecular-level study thus, we can understand how these mutations (S1C, R3C, G4D and G4S) cause significant depletion in catalytic efficiency of hNatD. Another, interesting observation is that enzymatic activity of hNatD is altered due to the considerably large deviation of acetyl-CoA from its original position (G4D). Further, simulation and correlation data suggest which regions of the hNatD are highly flexible and rigid and, which domains or residues have the correlation and anticorrelation. As hNatD is overexpressed in lung cancer, it is an important drug target for the cancer hence, our study provides structural information to target hNatD.


title: Dual blockade of misfolded alpha-sarcoglycan degradation by bortezomib and givinostat combination

abstract: Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the SGCA gene encoding alpha-sarcoglycan (-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C--SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C--SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis.


title: GIP receptor agonism improves dyslipidemia and atherosclerosis independently of body weight in obese mice

abstract: Agonism at the receptors for the glucose-dependent insulinotropic polypeptide (GIPR) is a key component of the novel unimolecular co-agonists which are among the most promising drugs in clinical development for the treatment of obesity and diabetes. The therapeutic effect of chronic GIPR agonism to treat dyslipidemia, and thus to reduce the cardiovascular disease risk, has not been explored yet. Herein we found that treatment with a long-acting acylated GIP analog (Acyl-GIP) reduced dyslipidemia and atherogenesis in male LDL receptor knockout mice. Acyl-GIP administration resulted in smaller adipocytes within the inguinal fat depot and RNAseq analysis of the latter revealed that Acyl-GIP may improve dyslipidemia by directly modulating lipid metabolism in this fat depot. This study identified an unanticipated efficacy of chronic GIPR agonist administration to improve dyslipidemia and cardiovascular disease.


title: Like host, like parasite: intraspecific divergence in a polystomatid flatworm parasite across South Africa echoes that of its frog host

abstract: The African Clawed Frog Xenopus laevis, a global invader, exhibits marked phylogeographic divergence among native populations in southern Africa, which enhances its invasive potential. The polystomatid flatworm Protopolystoma xenopodis, as the frogs most frequently co-introduced metazoan parasite, may be the ideal biological tag for the frogs movement, if corresponding divergence can be demonstrated. In an integrative approach, we utilised morphometrics and molecular markers to assess divergence in P. xenopodis in its native range. We measured twelve key morphological characters from 23 flatworms and compared these statistically between flatworms collected to the north and south of the Great Escarpment Mountain Range in South Africa. Phylogenetic analyses were based on three concatenated markers, namely 28S and 12S rDNA and COX1, from six flatworms. The combination of five morphological characters, which involve egg size, gut morphology and size of the attachment hooks, differentiated northern and southern populations of P. xenopodis in South Africa. The multilocus phylogenetic analyses supported these findings, showing a well-supported cluster of northern P. xenopodis. These findings suggest that taxonomic studies of polystomatid flatworms should make use of geographically representative data sets that consider both morphological and molecular evidence. Moreover, the findings demonstrate that the frog host and flatworm parasite exhibit corresponding phylogeographic structuring in the native range. Consequently, the phylogeography of P. xenopodis, both in the native and invasive range of its host, may act as a key piece of evidence to reconstruct past invasion pathways of X. laevis.

category: zoology


title: Electroencephalography-derived functional connectivity in sensorimotor networks in Stroke and Multiple Sclerosis Fatigue

abstract:
A common mechanism of altered sensory processing is the basis of chronic fatigue in neurological disorders. Here we test the hypothesis  Altered connectivity in sensory networks underlies chronic fatigue in stroke and multiple sclerosis.

In 46 non-depressed, minimally impaired stroke survivors (n=29) and multiple sclerosis patients (n=17), median disease duration of 5 years, resting state neuronal activity was measured using 64-channel electroencephalography. Graph theory-based network analysis measure of functional connectivity (small-world index) was calculated in right and left motor (Brodmann areas 4, 6, 8, 9, 24 and 32) and somatosensory (Brodmann areas 1, 2, 3, 5, 7, 40 and 43) networks, in 5 frequency bands: delta, theta, alpha, beta and gamma. Fatigue was measured using Fatigue Severity Scale (Stroke) and modified Fatigue Impact Scale (MS), with scores of >4 (FSS) and >38 (mFIS), defined as high fatigue.

Both stroke survivors and multiple sclerosis patients with high fatigue showed significantly more small-worldness in the right sensory networks in the beta band frequency. Additionally, only in stroke survivors with high fatigue, there was decreased small-worldness in the left motor network in the delta and theta bands.

Altered sensory network connectivity is common to both stroke and MS fatigue, indicating impaired sensory processing as a disease-independent mechanism of chronic fatigue in neurological conditions. Furthermore, such difference in functional connectivity emerges in beta band activity, further strengthening the idea of altered sensorimotor processing as the basis of chronic neurological fatigue.


title: Sst+ GPi output neurons provide direct feedback to key nodes of the basal ganglia and drive behavioral flexibility.

abstract: The GPi-LHb pathway is the main output of the basal ganglia suggested to shape motivated behaviors. We show here that Sst+ GPi-LHb neurons send direct feedback to key nodes of the basal ganglia: the GPe, the striatal striosomes, and dopamine neurons in the SNc. Chronic silencing of this pathway did not affect learning or execution of value-guided choices, but severely disrupted the ability to adapt choice-behavior and seek an alternative reward location after task reversal. Calcium imaging revealed that Sst+ GPi neurons did not signal outcome value or value updates during reversal learning. Instead, progressive suppression of the Sst+ GPi activity was linked to increased commitment to one choice, and activity increased during exploration of alternative choices. We propose that GPi Sst+ neurons drive behavioral flexibility through a direct feedback signal to balance the activity of key nodes in the basal ganglia.


title: Microglial P2Y12 mediates chronic stress-induced synapse loss in the prefrontal cortex and associated behavioral consequences in male mice

abstract: Recent studies demonstrate that chronic unpredictable stress (CUS) drives microglia-mediated neuronal remodeling, contributing to synapse loss in the prefrontal cortex (PFC) and cognitive-behavioral dysfunction. Nonetheless, it remains unclear what mechanisms guide microglia-neuron interactions in stress. Evidence indicates that neuronal activity-dependent purinergic signaling directs microglial processes and microglia-synapse interaction via P2Y12, a purinergic receptor exclusively expressed by microglia in the brain. Stress exposure alters excitatory neurotransmission in the PFC, thus we aimed to determine if P2Y12 signaling promotes functional changes in microglia in the context of chronic stress. Using an activating DREADD, our initial studies showed that PFC microglia adopt a CUS-associated phenotype after repeated pyramidal neuronal activation. To further investigate the role of purinergic signaling, we used genetic (P2ry12-/-) or pharmacological (clopidogrel, ticagrelor) approaches to block P2Y12 in the context of CUS. Various behavioral, physiological, and cytometric endpoints were analyzed. Both P2Y12-deletion and treatment with clopidogrel prevented increases in forced swim test immobility and attenuated deficits in temporal object recognition following CUS. Flow cytometry of PFC microglia revealed that both P2ry12-/- mice and those treated with clopidogrel have significantly different phenotypes (independent of CUS); with diminished P2Y12 expression and altered surface levels of CX3CR1, CSF1R, and CD11b. Immunohistology in Thy1-GFP(M) mice demonstrated that pharmacological blockade of P2Y12 prevented stress-induced increases in the proportion of microglia with GFP+ neuronal inclusions and limited dendritic spine loss in the PFC. Together, these findings indicate that microglial P2Y12 is a critical mediator of stress-induced neuronal remodeling in the PFC and subsequent behavioral deficits.


title: How Hippocampus Solves Inverse Problems: A Circuit Hypothesis

abstract: This paper proposes an explicit computational process which reproduces what are considered to be two essential experimentally observed hippocampus capabilities. These are path planning and determining the chain of ordered relationships which bind two concepts. These are both inverse problems. The paper presents algorithmic simulations of the process and a neuronal circuit (and variants) with the same dataflow, both consistent with the known neuroanatomy of the hippocampus. The process described here is purpose-built for hippocampus


title: Comparable theta phase coding dynamics along the transverse axis of CA1

abstract:
Topographical projection patterns from the entorhinal cortex to area CA1 of the hippocampus have led to a hypothesis that proximal CA1 (pCA1) is spatially more selective than distal CA1 (dCA1). While earlier studies have shown evidence supporting this hypothesis, we recently showed that this difference does not hold true under all experimental conditions. In a complex environment with distinct local texture cues on a circular track and global visual cues, pCA1 and dCA1 display comparable spatial selectivity. Correlated with the spatial selectivity differences, the earlier studies also showed differences in theta phase coding dynamics between pCA1 and dCA1 neurons. Here we show that there are no differences in theta phase coding dynamics between neurons in these two regions under the experimental conditions where pCA1 and dCA1 neurons are equally spatially selective. We also show that dCA1 local field potentials (LFPs) show higher power in theta range compared to pCA1 LFPs. These findings challenge the established notion of dCA1 being inherently less spatially selective and theta modulated than pCA1 and suggest that theta-mediated activation of the CA1 sub-networks to represent space is task-dependent.

New and NoteworthyProximal CA1 has been shown to be more spatially selective and influenced by theta oscillations than distal CA1. We demonstrate an experimental condition under which this functional distinction does not persist, and distal and proximal CA1 neurons show comparable spatial selectivity and influence of theta oscillations. We discuss how the observed results cannot be explained by the anatomical segregation of the lateral and the medial entorhinal cortical inputs as proposed earlier and suggest alternative mechanisms.


title: A flexible and versatile system for multicolor fiber photometry and optogenetic manipulation

abstract:
Fiber photometry is a technique of growing popularity in neuroscientific research. It is widely used to infer brain activity by recording calcium dynamics in genetically defined populations of neurons. Aside from the wide variety of calcium indicators, other genetically encoded biosensors have recently been engineered to measure membrane potential, neurotransmitter release, pH, or various cellular metabolites, such as ATP or cAMP. Due to the spectral characteristics of these molecular tools, different assemblies of optical hardware are usually needed to reveal the full potential of different biosensors. In addition, the combination of multiple biosensors in one experiment often requires the investment in more complex equipment, which limits the flexibility of the experimental design. Such constraints often hamper a straightforward implementation of new molecular tools, evaluation of their performance in vivo, and design of new experimental paradigms - especially if the financial budget is a limiting factor. Here, we propose a novel approach for fiber photometry recordings, based on a multimode optical fused-fiber coupler (FFC) for both light delivery and collection. Recordings can readily be combined with optogenetic manipulations in a single device without the requirement for dichroic beam-splitters. In combination with a multi-color light source and appropriate emission filters, our approach offers remarkable flexibility in experimental design and facilitates the implication of new molecular tools in vivo at minimal cost. The ease of assembly, operation, characterization, and customization of this platform holds the potential to foster the development of experimental strategies for multicolor fused fiber photometry (FFP) combined with optogenetics far beyond its current state.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/484590v1_ufig1.gif" ALT="Figure 1">
View larger version (45K):
org.highwire.dtl.DTLVardef@13abbdcorg.highwire.dtl.DTLVardef@10384d7org.highwire.dtl.DTLVardef@67938org.highwire.dtl.DTLVardef@1005aee_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIFused Fiber Photometry (FFP) enables straightforward monitoring and manipulation of brain activity
C_LIO_LIFFP allows monitoring of indicators with virtually any spectral characteristics
C_LIO_LIFFP is compatible with simultaneous optogenetic manipulation
C_LIO_LIEasy assembly, characterization, and customization
C_LI


title: Thermoregulatory Dynamics Reveal Sex-Specific Inflammatory Responses to Experimental Autoimmune Encephalomyelitis in Mice: Implications for Multiple Sclerosis-Induced Fatigue in Females

abstract: The course of multiple sclerosis (MS) is characterized by striking sex differences in symptoms such as fatigue and impaired thermal regulation, which are associated with aggravated systemic pro-inflammatory processes. The purpose of this study was to replicate these symptoms in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice in the quest to advance the preclinical study of non-motor symptoms of MS. Male and female C57BL/6 mice exposed to a mild form of EAE were evaluated for the progression of clinical, behavioural, thermal, and inflammatory processes. We show higher susceptibility in females to EAE than males based on greater clinical score and cumulative disease index (CDI), fatigue-like and anxiety-like behaviours. Accordingly, infrared (IR) thermography indicated higher cutaneous temperature in females from post-induction days 12 to 23. Females also responded to EAE with greater splenic and adrenal gland weights than males as well as sex-specific changes in pro- and anti-inflammatory cytokines. These findings provide the first evidence of a sex-specific thermal response to immune-mediated demyelination, thus proposing a non-invasive assessment approach of the psychophysiological dynamics in EAE mice. The results are discussed in relation to the thermoregulatory correlates of fatigue and how endogenously elevated body temperature without direct heat exposure may be linked to psychomotor inhibition in patients with MS.