title: Comparative infectivity and pathogenesis of emerging SARS-CoV-2 variants in Syrian hamsters

abstract: Within one year after its emergence, more than 108 million people contracted SARS-CoV-2 and almost 2.4 million succumbed to COVID-19. New SARS-CoV-2 variants of concern (VoC) are emerging all over the world, with the threat of being more readily transmitted, being more virulent, or escaping naturally acquired and vaccine-induced immunity. At least three major prototypic VoC have been identified, i.e. the UK (B.1.1.7), South African (B.1.351) and Brazilian (B.1.1.28.1), variants. These are replacing formerly dominant strains and sparking new COVID-19 epidemics and new spikes in excess mortality. We studied the effect of infection with prototypic VoC from both B.1.1.7 and B.1.351 lineages in Syrian golden hamsters to assess their relative infectivity and pathogenicity in direct comparison to two basal SARS-CoV-2 strains isolated in early 2020. A very efficient infection of the lower respiratory tract of hamsters by these VoC is observed. In line with clinical evidence from patients infected with these VoC, no major differences in disease outcome were observed as compared to the original strains as was quantified by (i) histological scoring, (ii) micro-computed tomography, and (iii) analysis of the expression profiles of selected antiviral and pro-inflammatory cytokine genes. Noteworthy however, in hamsters infected with VoC B.1.1.7, a particularly strong elevation of proinflammatory cytokines was detected. Overall, we established relevant preclinical infection models that will be pivotal to assess the efficacy of current and future vaccine(s) (candidates) as well as therapeutics (small molecules and antibodies) against two important SARS-CoV-2 VoC.


title: An anaphase surveillance mechanism prevents micronuclei formation from mitotic errors

abstract: Micronuclei are a hallmark of cancer and other human disorders and have recently been implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reformation to protect against micronuclei formation during human cell division. Using high-resolution live-cell imaging of human cancer and non-cancer cells we found that anaphase lagging chromosomes are often transient and rarely formed micronuclei. This strong bias against micronuclei formation relied on a midzone-based Aurora B phosphorylation gradient that assisted the mechanical transduction of spindle forces at the kinetochore-microtubule interface required for anaphase error correction, while delaying nuclear envelope reformation on lagging chromosomes, independently of microtubules. Our results uncover a new layer of protection against genomic instability and provide a strategy for the rational design of micronuclei-targeting therapies.

category: cell biology


title: Stability of ligand-induced protein conformation influences affinity in maltose-binding protein

abstract: Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MPB) from Escherichia coli. We used singlemolecule Forster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.

category: biochemistry


title: Allostery in Proteins as Point-to-Point Telecommunication in a Network: Frequency Decomposed Signal-to-Noise Ratio and Channel Capacity Analysis

abstract: AO_SCPLOWBSTRACTC_SCPLOWAllostery in proteins is a phenomenon in which the binding of a ligand induces alterations in the activity of remote functional sites. This can be conceptually viewed as point-to-point telecommunication in a networked communication medium, where a signal (ligand) arriving at the input (binding site) propagates through the network (interconnected and interacting atoms) to reach the output (remote functional site). The reliable transmission of the signal to distal points occurs despite all the disturbances (noise) affecting the protein. Based on this point of view, we propose a computational frequency-domain framework to characterize the displacements and the fluctuations in a region within the protein, originating from the ligand excitation at the binding site and noise, respectively. We characterize the displacements in the presence of the ligand, and the fluctuations in its absence. In the former case, the effect of the ligand is modeled as an external dynamic oscillatory force excitation, whereas in the latter, the sole source of fluctuations is the noise arising from the interactions with the surrounding medium that is further shaped by the internal protein network dynamics. We introduce the excitation frequency as a key factor in a Signal-to-Noise ratio (SNR) based analysis, where SNR is defined as the ratio of the displacements stemming from only the ligand to the fluctuations due to noise alone. We then employ an information-theoretic (communication) channel capacity analysis that extends the SNR based characterization by providing a route for discovering new allosteric regions. We demonstrate the potential utility of the proposed methods for the representative PDZ3 protein.

category: biophysics


title: Xenogeneic Skin Transplantation Promotes Angiogenesis and Tissue Regeneration Through Vitamin D-Activated Trem2+ Macrophages

abstract:
Skin allo- and xenotransplantation are the standard treatment for major burns when donor sites for autografts are not available and have been shown to significantly accelerate wound healing. Although the cellular elements of foreign grafts are rejected, the extracellular matrix components integrate into the wound and may underlie their beneficial effects on wound healing. The molecular mechanisms defining the relationship between the immune response to foreign grafts and their impact on wound healing have not been fully elucidated. Here, we investigated changes in collagen architecture after xenogeneic implantation of clinically available human biologic scaffolds. We show that collagen deposition in response to the implantation of human split-thickness skin grafts (hSTSG) containing live cells recapitulates normal skin architecture, whereas human acellular dermal matrix (ADM) grafts led to highly aligned collagen deposition, characteristic of fibrosis and scar. Using single-cell RNA-sequencing, we show that macrophage differentiation in response to hSTSG is driven by vitamin D (VD) signaling toward Trem2+ subpopulations with an enrichment of pro-angiogenic and anti-fibrotic transcriptomic programs. We subsequently induced this regenerative subpopulation in vitro by treating bone marrow-derived cells with vitamin D3 and found that hydrogel delivery of Trem2+ macrophages significantly accelerated wound closure in a human-like murine excisional wound model. Our study identifies the preclinical therapeutic potential of Trem2+ macrophages to mitigate fibrosis and promote wound healing and provides a novel effective strategy to develop advanced cell therapies for complex wounds.

One Sentence SummaryVitamin D-activated Trem2+ macrophages promote angiogenesis and mitigate fibrosis, providing a novel effective strategy to develop advanced cell therapies for complex wounds.

category: immunology


title: DENGUE VIRUS ALTERS SIALIC ACID RESIDUES CONFIGURATION IN MACROPHAGES

abstract: The activation of the innate immune response requires sialic acid residues removal. Nevertheless, it is unknown the role for these changes during the Dengue virus infection. We determine if during Dengue virus infection, the sialic acid residues alter on the macrophages. The human monocytic cell line THP-1 was differentiated into macrophages and were infected with Dengue virus. The changes in sialic acid were evaluated by lectin blot in the cellular lysate. The activity of neuraminidase was defined by RT-PCR and fluorescence assays. Macrophages infection with DENV-2 reduces -2,6 sialic acid residues at 24 h, and -2,3 sialic acid residues lower at 48 h in some proteins. Transcriptional profile and enzymatic activities of Neu-1 showed a narrow decrease. Sialic acid residues oscillation in varied conformations and times suggest it a role of a selective mechanism to remove these residues. The lesser participation of Neu-1 in this process could be concomitant to other similar enzymes such as sialyl-transferases, or the phenomenon requires minimal activity to have a relevant biological function.


title: Growth response and recovery of Corynebacterium glutamicum colonies on single-cell level upon defined pH stress pulses

abstract: Bacteria respond to pH changes in their environment via pH homeostasis to keep the intracellular pH as constant as possible within a small range. A change of the intracellular pH value influences e.g., the enzyme activity, protein stability, solubility of trace elements and the proton motive force. Here, the species Corynebacterium glutamicum has been chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 {+/-} 0.5 in environments with an external pH between 5.5 and 9. In the recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell responding to defined short-term pH perturbations/ pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyse the physiological growth response of C. glutamicum upon precise pH stress pulses at a single-cell level. Analysis of the growth behaviour at the colony level by dMSCC exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in the viability with increasing stress duration. Colony regrowth was possible after increasing lag phases when stress durations were increased from 5 min to 9 h for all tested pH values. Furthermore, the single-cell analysis revealed heterogeneous regrowth of cells after pH stress, which can be distinguished into two distinct behaviours firstly, cells continue to grow without interruption after the pH stress, and secondly, some cells rest for several hours after the pH stress before they start to grow again after this lag phase. This study provides the first insights into the single-cell response to acidic and alkaline pH stress adaptation of C. glutamicum.

category: microbiology


title: Organotypic endothelial adhesion molecules are key for Trypanosoma brucei tropism and virulence

abstract: Trypanosoma brucei is responsible for lethal diseases in humans and cattle in Sub-Saharan Africa. These extracellular parasites extravasate from the blood circulation into several tissues. The importance of the vasculature in tissue tropism is poorly understood. Using intravital imaging and bioluminescence, we found that gonadal white adipose tissue and pancreas are the two main parasite reservoirs. We show that reservoir establishment happens before vascular permeability is compromised, suggesting that extravasation is an active mechanism. Blocking endothelial surface adhesion molecules (E-selectin, P-selectins, or ICAM2) significantly reduced extravascular parasite load in all organs and delayed host lethality. Remarkably, blocking CD36 had a specific effect on adipose tissue tropism that was sufficient to delay lethality, suggesting that establishment of the adipose tissue reservoir is necessary for parasite virulence. This works demonstrates the importance of the vasculature in a T. brucei infection and identifies organ-specific adhesion molecules as key players for tissue tropism.


title: Calprotectin-mediated zinc chelation inhibits Pseudomonas aeruginosa protease activity in cystic fibrosis sputum

abstract:
Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to control cultures, but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc-starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to post-translationally inhibit zinc metalloprotease activity and thereby impact the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment.

ImportanceThe factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc-starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.


title: Transcriptional regulation of HSFA7 and post-transcriptional modulation of HSFB4a by miRNA4200 govern general and varietal thermotolerance in tomato

abstract: Heat shock factors (HSFs) are at the core of heat stress (HS) response in plants. However, the contribution of HSFs governing the inherent thermotolerance mechanism in tomato from sub-tropical hot climates is poorly understood. With the above aim, comparative expression profiles of the HSF family in a HS tolerant (CLN1621L) and a sensitive cultivar (CA4) of tomato under HS revealed cultivar-biased regulation of an activator (HSFA7a) and repressor (HSFB4a) class HSF. Functional characterization of HSFA7a that was strongly up-regulated in the tolerant cultivar by VIGS-based silencing and overexpression established it as a positive regulator of HS-tolerance. While knock-down and overexpression analyses of HSFB4a that was down-regulated in CLN1621L in HS, showed it as a negative regulator of thermotolerance. Promoter:GUS reporter assays and promoter sequence analyses suggest heat-mediated transcriptional control of both the HSF genes in the contrasting cultivars. Moreover, we show HSFB4a is also regulated post-transcriptionally by microRNA Sly-miR4200 using degradome, short-tandem-target-mimic of Sly-miR4200 and transient in-planta Sly-miR4200-effector:HSFB4a-reporter assays. This miRNA is induced several folds upon HS in the tolerant variety thereby reducing HSFB4a levels. We thus propose that the alleviation of HSFB4a repressor governs thermotolerance in the tolerant cultivar by regulating downstream heat stress responsive genes.

category: molecular biology


title: Big fish, little fish: N-terminal acetyltransferase Naa40p proteoforms caught in the act

abstract: NatD consist of the evolutionary conserved N-alpha acetyltransferase catalytic subunit Naa40p and is among the most selective N-terminal acetyltransferase (NAT) complexes identified to date. Here we identified a conserved 22 amino acid N-terminally truncated Naa40 proteoform named Naa40p25 or short Naa40 (Naa40S). Intriguingly, while upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the well characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40 (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Further, since Naa40L was identified as being N-terminally myristoylated and shown to interact with N-myristoyltransferase 1 (NMT1), NMT1 may steer Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.


title: Microtubule-based Transport is Essential to Distribute RNA and Nascent Protein in Skeletal Muscle

abstract:
While the importance of RNA localization in highly differentiated cells is well appreciated, basic principles of RNA localization in skeletal muscle remain poorly characterized. Here, we develop a method to detect single RNA molecules and quantify localization patterns in skeletal myofibers, and we uncover a critical and general role for directed transport of RNPs in muscle. We find that RNAs are localized and translated along cytoskeletal filaments, and we identify the Z-disk as a biological hub for RNA localization and protein synthesis. We show that muscle development triggers complete reliance on the lattice-like microtubule network to transport RNAs and that disruption of microtubules leads to striking accumulation of RNPs and nascent protein around myonuclei. Our observations suggest that active transport may be globally required to distribute RNAs in highly differentiated cells and reveal fundamental mechanisms relevant to myopathies caused by perturbations to RNPs, microtubules, and the nuclear envelope.

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title: Identification of Human CD4+ Sub-population of Resident Cardiac Fibroblasts Linked to Inflammation-Mediated Cardiac Fibrosis

abstract: Infiltration with inflammatory T-cells and accumulation of cardiac myofibroblasts are hallmarks of cardiac fibrosis and maladaptive remodeling. The origin, identity, and functions of the resident cardiac cells involved in this process are, however, unclear. To determine the identity of cells contained in regions exhibiting fibrosis, mass cytometry profiling was performed using resident human ventricular cardiac fibroblasts and right ventricle autopsy tissues from individuals diagnosed with pulmonary hypertension and SUGEN/hypoxia rats. Results showed that a subpopulation of resident myocardial fibroblasts expresses increased levels of CD4+, a helper T-cell surface marker, in addition to mesenchymal markers in humans and rats. Characterization of the resident cardiac fibroblast subpopulation, both structurally and functionally, using transcriptome and secretome analysis of the secreted cytokines, chemokines, proteins, and metabolites, evidenced that IL-1{beta} induces a phenotypic switch of human cardiac fibroblasts from mesenchymal to CD4+ lymphoidal lineage in vitro. RNA sequencing (RNA-seq) analysis of FACS-sorted CD4-expressing cardiac fibroblasts further revealed that the transcriptome of such IL-1{beta}-induced CD4+ fibroblast population exhibited classical lymphoidal and stem cell-like signatures. Lastly, reversal of cell clustering, phosphorylation of MAPK p38 and NF-{kappa}B p65, and phenotypic switching was achieved with the administration of an IL-1R antagonist. In conclusion, we have identified a subpopulation of cardiac fibroblasts which exhibits structural and functional attributes of both mesenchymal and lymphoid cells which is induced by IL-1{beta}-IL-1R-NFkB pathway for differentiation of cardiac fibroblast cells. These data suggest that cardiac fibroblast transdifferentiation during inflammation may form the basis for maladaptive remodeling during myocardial fibrosis.


title: Identification of CD4+ Sub-population of Resident Cardiac Fibroblasts Linked to Myocardial Fibrosis


title: Tracking cell lineages in 3D by incremental deep learning

abstract: Deep learning is emerging as a powerful approach for bioimage analysis. Its use in cell tracking is limited by the scarcity of annotated data for the training of deep learning models. Moreover, annotation, training, prediction and proofreading currently lack a unified user interface. We present ELEPHANT, an interactive platform for 3D cell tracking that addresses these challenges by taking an incremental approach to deep learning. ELEPHANT provides an interface that seamlessly integrates cell track annotation, deep learning, prediction and proofreading. This enables users to implement cycles of incremental learning starting from a few annotated nuclei. Successive prediction-validation cycles enrich the training data, leading to rapid improvements in tracking performance. We test the softwares performance against state-of-the-art methods and track lineages spanning the entire course of leg regeneration in a crustacean over 1 week (504 timepoints). ELEPHANT yields accurate, fully-validated cell lineages with a modest investment in time and effort.

category: developmental biology


title: Global genetic heterogeneity in adaptive traits

abstract: Understanding the genetic architecture of complex traits is a major objective in biology. The standard approach for doing so is genome-wide association studies (GWAS), which aim to identify genetic polymorphisms responsible for variation in traits of interest. In human genetics, consistency across studies is commonly used as an indicator of reliability. However, if traits are involved in adaptation to the local environment, we do not necessarily expect reproducibility. On the contrary, results may depend on where you sample, and sampling across a wide range of environments may decrease the power of GWAS because of increased genetic heterogeneity. In this study, we examine how sampling affects GWAS for a variety of phenotypes in the model plant species Arabididopsis thaliana. We show that traits like flowering time are indeed influenced by distinct genetic effects in local populations. Furthermore, using gene expression as a molecular phenotype, we show that some genes are globally affected by shared variants, while others are affected by variants specific to subpopulations. Remarkably, the former are essentially all cis-regulated, whereas the latter are predominately affected by trans-acting variants. Our result illustrate that conclusions about genetic architecture can be incredibly sensitive to sampling and population structure.

category: evolutionary biology


title: NMR Hydrogen Exchange and Relaxation Reveal Positions Stabilized by p53 Rescue Mutants N239Y and N235K

abstract:
Inactivation of p53 is found in over 50% of all cancers; p53 disfunction is often caused by a single missense mutation localized in the DNA binding domain (DBD). Rescue mutants N235K and N239Y stabilize and restore function to multiple p53 cancer mutants. Here, we use NMR to compare protein dynamics between WT and rescue mutants to understand the mechanism of stabilization. We measured and compared folding dynamics by calculating protection factors (PFs) from NMR hydrogen exchange rates of backbone amides. We find that both rescue mutants impose a global stabilizing effect that dampens their motions compared to WT DBD, predominantly in the {beta}-sandwich. However, a few regions become more flexible in rescue mutants. Notably, positions that have increased PFs map to cancer mutants rescued by each mutant. We also compared relaxation results to obtain flexibility information in the ps to ns timescale regime. Protein sequence analysis was used to determine the occurrence of these rescue mutants in nature and showed that 235K is found in mice and rats, but there is no evidence of 239Y occurring naturally in any species. Understanding the mechanism by which stabilizing mutants rescue p53 may reveal novel avenues for the development of cancer therapeutics. Our findings suggest that cancer therapeutics aimed at restoring p53 function could consider protein dynamics as a metric of drug efficacy.

Statement of SignificanceTwo mutations (N235K and N239Y) within the DNA binding domain of p53 are known to reverse the effects of multiple cancer mutants. However, the mechanism of rescue is not clear since the crystal structures of these mutants are virtually identical to that of the WT. Here we use NMR methods to show that the protein dynamics of the rescue mutants are significantly different compared to WT, allowing us to describe the stability on a per-residue basis. Although these mutations are only four residues apart, they stabilize the structure distinctly in different regions, consistent with the specific cancer mutations they to which they restore function.


title: Cyclocreatine suppresses prostate tumorigenesis through dual effects on SAM and creatine metabolism

abstract: Prostate cancer is highly prevalent, being the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of PTEN and SPRY2 (Sprouty 2) tumour suppressors, we identified enhanced creatine metabolism within the phosphagen system in progressive disease. Altered creatine metabolism was validated in in vitro and in vivo prostate cancer models and in clinical cases. Upregulated creatine levels were due to increased uptake through the SLC6A8 creatine transporter and de novo synthesis, resulting in enhanced cellular basal respiration. Treatment with cyclocreatine (a creatine analogue that potently and specifically blocks the phosphagen system) dramatically reduces creatine and phosphocreatine levels. Blockade of creatine biosynthesis by cyclocreatine leads to cellular accumulation of S-adenosyl methionine (SAM), an intermediary of creatine biosynthesis, and suppresses prostate cancer growth in vitro. Furthermore, cyclocreatine treatment impairs cancer progression in our GEMM and in a xenograft liver metastasis model. Hence, by targeting the phosphagen system, cyclocreatine results in anti-tumourigenic effects from both SAM accumulation and suppressed phosphagen system.

category: cancer biology


title: Acyloxyacyl Hydrolase Regulates Microglia-Mediated Pelvic Pain Through Toll-Like Receptor-4

abstract: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a devastating condition of chronic pelvic pain and urinary dysfunction. We have shown that mice deficient for the lipase acyloxyacyl hydrolase (AOAH) develop pelvic allodynia and exhibit symptoms and comorbidities consistent with IC/BPS, as well as gut dysbiosis. Microglia are resident immune cells of the central nervous system (CNS) that respond to changes in the gut microbiome, and studies have linked microglial activation to neuropathic pain. Additionally, microglia express toll-like receptors (TLRs), including TLR4, which are activated by microbial components. We have previously shown that AOAH-deficient mice exhibit increased gut permeability, suggesting a possible mechanism of microglial TLR4 activation via translocation of microbial products across the intestinal barrier to the brain. Here, we assessed the role of AOAH and TLR4 in microglial activation and pelvic pain. AOAH immunoreactivity co-localized with the microglial marker P2YR12 but not astrocytes, suggesting a functional role for AOAH in microglia. Pharmacologic ablation of CNS microglia with PLX5622 resulted in decreased pelvic allodynia in AOAH-deficient mice and resurgence of pelvic pain upon drug washout. Aligned with microglial activation, we observed altered cytokine abundance in Aoah-/- cortex that was reduced in Aoah/Tlr4-/- cortex. Consistent with our hypothesis of TLR4 activation by gut microbes, we observed microbiome-dependent activation of cultured BV2 microglial cells. Skeletal analyses revealed that AOAH-deficient mice have an activated microglia morphology in brain regions associated with neuropathic pain, independent of TLR4. Compared to Aoah-/- mice, Aoah/Tlr4-/- mice exhibited decreased pelvic pain and microglial cytokine expression. Together, these findings demonstrate differential roles for AOAH and TLR4 in microglial activation and pelvic pain and thus identify novel therapeutic targets for IC/BPS.

category: neuroscience


title: Calbindin regulates Kv4.1 trafficking and excitability of dentate granule cells via CaMKII-dependent phosphorylation

abstract: Calbindin, a major Ca2+ buffer in dentate granule cells (GCs), plays a critical role in shaping Ca2+ signals, yet how it regulates neuronal functions remains largely unknown. Here, we found that calbindin knock-out mice (CBKO) exhibited hyperexcitability in dentate GCs and impaired pattern separation, which was concurrent with reduced K+ current due to downregulated surface expression of Kv4.1. Consistently, manipulation of the calbindin expression in HT22 led to changes in CaMKII activation and the level of surface localization of Kv4.1 through phosphorylation at serine 555, confirming the mechanism underlying neuronal hyperexcitability in CBKO. We also discovered that Ca2+ buffering capacity was significantly reduced in the GCs of Tg2576 to the level of CBKO GCs, and this reduction was restored to normal levels by antioxidants, suggesting that calbindin is a target of oxidative stress. Our data suggest that regulation of CaMKII signaling by Ca2+ buffer is crucial for neuronal excitability regulation.


title: Microglia become hypofunctional and release metalloproteases and tau seeds after phagocytosing live neurons with P301S tau aggregates

abstract: The microtubule-associated protein tau aggregates in multiple neurodegenerative diseases, causing inflammation and changing the inflammatory signature of microglia by unknown mechanisms. We have shown that microglia phagocytose live neurons containing tau aggregates cultured from P301S tau transgenic mice due to neuronal tau aggregate-induced exposure of the  eat me signal phosphatidylserine. Here we show that after phagocytosis, microglia become hypophagocytic while releasing seed-competent insoluble tau aggregates. These microglia activate acidic {beta}-galactosidase, and release senescence-associated cytokines and matrix remodeling enzymes alongside tau, indicating a senescent phenotype. In particular, the marked NF{kappa}B-induced activation of matrix metalloprotease 3 (MMP3/stromelysinl) was replicated in the brains of P301S mutant tau transgenic mice, and in human brains from tauopathy patients. These data show that microglia that have been activated to ingest live neurons with tau aggregates behave hormetically, becoming hypofunctional while acting as vectors of tau aggregate spreading.


title: Incomplete recruitment of protective T cells facilitates Trypanosoma cruzi persistence in the mouse colon

abstract: Trypanosoma cruzi is the etiological agent of Chagas disease. Following T cell mediated suppression of the acute phase infection, this intracellular eukaryotic pathogen persists in a limited sub-set of tissues at extremely low-levels. The reasons for this tissue-specific chronicity are not understood. Using a dual bioluminescent:fluorescent reporter strain, which allows experimental infections to be imaged at single-cell resolution, we have characterised the  hyper-local immunological microenvironment of rare parasitized cells in the mouse colon, a key site of persistence. We demonstrate that incomplete recruitment of T cells to infection foci permits the occurrence of repeated cycles of intracellular parasite replication and differentiation to motile trypomastigotes at a frequency sufficient to perpetuate chronic infections. The life-long persistence of parasites in this tissue site continues despite the presence, at a systemic level, of a highly effective T cell response. Overcoming this low-level dynamic host:parasite equilibrium represents a major challenge for vaccine development.


title: The Aurora B gradient sustains kinetochore stability in anaphase

abstract: Kinetochores assemble on chromosomes in mitosis to allow microtubules to attach and bring about accurate chromosome segregation. The kinases Cyclin B-Cdk1 and Aurora B are crucial for the formation of stable kinetochores. However, the activity of these two kinases appears to decline dramatically at centromeres during anaphase onset, precisely when microtubule attachments are required to move chromosomes towards opposite poles of the dividing cell. We find that, although Aurora B leaves centromeres at anaphase, a gradient of Aurora B activity centred on the central spindle is still able to phosphorylate kinetochore substrates such as Dsn1 to modulate kinetochore stability in anaphase and to regulate kinetochore disassembly as cells enter telophase. We provide a model to explain how Aurora B co-operates with Cyclin B-Cdk1 to maintain kinetochore function in anaphase.


title: Asymmetric Contraction of Adherens Junctions arises through RhoA and E-cadherin feedback

abstract: Tissue morphogenesis often arises from the culmination of discrete changes in cell-cell junction behaviors, namely ratcheted junction contractions that lead to collective cellular rearrangements. Mechanochemical signaling in the form of RhoA underlies these ratcheted contractions, which occur asymmetrically as one highly motile vertex contracts toward a relatively less motile tricellular vertex. The underlying mechanisms driving asymmetric vertex movement remains unknown. Here, we use optogenetically controlled RhoA in model epithelia together with biophysical modeling to uncover the mechanism lending to asymmetric vertex motion. We find that both local and global RhoA activation leads to increases in junctional tension, thereby facilitating vertex motion. RhoA activation occurs in discrete regions along the junction and is skewed towards the less-motile vertex. At these less-motile vertices, E-cadherin acts as an opposing factor to limit vertex motion through increased frictional drag. Surprisingly, we uncover a feedback loop between RhoA and E-cadherin, as regional optogenetic activation of specified junctional zones pools E-cadherin to the location of RhoA activation. Incorporating this circuit into a mathematical model, we find that a positive feedback between RhoA-mediated tension and E-cadherin-induced frictional drag on tricellular vertices recapitulates experimental data. As such, the location of RhoA determines which vertex is under high tension, pooling E-cadherin and increasing the frictional load at the tricellular vertex to limit its motion. This feedback drives a tension-dependent intercellular "clutch" at tricellular vertices which stabilizes vertex motion upon tensional load.


title: PI3K Signaling Specifies Proximal-Distal Fate by Driving a Developmental Gene Regulatory Network in Sox9+ Lung Progenitors

abstract: The tips of the developing respiratory buds are home to important progenitor cells marked by the expression of SOX9 and ID2. Early in embryonic development (prior to E13.5), SOX9+ progenitors are multipotent, generating both airway and alveolar epithelium, but are selective progenitors of alveolar epithelial cells later in development. Transcription factors, including Sox9, Etv5, Irx, Mycn, and Foxp1/2 interact in complex gene regulatory networks to control proliferation and differentiation of SOX9+ progenitors. Molecular mechanisms by which these transcription factors and other signaling pathways control chromatin state to establish and maintain cell-type identity are not well-defined. Herein, we analyze paired gene expression (RNA-Seq) and chromatin accessibility (ATAC-Seq) data from SOX9+ epithelial progenitor cells (EPCs) during embryonic development. Widespread changes in chromatin accessibility were observed between E11.5 and E16.5, particularly at distal cis-regulatory elements (e.g. enhancers). Gene regulatory network (GRN) inference identified a common SOX9+ progenitor GRN, implicating phosphoinositide 3-kinase (PI3K) signaling in the developmental regulation of SOX9+ progenitor cells. Consistent with this model, conditional ablation of PI3K signaling in the developing lung epithelium in vivo and in lung explants in vitro resulted in an expansion of the SOX9+ EPC population and impaired epithelial cell differentiation. These data demonstrate that PI3K signaling is required for epithelial patterning during lung organogenesis, and emphasize the combinatorial power of paired RNA-Seq and ATAC-Seq in defining regulatory networks in development.


title: High-throughput functional analysis of natural variants in yeast

abstract: How natural variation affects phenotype is difficult to determine given our incomplete ability to deduce the functional impact of the polymorphisms detected in a population. Although current computational and experimental tools can predict and measure allele function, there has previously been no assay that does so in a high-throughput manner while also representing haplotypes derived from wild populations. Here, we present such an assay that measures the fitness of hundreds of natural alleles of a given gene without site-directed mutagenesis or DNA synthesis. With a large collection of diverse Saccharomyces cerevisiae natural isolates, we piloted this technique using the gene SUL1, which encodes a high-affinity sulfate permease that, at increased copy number, can improve the fitness of cells grown in sulfate-limited media. We cloned and barcoded all alleles from a collection of over 1000 natural isolates en masse and matched barcodes with their respective variants using PacBio long-read sequencing and a novel error-correction algorithm. We then transformed the reference S288C strain with this library and used barcode sequencing to track growth ability in sulfate limitation of lineages carrying each allele. We show that this approach allows us to measure the fitness conferred by each allele and stratify functional and nonfunctional alleles. Additionally, we pinpoint which polymorphisms in both coding and noncoding regions are detrimental to fitness or are of small effect and result in intermediate phenotypes. Integrating these results with a phylogenetic tree, we observe how often loss-of-function occurs and whether or not there is an evolutionary pattern to our observable phenotypic results. This approach is easily applicable to other genes. Our results complement classic genotype-phenotype mapping strategies and demonstrate a high-throughput approach for understanding the effects of polymorphisms across an entire species which can greatly propel future investigations into quantitative traits.

category: genetics


title: Ovarian Tumor Mitochondria Exhibit Abnormal Phenotypes and Blunted Associations with Biobehavioral Factors

abstract: Tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and tumor mitochondria remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign and cancerous ovarian tissue in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n=1,435) showed that gene expression of specific mitochondrial proteins in ovarian tumors is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in 51 benign and 128 high-grade epithelial ovarian tumors revealed that compared to benign tissue, tumors exhibit 3.3-8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P<0.001) but similar mitochondrial DNA levels (-3.1%), documenting abnormal mitochondrial phenotypes in tumors. Mitochondrial respiratory chain activity was also associated with interleukin-6 (IL-6) levels in ascites. In benign tissue, negative biobehavioral factors were inversely correlated with mitochondrial content and respiratory chain activities, whereas positive biobehavioral factors tended to be positively correlated with mitochondrial measures, although effect sizes were small to medium (r=-0.43 to 0.47). In contrast, serous tumors showed less pronounced biobehavioral-mitochondrial correlations. These results document abnormal mitochondrial functional phenotypes in ovarian tumors and warrant further research on the link between biobehavioral factors and mitochondria in cancer.


title: Multiple metabolic signals including AMPK and PKA regulate glucose-stimulated double strand break resection in yeast

abstract: DNA double strand breaks (DSBs) are cytotoxic lesions repaired by non-homologous end joining (NHEJ) and homologous recombination (HR), with 5 strand resection being the committed step in transition from NHEJ to HR. We previously discovered that gal1 yeast, which cannot metabolize galactose, were unable to perform efficient 5 resection even though DSBs were formed. Adding glucose or restoring GAL1 restored resection, suggesting that carbon source metabolism signals to DSB repair. Here we demonstrate that any fermentable carbon source, including raffinose, can stimulate resection and that the stimulatory effect of glucose was associated with decreased, not increased, cellular ATP. The effect was cell cycle dependent and did not occur in G1, while glucose augmented the G2/M checkpoint arrest even in cells deficient in resection. AMP-activated protein kinase pathway mutants showed only low basal resection despite glucose addition but had normal checkpoint arrest, indicating a primary role for Snf1 specifically in glucose-stimulated resection. The metabolic inputs to resection were multifactorial, however, with loss of the transcriptional repressor Mig1 leading to increased basal resection, three distinct patterns of deficiency with loss of the protein kinase A catalytic subunits, Tpk1, Tpk2 andTpk3, and a resection delay in yeast lacking the lysine demethylase Rph1 that helped separate early and late phase responses to glucose. These results reveal multiple interrelated metabolic signals that optimize DSB resection efficiency while independently amplifying the G2/M checkpoint response.


title: Biomechanical evaluation of a novel orthogonal angle-stable interlocking nail in a canine femur model

abstract: Interlockings nails (IN) are orthopedic implants with superior mechanical and, potentially, biological qualities. Despite the countless and indisputable advantages of current angle-stable models, there are still limitations for their use in certain scenarios. The objective of the present study was to describe and biomechanically test a new orthogonal angle-stable intramedullary nail model for veterinary use. The proposed orthogonal angle-stable nail has two 3.8-mm threaded cylindrical holes in each of its portions: in the proximal portion, the holes are 11 mm apart; in the distal portion, the penultimate orifice is positioned at 90 degrees in relation to the last one, with a distance of 5.5 mm between them. The novel orthogonal nail (Group 3 - G3) was evaluated and compared biomechanically with the conventional interlocking nail (Group 1 - G1) and the uniplanar angle-stable nail (Group 2 - G2) by means of destructive torsion and axial compression tests. No statistically significant differences were observed in torsion resistance between the groups in the destructive tests. However, statistical differences were found in stiffness values in the compression tests between the orthogonal (G3) and conventional interlocking (G1) nails (p=0.01) and also between the uniplanar (G2) and interlocking (G1) nails (p=0.001). The new orthogonal nail proved to be biomechanically similar to the uniplanar angle-stable model and superior to the conventional nail. This new arrangement of interlocking screws (orthogonal and closer to each other) potentially enables the fixation of small fragments and at the extremities of long bones in dogs. Nonetheless, further clinical studies are necessary to validate such hypotheses.

category: bioengineering


title: Identification of Hsp90 inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

abstract: Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.


title: Increasing central serotonin with 5-HTP disrupts the inhibition of social gaze in non-human primates

abstract: To competently navigate the world, individuals must flexibly balance distinct aspects of social gaze, orienting toward others and inhibiting orienting responses, depending on the context. These behaviors are often disrupted in patient populations treated with serotonergic drugs. However, the field lacks a clear understanding of how the serotonergic system mediates social orienting and inhibiting behaviors. Here, we tested how increasing central concentrations of serotonin with the direct precursor 5-Hydroxytryptophan (5-HTP) would modulate the ability of rhesus macaques to use eye movements to flexibly orient to, or inhibit orienting to, faces. Systemic administrations of 5-HTP effectively increased central serotonin levels and impaired flexible orientation and inhibition. Critically, 5-HTP selectively impaired the ability of monkeys to inhibit orienting to face images, whereas it similarly impaired orienting to face and control images. 5-HTP also caused monkeys to perseverate on their gaze responses, making them worse at flexibly switching between orientating and inhibiting behaviors. Furthermore, the effects of 5-HTP on performance correlated with a constriction of the pupil, an increased time to initiate trials, and an increased reaction time, suggesting that the disruptive effects of 5-HTP on social gaze behaviors are likely driven by a downregulation of arousal and motivational states. Taken together, these findings provide causal evidence for a modulatory relationship between 5-HTP and social gaze behaviors in non-human primates and offer translational insights for the role of the serotonergic system in social gaze.


title: Choice of method of place cell classification determines the population of cells identified.

abstract:
Place cells, spatially responsive hippocampal cells, provide the neural substrate supporting navigation and spatial memory. Historically most studies of these neurons have used electrophysiological recordings from implanted electrodes but optical methods, measuring intracellular calcium, are becoming increasingly common. Several methods have been proposed as a means to identify place cells based on their calcium activity but there is no common standard and it is unclear how reliable different approaches are. Here we tested three methods that have previously been applied to two-photon hippocampal imaging or electrophysiological data, using both model datasets and real imaging data. These methods use different parameters to identify place cells, including the peak activity in the place field, compared to other locations (the Peak method); the stability of cells activity over repeated traversals of an environment (Stability method); and a combination of these parameters with the size of the place field (Combination method). The three methods performed differently from each other on both model and real data. The Peak method showed high sensitivity and specificity for detecting model place cells and was the most robust to variations in place field width, reliability and field location. In real datasets, vastly different numbers of place cells were identified using the three methods, with little overlap between the populations identified as place cells. Therefore, choice of place cell detection method dramatically affects the number and properties of identified cells. We recommend the Peak method be used in future studies to identify place cell populations, unless there is an explicit theoretical reason for detecting cells with more narrowly defined properties.

Author SummaryPlace cells are hippocampal cells that have spatially constrained receptive fields, the place field. These cells have been widely studied in the context of navigation, more recently using virtual reality environments in combination with optical methods of recording neuronal activity. However, there is a lack of consensus regarding how to identify place cells in these data. In this study we tested the sensitivity and specificity of three methods of identifying place cells. By comparing these methods and quantifying the populations of place cells they identify, we aimed to increase our understanding of exactly the populations that are currently being studied under the name "place cells". Although the appropriate method may depend on the experimental design, we generally recommend a single method going forward, which will increase consensus within the field about what should be included in a place cell population, and allow us to better compare results between studies.


title: Sparse balance: excitatory-inhibitory networks with small bias currents and broadly distributed synaptic weights

abstract: Cortical circuits generate excitatory currents that must be cancelled by strong inhibition to assure stability. The resulting excitatory-inhibitory (E-I) balance can generate spontaneous irregular activity but, in standard balanced E-I models, this requires that an extremely strong feedforward bias current be included along with the recurrent excitation and inhibition. The absence of experimental evidence for such large bias currents inspired us to examine an alternative regime that exhibits asynchronous activity without requiring unrealistically large feedforward input. In these networks, irregular spontaneous activity is supported by a continually changing sparse set of neurons. To support this activity, synaptic strengths must be drawn from high-variance distributions. Unlike standard balanced networks, these sparse balance networks exhibit robust nonlinear responses to uniform inputs and non-Gaussian statistics. In addition to simulations, we present a mean-field analysis to illustrate the properties of these networks.


title: A cytofluorimetric analysis of a Saccharomyces cerevisiae population cultured in a fed-batch bioreactor

abstract: The yeast Saccharomyces cerevisiae is a reference model system and one of the widely used microorganisms in many biotechnological processes. In industrial yeast applications, combined strategies aim to maximize biomass/product yield, with the fed-batch culture being one of the most frequently used. Flow cytometry (FCM) is widely applied in biotechnological processes and represents a key methodology to monitor cell population dynamics. We propose here an application of FCM in the analysis of yeast cell cycle along the time course of a typical S. cerevisiae fed-batch culture. We used two different dyes, SYTOX Green and SYBR Green, with the aim to better define each stage of cell cycle during S. cerevisiae fed-batch culture. The results provide novel insights in the use of FCM cell cycle analysis for the real-time monitoring of S. cerevisiae bioprocesses.


title: THz irradiation inhibits cell division by affecting actin dynamics

abstract: Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.


title: Extended numerical analysis of an eyeball injury under direct impact

abstract: The objective of this study was to develop a numerical model of the eyeball and orbit to simulate a blunt injury to the eyeball leading to its rupture, as well as to conduct a comparative analysis of the results obtained using the finite element method against the clinical material concerning patients who had suffered an eyeball rupture due to a blunt force trauma. Using available sclera biometric and strength data, a numerical model of the eyeball, the orbital contents, and the bony walls were developed from the ground up. Then, eight different blunt force injury scenarios were simulated. The results of numerical analyses made it possible to identify possible locations and configurations of scleral rupture. The obtained results were compared against the clinical picture of patients hospitalized at the Clinic of Ophthalmology in 2010-2016 due to isolated blunt force trauma to the eyeball. It has been demonstrated that the extent of damage observed on the numerical model that indicated a possible location of eyeball rupture did not differ from the clinically observed configurations of the scleral injuries, however this applies to injuries the extend of which did not exceed 2-3 clock-hours on the eyeball. It has been found that the direction of the impact applied determines the location of eyeball rupture. Most often the rupture occurs at the point opposite to the clock-hour of the impact application. The eyeball rupture occurs in the first 7-8 milliseconds after the contact with the striking object, assuming that it strikes at a speed close to the speed of a human fist. It has been established that the injuries most often affected the upper sectors of the eyeball. Men are definitely more likely to sustain such injuries. Eyeball ruptures lead to significant impairment of visual acuity being most often degraded to light perception in front of the eye. Despite immediate specialist treatment, it is possible to achieve, on average, a visual acuity of 0.26 as per the Snellen scale. This study may contribute to a better understanding of injury mechanisms and better treatment planning. It may also contribute to the development of eyeball protection methods for persons exposed to the above-mentioned injuries.

category: pathology


title: A public broadly neutralizing antibody class targets a membrane-proximal anchor epitope of influenza virus hemagglutinin

abstract: Broadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is "anchored" to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an underappreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.


title: Gene flux and acid-imposed selection are the main drivers of antimicrobial resistance in broiler chicks infected with Salmonella enterica serovar Heidelberg

abstract:
Antimicrobial resistance (AR) spread is a worldwide health challenge, stemming in large part, from the ability of microbes to share their genetic material through horizontal gene transfer (HGT). Overuse and misuse of antibiotics in clinical settings and in food production have been linked to this increased prevalence and spread of AR. Consequently, public health and consumer concerns have resulted in a remarkable recent reduction in antibiotics used for food animal production. This is driven by the assumption that removing this selective pressure will favor the recovery of antibiotic susceptible taxa and will limit AR sharing through HGT, allowing the currently available antibiotic arsenal to be effective for a longer period. In this study we used broiler chicks raised antibiotic-free and Salmonella enterica serovar Heidelberg (SH), as a model food pathogen, to test this hypothesis. Our results show that neonatal broiler chicks challenged with an antibiotic susceptible SH strain and raised without antibiotics carried susceptible and multidrug resistance SH strains 14 days after challenge. SH infection perturbed the microbiota of broiler chicks and gavaged chicks acquired antibiotic resistant SH at a higher rate. We determined that the acquisition of a plasmid from commensal Escherichia coli population conferred multidrug resistance phenotype to SH recipients and carriage of this plasmid increased the fitness of SH under acidic selection pressure. These results suggest that HGT of AR shaped the evolution of SH and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella.

ImportanceThe reported increase in antibiotic resistant bacteria in humans have resulted in a major shift away from antibiotics use in food animal production. This has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, and this in turn will allow the currently available antibiotic arsenal to be more effective. This shift in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic susceptible Salmonella Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggests that simply removing antibiotics from food-animal production might not be sufficient to limit the spread of antimicrobial resistance.


title: Analyses of the complete genome sequence of 2,6-dichlorobenzamide (BAM) degrader Aminobacter sp. MSH1 suggests a polyploid chromosome, phylogenetic reassignment, and functions of (un)stable plasmids.

abstract:
Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its transformation product, the recalcitrant groundwater micropollutant, 2,6-dichlorobenzamide (BAM) as sole source of carbon, nitrogen, and energy. The concentration of BAM in groundwater often exceeds the threshold limit for drinking water, resulting in the use of additional treatment in drinking water treatment plants (DWTPs) or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. Combining Illumina and Nanopore sequencing, we here present the complete genome of MSH1, which was determined independently in two different laboratories. Unexpectedly, divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism pBAM1 (41 kb) and pBAM2 (54 kb), we observe that the genome of MSH1 contains two megaplasmids pUSP1 (367 kb) and pUSP2 (366 kb) and three smaller plasmids pUSP3 (97 kb), pUSP4 (64 kb), and pUSP5 (32 kb). The MSH1 substrain from KU Leuven showed a reduced genome lacking plasmids pUSP2 and the three smaller plasmids and was designated substrain MK1, whereas the variant with all plasmids was designated as substrain DK1. Results of a plasmid stability experiment, indicate that strain MSH1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Based on phylogenetic analyses, strain MSH1 is reassigned as Aminobacter niigataensis MSH1.

ImportanceThe complete genomes of the two MSH1 substrains, DK1 and MK1, provide further insight into this already well-studied organism with bioremediation potential. The varying plasmid contents in the two substrains suggest that some of the plasmids are unstable, although this is not supported by the herein described plasmid stability experiment. Instead, results suggest that MSH1 is polyploid with respect to its chromosome, at least under some growth conditions. As the essential BAM-degradation genes are found on some of these plasmids, stable inheritance is essential for continuous removal of BAM. Finally, Aminobacter sp. MSH1 is reassigned as Aminobacter niigataensis MSH1, based on phylogenetic evidence.


title: A Tad-like apparatus is required for contact-dependent prey killing in predatory social bacteria

abstract: Myxococcus xanthus, a soil bacterium, predates collectively using motility to invade prey colonies. Prey lysis is mostly thought to rely on secreted factors, cocktails of antibiotics and enzymes, and direct contac with Myxococcus cells. In this study, we show that on surfaces the coupling of A-motility and contact-dependent killing is the central predatory mechanism driving effective prey colony invasion and consumption. At the molecular level, contact-dependent killing involves a newly discovered type IV filament-like machinery (Kil) that both promotes motility arrest and prey cell plasmolysis. In this process, Kil proteins assemble at the predator-prey contact site, suggesting that they allow tight contact with prey cells for their intoxication. Kil-like systems form a new class of Tad-like machineries in predatory bacteria, suggesting a conserved function in predator-prey interactions. This study further reveals a novel cell-cell interaction function for bacterial pili-like assemblages.


title: Cell-to-cell heterogeneities during extrinsic apoptosis arise from cell cycle progression and transmitotic apoptosis resistance

abstract: Extrinsic apoptosis relies on TNF-family receptor activation by immune cells or receptor-activating biologics. Here, we monitored cell cycle progression at minutes resolution to relate apoptosis kinetics and cell-to-cell heterogeneities in death decisions to cell cycle phases. Interestingly, we found that cells in S phase delay TRAIL receptor-induced death in favour for mitosis, thereby passing on an apoptosis-primed state to their offspring. This translates into two distinct fates, apoptosis execution post mitosis or cell survival from inefficient apoptosis. Transmitotic resistance is linked to Mcl-1 upregulation from mid S phase onwards, which allows cells to pass through mitosis with activated caspase-8, and with cells escaping apoptosis after mitosis sustaining sublethal DNA damage. Antagonizing Mcl-1 by BH3-mimetics suppresses cell cycle-dependent delays in apoptosis, prevents apoptosis-resistant progression through mitosis and averts unwanted survival from apoptosis induction. Cell cycle progression therefore modulates signal transduction during extrinsic apoptosis, with Mcl-1 governing decision making between death, proliferation and survival from inefficient apoptosis induction. Cell cycle progression thus is a crucial process from which cell-to-cell heterogeneities in fates and treatment outcomes emerge in isogenic cell populations during extrinsic apoptosis signalling.


title: LSTM-PHV: Prediction of human-virus protein-protein interactions by LSTM with word2vec

abstract: Viral infection involves a large number of protein-protein interactions (PPIs) between human and virus. The PPIs range from the initial binding of viral coat proteins to host membrane receptors to the hijacking of host transcription machinery. However, few interspecies PPIs have been identified, because experimental methods including mass spectrometry are time-consuming and expensive, and molecular dynamic simulation is limited only to the proteins whose 3D structures are solved. Sequence-based machine learning methods are expected to overcome these problems. We have first developed the LSTM model with word2vec to predict PPIs between human and virus, named LSTM-PHV, by using amino acid sequences alone. The LSTM-PHV effectively learnt the training data with a highly imbalanced ratio of positive to negative samples and achieved an AUC of 0.976 with an accuracy of 98.4% using 5-fold cross-validation. By using independent test dataset, we compared the LSTM-PHV with existing state-of-the-art PPI predictors including DeepViral. In predicting PPIs between human and unknown or new virus, the LSTM-PHV presented higher performance than the existing predictors when they were trained by multiple host protein-including datasets. LSTM-PHV learnt multiple host protein sequence contexts more efficiently than the DeepViral. Interestingly, learning of only sequence contexts as words presented remarkably high performances. Use of uniform manifold approximation and projection demonstrated that the LSTM-PHV clearly distinguished the positive PPI samples from the negative ones. We presented the LSTM-PHV online web server that is freely available at http://kurata35.bio.kyutech.ac.jp/.

category: bioinformatics


title: IHRW: An Improved Hypergraph Random Walk Model for Predicting Three-Drug Therapy

abstract:
Drug combination therapy is a well-established concept in the treatment of complex diseases due to its fewer side effects, lower toxicity, and better efficacy. However, it is challenging to identify efficacious drug combinations from many drug candidates. Computational models could greatly reduce the cost, but most models did not use data for more than two-drug combinations and could not predict three-drug therapy. However, three-drug combinations account for about 21% of the known combinations, which is a very important type of treatment. Here, we utilized higher-order information and developed an improved hypergraph random walk model (IHRW) for three-drug therapy prediction. This is the first method to explore the combination of three drugs.

As a result, the case studies of breast cancer, lung cancer, and colon cancer showed that IHRW had a powerful ability to predict potential efficacious three-drug combinations, which provides new prospects for complex disease treatment. The code of IHRW is freely available at https://github.com/wangqi27/IHRW.


title: AMBIENT: Accelerated Convolutional Neural Network Architecture Search for Regulatory Genomics

abstract: Convolutional neural networks (CNN) have become a standard approach for modeling genomic sequences. CNNs can be effectively built by Neural Architecture Search (NAS) by trading computing power for accurate neural architectures. Yet, the consumption of immense computing power is a major practical, financial, and environmental issue for deep learning. Here, we present a novel NAS framework, AMBIENT, that generates highly accurate CNN architectures for biological sequences of diverse functions, while substantially reducing the computing cost of conventional NAS.


title: TRPV1 in arteries enables a rapid myogenic tone

abstract: Arterioles maintain blow flow by adjusting their diameter in response to changes in local blood pressure. In this process called the myogenic response, a vascular smooth muscle mechanosensor controls tone predominantly through altering the membrane potential. In general, myogenic responses occur slowly, reaching a plateau in minutes. In the heart and skeletal muscle, however, myogenic tone is rapid; activation occurs in tens of seconds and arterial constrictions or raised extravascular pressure as brief as 100 ms remove tone. Previously, we identified extensive expression of TRPV1 in the smooth muscle of arterioles supplying skeletal muscle, heart and the adipose. Here we reveal a critical role for TRPV1 in the myogenic tone of these tissues. TRPV1 antagonists dilated skeletal muscle arterioles in vitro and in vivo, increased coronary flow in isolated hearts, and transiently decreased blood pressure. All of these effects of TRPV1 antagonists were abolished by genetic disruption of TRPV1. Stretch of isolated vascular smooth muscle cells, or raised intravascular pressure in arteries (with or without endothelium), triggered Ca2+ signaling and vasoconstriction. The majority of these stretch-responses were TRPV1-mediated, with the remaining tone being inhibited by the TRPM4 antagonist, 9-phenantrol. Notably, tone developed more quickly in arteries from wild-type compared with TRPV1-null mice. Furthermore, the rapid vasodilation following brief constriction of arterioles was also dependent on TRPV1, consistent with a rapid deactivation or inactivation of TRPV1. Pharmacologic experiments revealed that membrane stretch activates a phospholipase C/protein kinase C signaling pathway to activate TRPV1, and in turn, L-type Ca2+ channels. These results suggest a critical role, for TRPV1 in the dynamic regulation of myogenic tone and blood flow in the heart and skeletal muscle.

category: physiology


title: UV-B exposure and exogenous hydrogen peroxide application leads to cross-tolerance toward drought in Nicotiana tabacum L.

abstract: Acclimation of plants to water deficit involves biochemical and physiological adjustments. Here, we studied how UV-B exposure and exogenously applied hydrogen peroxide (H2O2) potentiates drought tolerance in tobacco (Nicotiana tabacum L.). Separate and combined applications for 14 days of 1.75 kJ m-2 day-1 UV-B radiation and 0.2 mM H2O2 were assessed. Both factors, individually and combined, resulted in inhibition of growth. Furthermore, the combined treatment led to the most compacted plants. UV-B- and UV-B+H2O2-treated plants increased total antioxidant capacity and foliar epidermal flavonol content. H2O2- and UV-B+H2O2-pre-treated plants showed cross-tolerance to a subsequent 7-day drought treatment. Plant responses to the pre-treatment were notably different: i) H2O2 increased the activity of catalase, phenylalanine ammonia lyase and peroxidase activities, and ii) the combined treatment induced epidermal flavonols which were key to drought tolerance. We report synergistic effects of UV-B and H2O2 on transcription accumulation of UV RESISTANCE LOCUS 8, NAC DOMAIN PROTEIN 13 (NAC13), and BRI1-EMS-SUPPRESSOR 1 (BES1). Our data demonstrate a pre-treatment-dependent response to drought for NAC13, BES1 and CHALCONE SYNTHASE transcript accumulation. This study highlights the potential of combining UV-B and H2O2 to improve drought tolerance which could become a useful tool to reduce water use.

category: plant biology


title: Alternative electron pathways of photosynthesis drive the algal CO2 concentrating mechanism

abstract:
Global photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption1. The great efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, thus enhancing CO2 fixation2. While many cellular components involved in the transport and sequestration of inorganic carbon (Ci) have been uncovered3,4, the way microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains elusive4-6. Here, by monitoring dissolved CO2 consumption, unidirectional O2 exchange and the chlorophyll fluorescence parameter NPQ in the green alga Chlamydomonas, we show that the complementary effects of cyclic electron flow and O2 photoreduction, respectively mediated by PGRL1 and flavodiiron proteins, generate the proton motive force (pmf) required by Ci transport across thylakoid membranes. We demonstrate that the trans-thylakoid pmf is used by bestrophin-like Ci transporters and further establish that a chloroplast-to-mitochondria electron flow contributes to energize non-thylakoid Ci transporters, most likely by supplying ATP. We propose an integrated view of the CCM energy supply network, describing how algal cells distribute photosynthesis energy to power different Ci transporters, thus paving the way to the transfer of a functional algal CCM in plants towards improving crop productivity.

One sentence summaryPhotosynthetic alternative electron flows and mitochondrial respiration drive the algal CO2 concentrating mechanism


title: A fungal powdery mildew pathogen induces extensive local and marginal systemic changes in the Arabidopsis thaliana microbiota

abstract:
O_LIPowdery mildew is a foliar disease caused by epiphytically growing obligate biotrophic ascomycete fungi. How powdery mildew colonization affects host resident microbial communities locally and systemically remains poorly explored.
C_LIO_LIWe performed powdery mildew (Golovinomyces orontii) infection experiments with Arabidopsis thaliana grown in either natural soil or a gnotobiotic system and studied the influence of pathogen invasion into standing natural multi-kingdom or synthetic bacterial communities (SynComs).
C_LIO_LIWe found that after infection of soil-grown plants, G. orontii outcompetes numerous resident leaf-associated fungi. We further detected a significant shift in foliar but not root-associated bacterial communities in this setup. Pre-colonization of germ-free A. thaliana leaves with a bacterial leaf-SynCom, followed by G. orontii invasion, induced an overall similar shift in the foliar bacterial microbiota and minor changes in the root-associated bacterial assemblage. However, a standing root SynCom in root samples remained robust against foliar infection with G. orontii. Although pathogen growth was unaffected by the leaf SynCom, fungal infection caused a more than two-fold increase in leaf bacterial load.
C_LIO_LIOur findings indicate that G. orontii infection affects mainly microbial communities in local plant tissue, possibly driven by pathogen-induced changes in source-sink relationships and host immune status.
C_LI


title: NiftyTorch: A Deep Learning framework for NeuroImaging

abstract: We present NiftyTorch a Deep Learning Framework for NeuroImaging. The motivation behind the development of such a library is that there are scant amount of centralized tool for deploying 3D deep learning for NeuroImaging. In addition, most of the existing tools require expert technical knowledge in Deep Learning or programming, creating a barrier for entry. The goal is to provide a one stop package using which the users can perform classification tasks, Segmentation tasks and Image Transformation tasks. The intended audience are the members of NeuroImaging who would like to explore deep learning but have no background in coding. In this article we explore the capabilities of the framework, the performance of the framework and the future work for the framework.


title: High and low expression of the hyperpolarization activated current (Ih) in mouse CA1 stratum oriens interneurons.

abstract:
Inhibitory interneurons are among the most diverse cell types in the brain; the hippocampus itself contains more than 28 different inhibitory interneurons. Interneurons are typically classified using a combination of physiological, morphological and biochemical observations. One broad separator is action potential firing: low threshold, regular spiking vs. higher threshold, fast spiking. We found that spike frequency adaptation (SFA) was highly heterogeneous in low threshold interneurons in the mouse stratum oriens region of area CA1. Analysis with a k-means clustering algorithm parsed the data set into two distinct clusters based on a constellation of physiological parameters and reliably sorted strong and weak SFA cells into different groups. Interneurons with strong SFA fired fewer action potentials across a range of current inputs and had lower input resistance compared to cells with weak SFA. Strong SFA cells also had higher sag and rebound in response to hyperpolarizing current injections. Morphological analysis shows no difference between the two cell types and the cell types did not segregate along the dorsal-ventral axis of the hippocampus. Strong and weak SFA cells were labeled in hippocampal slices from SST:cre Ai14 mice suggesting both cells express somatostatin. Voltage-clamp recordings showed hyperpolarization activated current Ih was significantly larger in strong SFA cells compared to weak SFA cells. We suggest that the strong SFA cell represents a previously uncharacterized type of CA1 stratum oriens interneuron. Due to the combination of physiological parameters of these cells, we will refer to them as Low Threshold High Ih (LTH) cells.

Key PointsO_LISpike frequency adaptation (SFA) was highly variable among stratum oriens interneurons
C_LIO_LIAdapting stratum oriens interneurons were separated into two cell groups using multiple subthreshold and action potential parameters by cluster analysis
C_LIO_LICells with strong SFA fired fewer action potentials for a given current injection, had lower input resistance and more sag and rebound compared to weak SFA cells
C_LIO_LIThe physiological differences were not correlated with neuron morphology, location in stratum oriens, or anatomical location along the dorsal-ventral axis of the hippocampus
C_LIO_LIVoltage-clamp recordings revealed that strong SFA cells had higher density of the hyperpolarization activated current Ih compared to weak SFA cells
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title: 3D Neuronal Mitochondrial Morphology in Axons, Dendrites, and Somata of the Aging Mouse Hippocampus

abstract: The brains ability to process complex informations relies on the constant supply of energy through aerobic respiration by mitochondria. Neurons contain three anatomically distinct compartments - the soma, dendrites, and projecting axons - which have different energetic and biochemical requirements, as well as different mitochondrial morphologies in cultured systems. Here we apply a quantitative three-dimensional electron microscopy approach to map mitochondrial network morphology and complexity in the mouse brain. We examine three neuronal sub-compartments - the soma, dendrites, myelinated axons - in the dentate gyrus and CA1 of the mouse hippocampus, two subregions with distinct principal cell types and functions. We also establish compartment-specific differences in mitochondrial morphology across these cell types between young and old mice, highlighting differences in age-related morphological recalibrations. Overall, these data define the nature of the neuronal mitochondrial network in the mouse hippocampus, providing a foundation to examine the role of mitochondrial morpho-function in the aging brain.


title: A Regression Framework for Brain Network Distance Metrics

abstract:
Analyzing brain networks has long been a prominent research topic in neuroimaging. However, statistical methods to detect differences between these networks and relate them to phenotypic traits are still sorely needed. Our previous work developed a novel permutation testing framework to detect differences between two groups. Here we advance that work to allow both assessing differences by continuous phenotypes and controlling for confounding variables. To achieve this, we propose an innovative regression framework to relate distances between brain network features to functions of absolute differences in continuous covariates and indicators of difference for categorical variables. We explore several similarity metrics for comparing distances between connection matrices, and adapt several standard methods for estimation and inference within our framework: Standard F-test, F-test with individual level effects (ILE), Feasible Generalized Least Squares (FGLS), and Permutation. Via simulation studies, we assess all approaches for estimation and inference while comparing them with existing Multivariate Distance Matrix Regression (MDMR) methods. We then illustrate the utility of our framework by analyzing the relationship between fluid intelligence and brain network distances in Human Connectome Project (HCP) data.

HighlightsO_LIRelated distances between connection matrices to differences in covariates.
C_LIO_LIAdapted methods for estimation and inference in this framework.
C_LIO_LIAssessment of methods and distance metrics via simulation.
C_LIO_LICompared our methods to existing MDMR methods via simulation.
C_LIO_LIAnalysis of the HCP data with the best approach for each distance metric.
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title: Towards a two-stage model of action-stopping: Attentional capture explains motor inhibition during early stop-signal processing

abstract: The ability to stop an already initiated action is paramount to adaptive behavior. Most scientific debate in the field of human action-stopping currently focuses on two interrelated questions. First: Which mental and neural processes underpin the implementation of inhibitory control, and which reflect the attentional detection of salient stop-signals instead? Second: Why do physiological signatures of inhibition occur at two different latencies after stop-signals (for visual signals, either before or after [~]150ms)? Here, we address both questions via two pre-registered experiments that combined transcranial magnetic stimulation, electromyography, and multi-variate pattern analysis of whole-scalp electroencephalography. Using a stop-signal task that also contained a second type of salient signal that did not require stopping, we found that both signals induced equal amounts of early-latency inhibitory activity, whereas only later signatures (after 175ms) distinguished the two. These findings resolve ongoing debates in the literature and strongly suggest a two-step model of action-stopping.


title: Perineuronal net receptor PTPσ regulates retention of memories

abstract:
Perineuronal nets (PNNs) have an important physiological role in retention of learning by restricting cognitive flexibility. Their deposition peaks after developmental periods of intensive learning, usually in late childhood, and they help in long-term preservation of new acquired skills and information. Modulation of PNN function by various techniques enhances plasticity and regulates retention of memories, which may be beneficial when memory persistence entails negative symptoms such as post-traumatic stress disorder (PTSD). In this study, we investigated the role of PTP{sigma} (receptor-type tyrosine-protein phosphatase S, a phosphatase that is activated by binding of chondroitin sulfate proteoglycans from PNNs) in retention of memories using novel object recognition and fear conditioning rodent models. We observed that mice haploinsufficient for PTPRS gene (PTP{sigma}+/-), although having improved short-term object recognition memory, display impaired long-term memory in both novel object recognition and fear conditioning paradigm, as compared to WT littermates. However, PTP{sigma}+/- mice didnt show any differences in behavioral tests that do not heavily rely on cognitive flexibility, such as elevated plus maze, open field, marble burying and forced swimming test. Since PTP{sigma} has been shown to interact with and dephosphorylate TRKB, we investigated activation of this receptor and its downstream pathways in limbic areas known to be associated with memory. We found that phosphorylation of TRKB and PLC{gamma} are increased in the hippocampus, prefrontal cortex and amygdala of PTP{sigma}+/- mice, but other TRKB-mediated signaling pathways are not affected. Our data suggest that disruption of PNN-PTP{sigma} complex facilitates short-term memory by promoting TRKB phosphorylation in different brain areas, but that PTP{sigma} activity is required for the retention of long-term memories. Inhibition of PTP{sigma} or disruption of PNN-PTP{sigma}-TRKB complex might be a potential target for disorders where negative modulation of the acquired memories can be beneficial.

Conflict of interestThe authors declare no conflict of interest.

FundingThis work was supported by Doctoral Program in Integrative Life Science, Jalmari ja Rauha Ahokkaan Saatio grant, Centre for International Mobility (CIMO) Grant TM-16-10112, and by grants from European Research Council (#322742), EU Joint Programme - Neurodegenerative Disease Research (JPND) CircProt (#301225 and #643417), Sigrid Juselius Foundation, Jane and Aatos Erkko Foundation, and the Academy of Finland (#294710 and #307416). None of the funders had a role in the data acquisition, analysis or manuscript preparation.

Author contributionsPC, CB and EC designed the study; AL, PC and CB performed the experiments and analyzed the data; AL wrote the manuscript draft; AL, PC, CB and EC revised the manuscript.

Contribution to the Field StatementPlasticity of neuronal networks increases brains ability to adapt and it is compromised in various conditions including psychiatric diseases, brain injuries, neurodevelopmental and neurodegenerative disorders. Perineuronal nets, through their receptor PTP{sigma} restrict neuronal plasticity in adult brain, which is considered important for the retention of long-term memories. We have previously shown that this restricted plasticity is mediated by inhibition by PTP{sigma} of the TRKB neurotrophin receptor. We have now investigated the biochemical and behavioral phenotype of mice with reduced PTP{sigma} expression. We observed that PTP{sigma}+/- mice have increased phosphorylation of TRKB and its downstream partner PLC{gamma}1 in the prefrontal cortex, hippocampus and amygdala, demonstrating chronic overactivation of plasticity-related pathways. Consistently, PTP{sigma}+/- mice demonstrated facilitated learning but impaired long-term memory retention. Unexpectedly, long-term memory of PTP{sigma}+/- mice was impaired, suggesting that perneuronal net-stimulated PTP{sigma} activity is important in memory retention. This effect of neuronal "hyperplasticity" induced by PTP{sigma} knockout may be both beneficial and harmful in the treatment of human patients, which should be taken into account when developing therapeutic strategies.


title: Functional characterization of ovine dorsal root ganglion neurons reveals peripheral sensitization after osteochondral defect

abstract:
ObjectiveKnee joint trauma can cause an osteochondral defect (OD), a risk factor for osteoarthritis and cause of debilitating pain in patients. Modelling OD in rodents is difficult due to their smaller joint size. This study proposes sheep as a translationally relevant model to understand the neuronal basis of OD pain.

MethodsUnilateral 6 mm deep OD was induced in adult sheep, 2-6 weeks after which dorsal root ganglion neurons (DRG neurons) were cultured from the control and OD side. Functional assessment of neuronal excitability and activity of the pain-related ion channels, TRPV1 and P2X3, was carried out using electrophysiology and Ca2+-imaging. Immunohistochemistry was utilized to verify expression of pain-related proteins.

ResultsAn increased proportion of OD DRG neurons (sheep, n = 3, Ctrl neurons, n =15, OD neurons, n = 16) showed spontaneous electrical excitability (p = 0.009, unpaired t-test) and hyperexcitability upon TRPV1 agonist (capsaicin) application (p = 0.04, chi-sq test). Capsaicin also produced Ca2+ influx in an increased proportion of OD DRG neurons isolated (p = 0.001, chi-sq test). By contrast, neither protein expression, nor functionality of the P2X3 ion channel were altered in OD neurons.

ConclusionsWe provide evidence of increased excitability of DRG neurons (which is an important neural correlate of pain) and TRPV1 function in an OD sheep model. Our data show that functional assessment of sheep DRG neurons can provide important insights into the neural basis of OD pain and thus potentially prevent its progression into arthritic pain.


title: Alpha Event-related Desynchronization During Reward Processing in Schizophrenia

abstract:
BackgroundDeficits in the way the brain processes rewards may contribute to negative symptoms in schizophrenia. Synchronization of alpha band neural oscillations is a dominant EEG signal when people are awake, but at rest. In contrast, alpha desynchronization to salient events is thought to direct allocation of information processing resources away from the internal state, to process salient stimuli in the external environment. Here, we hypothesize that alpha event-related desynchronization (ERD) during reward processing is altered in schizophrenia, leading to less difference in alpha ERD magnitude between winning and losing outcomes.

MethodsEEG was recorded while participants (patients with schizophrenia (SZ)=54; healthy controls (HC) = 54) completed a casino-style slot machine gambling task. Total power, a measure of neural oscillation magnitude was measured in the alpha frequency range (8-14 Hz), time-locked to reward delivery, extracted via principal components analysis, and then compared between groups and equiprobable win and near miss loss reward outcomes. Associations between alpha power and negative symptoms and trait rumination were examined.

ResultsA significant Group X Reward Outcome interaction (p=.018) was explained by differences within the HC group, driven by significant posterior-occipital alpha desynchronization to wins, relative to near miss losses (p<.001). In contrast, SZ did not modulate alpha power to wins vs. near miss losses (p>.1), nor did alpha power relate to negative symptoms (p>.1). However, across all participants, less alpha ERD to reward outcomes was related to more trait rumination, for both wins (p=.005) and near-miss losses (p=.002), with no group differences observed in the slopes of these relationships.

ConclusionThese findings suggest that event-related modulation of alpha power is altered in schizophrenia during reward outcome processing, even when reward attainment places minimal demands on higher-order cognitive processes during slot machine play. In addition, high trait rumination is associated with less event-related desynchronization to reward feedback, suggesting that rumination covaries with less external attentional allocation to reward processing, regardless of reward outcome valence and group membership.


title: Chronic wireless neural population recordings with common marmosets

abstract:
Marmosets are an increasingly important model system for neuroscience in part due to genetic tractability and enhanced cortical accessibility, due to a lissencephalic neocortex. However, many of the techniques generally employed to record neural activity in primates inhibit the expression of natural behaviors in marmosets precluding neurophysiological insights. To address this challenge, we developed methods for recording neural population activity in unrestrained marmosets across multiple ethological behaviors, multiple brain states, and over multiple years. Notably, our flexible methodological design allows for replacing electrode arrays and removal of implants providing alternative experimental endpoints. We validate the method by recording sensorimotor cortical population activity in freely moving marmosets across their natural behavioral repertoire and during sleep.

HIGHLIGHTS- Simultaneous and chronic wireless neural population recordings in multiple freely moving marmosets
- Neural recording approach enables studies of natural repertoire of behaviors and sleep
- Methyl-methacrylate free surgical approach designed to promote biocompatibility and longitudinal success of the implant
- Modular headstage configuration requires minimal daily animal handling for daily neural recordings
- Alternative experimental endpoints: implant removal, healing, and electrode array replacement


title: The Epstein-Barr virus ubiquitin deconjugase BPLF1 regulates the activity of Topoisomerase II during virus replication

abstract: Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 selectively inhibited the ubiquitination of TOP2 following treatment with topoisomerase poisons, interacted with TOP2 and TOP2{beta} in co-immunoprecipitation and in vitro pull-down, stabilized Etoposide-trapped TOP2 cleavage complexes (TOP2cc) and promoted TOP2 SUMOylation, which halted the DNA-damage response and reduced Etoposide toxicity. Induction of the productive virus cycle promoted the accumulation of TOP2{beta}cc, enhanced TOP2{beta} SUMOylation, and reduced Etoposide toxicity in lymphoblastoid cell lines carrying recombinant EBV encoding the active enzyme. Attenuation of this phenotype upon expression of a catalytic mutant BPLF1-C61A impaired viral DNA synthesis and virus release. These findings highlight a previously unrecognized function of BPLF1 in promoting non-proteolytic pathways for TOP2cc debulking that favor cell survival and virus production.


title: Use of botanical gardens as arks for conserving pollinators and plant-pollinator interactions: A case study from the US Northern Great Plains

abstract: Botanical gardens have contributed to plant conservation through the maintenance of both living and preserved plant specimens for decades. However, there is still a large gap in the literature with regards to understanding the potential conservation value botanical gardens could provide for local pollinators. We investigated how plant-pollinator community structure and diversity may differ between botanical gardens and native habitats by sampling and comparing between two environments: a restored native grassland patch within a local botanical garden and fifteen native, remnant temperate grassland sites in the Northern Great Plains. We found pollinator diversity within the restored native grassland patch was greater than 55% of total remnant temperate grassland transects throughout the entire flowering season, while plant diversity and network community metrics between the two environments remained similar throughout, except that remnant prairies have more links (higher connectance) with pollinators than the garden patch. Overall, our findings demonstrate the promising role restored native grassland patches in botanical gardens could play as reservoirs for local pollinator communities by supporting plant-pollinator interactions comparable to those found in native habitat remnants in the same region.

category: ecology