title: PHIST: fast and accurate prediction of prokaryotichosts from metagenomic viral sequences

abstract:
SummaryPHIST (Phage-Host Interaction Search Tool) predicts prokaryotic hosts of viruses from their genomic sequences. It improves host prediction accuracy at species level over current alignment-based tools (on average by 3 percentage points) as well as alignment-free and CRISPR-based tools (by 14-20 percentage points). PHIST is also two orders of magnitude faster than alignment-based tools making it suitable for metagenomics studies.

Availability and implementationGNU-licensed C++ code wrapped in Python API available at: https://github.com/refresh-bio/phist

Contactandrzej.zielezinski@amu.edu.pl, adam.gudys@polsl.pl

Supplementary informationSupplementary data are available at publisher Web site.


title: Machine learning enables accurate and rapid prediction of active molecules against breast cancer cells

abstract:
SummaryBreast cancer (BC) has surpassed lung cancer as the most frequently occurring cancer, and it is the leading cause of cancer-related death in women. Therefore, there is an urgent need to discover or design new drug candidates for BC treatment. In this study, we first collected a series of structurally diverse datasets consisting of 33,757 active and 21,152 inactive compounds for 13 breast cancer cell lines and one normal breast cell line commonly used in in vitro antiproliferative assays. Predictive models were then developed using five conventional machine learning algorithms, including naive Bayesian, support vector machine, k-Nearest Neighbors, random forest, and extreme gradient boosting, as well as five deep learning algorithms, including deep neural networks, graph convolutional networks, graph attention network, message passing neural networks, and Attentive FP. A total of 476 single models and 112 fusion models were constructed based on three types of molecular representations including molecular descriptors, fingerprints, and graphs. The evaluation results demonstrate that the best model for each BC cell subtype can achieve high predictive accuracy for the test sets with AUC values of 0.689-0.993. Moreover, important structural fragments related to BC cell inhibition were identified and interpreted. To facilitate the use of the model, an online webserver called ChemBC and its local version software were developed to predict potential anti-BC agents.

AvailabilityChemBC webserver is available at http://chembc.idruglab.cn/ and its local version Python software is maintained at a GitHub repository (https://github.com/idruglab/ChemBC).

Contactzjqgmc@163.com or lingwang@scut.edu.cn

Supplementary informationSupplementary data are available at Bioinformatics online.

category: bioinformatics


title: Enzyme Catalysis Enhances Lateral Lipid Motility and Directional Particle Transport on Membranes

abstract: The dynamic interplay between the composition of lipid membranes and the behavior of membrane-bound enzymes is critical to the understanding of cellular function and viability, and the design of membrane-based biosensing platforms. While there is a significant body of knowledge on how lipid composition and dynamics affect membrane-bound enzymes, little is known about how enzyme catalysis influences the motility and lateral transport in lipid membranes. Using enzymes-attached lipids in supported bilayers (SLB), we show catalysis-induced enhanced lateral diffusion of lipids in the bilayer. Enhancing the membrane viscosity by increasing the cholesterol content in the bilayer suppresses the overall diffusion but not the relative diffusion enhancement of the enzyme-attached lipids. We also provide direct evidence of catalysis-induced membrane fluctuations leading to the enhanced diffusion of passive tracers resting on the SLB. Additionally, by using active enzyme patches, we demonstrate the directional transport of tracers on SLBs. These are first steps in understanding diffusion and transport in lipid membranes due to active, out-of-equilibrium processes that are the hallmark of living systems. In general, our study demonstrates how active enzymes can be used to control diffusion and transport in confined 2-D environments.

category: biophysics


title: Non-Gaussian, transiently anomalous and ergodic self-diffusion of flexible dumbbells in crowded two-dimensional environments: coupled translational and rotational motions

abstract: We employ Langevin-dynamics simulations to unveil non-Brownian and non-Gaussian center-of-mass self-diffusion of massive flexible dumbbell-shaped particles in crowded two-dimensional solutions. We also study the intra-dumbbell dynamics due to the relative motion of the two constituent elastically-coupled disks. Our main focus is on effects of the crowding fraction{phi} and the particle structure on the diffusion characteristics. We evaluate the time-averaged mean-squared displacement (TAMSD), the displacement probability-density function (PDF) and the displacement autocorrelation function (ACF) of the dimers. For the TAMSD at highly crowded conditions of dumbbells, e.g., we observe a transition from the short-time ballistic behavior, via an intermediate subdiffusive regime, to long-time Brownian-like spreading dynamics. The crowded system of dimers exhibits two distinct diffusion regimes distinguished by the scaling exponent of the TAMSD, the dependence of the diffusivity on{phi} , and the features of the displacement-ACF. We attribute these regimes to a crowding-induced transition from a viscous to a viscoelastic diffusion medium upon growing{phi} . We also analyze the relative motion in the dimers, finding that larger{phi} suppress their vibrations and yield strongly non-Gaussian PDFs of rotational displacements. For the diffusion coefficients D({phi}) of translational and rotational motion of the dumbbells an exponential decay with{phi} for weak and a power-law D({phi}) {propto} ({phi} -{phi}[*] )2.4 for strong crowding is found. A comparison of simulation results with theoretical predictions for D({phi}) is discussed and some relevant experimental systems are overviewed.


title: Interpretable and tractable models of transcriptional noise for the rational design of single-molecule quantification experiments

abstract:
The question of how cell-to-cell differences in transcription rate affect RNA count distributions is fundamental for understanding biological processes underlying transcription. We argue that answering this question requires quantitative models that are both interpretable (describing concrete biophysical phenomena) and tractable (amenable to mathematical analysis). This enables the identification of experiments which best discriminate between competing hypotheses. As a proof of principle, we introduce a simple but flexible class of models involving a stochastic transcription rate coupled to a discrete stochastic RNA transcription and splicing process, and compare and contrast two biologically plausible hypotheses about transcription rate variation. One assumes variation is due to DNA experiencing mechanical strain, while the other assumes it is due to regulator number fluctuations. Although biophysically distinct, these models are mathematically similar, and we show they are hard to distinguish without comparing whole predicted probability distributions. Our work illustrates the importance of theory-guided data collection, and introduces a general framework for constructing and solving mathematically nontrivial continuous-discrete stochastic models.

Significance StatementThe interpretation of transcriptomic observations requires detailed models of biophysical noise that can be compared and fit to experimental data. Models of intrinsic noise, describing stochasticity in molecular reactions, and extrinsic noise, describing cell-to-cell variation, are particularly common. However, integrating and solving them is challenging, and previous results are largely limited to summary statistics. We examine two mechanistically grounded stochastic models of transcriptional variation and demonstrate that (1) well-known regimes naturally emerge in limiting cases, and (2) the choice of noise model significantly affects the RNA distributions, but not the lower moments, offering a route to model identification and inference. This approach provides a simple and biophysically interpretable means to construct and unify models of transcriptional variation.


title: Determinants of directionality and efficiency of the ATP synthase Fo motor at atomic resolution

abstract: Fo subcomplex of ATP synthase is an membraneembedded rotary motor that converts proton motive force into mechanical energy. Despite a rapid increase in the number of high-resolution structures, the mechanism of tight coupling between proton transport and motion of the rotary c-ring remains elusive. Here, using extensive all-atom free energy simulations, we show how the motors directionality naturally arises from the interplay between intra-protein interactions and energetics of protonation of the c-ring. Notably, our calculations reveal that the strictly conserved arginine in the a-subunit (R176) serves as a jack-of-all-trades: it dictates the direction of rotation, controls the protonation state of the proton-release site and separates the two proton-access half-channels. Therefore, arginine is necessary to avoid slippage between the proton flux and the mechanical output and guarantees highly efficient energy conversion. We also provide mechanistic explanations for the reported defective mutations of R176, reconciling the structural information on the Fo motor with previous functional and single-molecule data.


title: Immunosuppression and Outcomes in Acute Myeloid Leukemia

abstract: Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T-cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T-cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ T-cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

category: cancer biology


title: USP28 enables oncogenic transformation of respiratory cells and its inhibition potentiates molecular therapy targeting mutant EGFR, BRAF and PI3K.

abstract: Oncogenic transformation of lung epithelial cells is a multi-step process, frequently starting with the inactivation of tumor suppressors and subsequent activating mutations in proto-oncogenes, such as members of the PI3K or MAPK family. Cells undergoing transformation have to adjust to changes, such as metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors, which manifest these adjustments. Here, we report that the deubiquitylase USP28 enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes, such as c-JUN, c-MYC, NOTCH and {Delta}NP63, at early stages of malignant transformation. USP28 is increased in cancer compared to normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors, such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small molecule inhibitor reset the proteome of transformed cells towards a  pre-malignant state, and its inhibition cooperated with clinically established compounds used to target EGFRL858R, BRAFV600E or PI3KH1047R driven tumor cells. Targeting USP28 protein abundance already at an early stage via inhibition of its activity therefore is a feasible strategy for the treatment of early stage lung tumours and the observed synergism with current standard of care inhibitors holds the potential for improved targeting of established tumors.


title: SETDB1 Fuels the Lung Cancer Phenotype by Modulating Epigenome, 3D Genome Organization and Chromatin Mechanical Properties

abstract: Imbalance in the finely orchestrated system of chromatin-modifying enzymes is a hallmark of many pathologies such as cancers, since causing the affection of the epigenome and transcriptional reprogramming. Here, we demonstrate that a loss-of-function mutation (LOF) of the major histone lysine methyltransferase SETDB1 possessing oncogenic activity in lung cancer cells leads to broad changes in the overall architecture and mechanical properties of the nucleus through genome-wide redistribution of heterochromatin, which perturbs chromatin spatial compartmentalization. Together with the enforced activation of the epithelial expression program, cytoskeleton remodeling, reduced proliferation rate and restricted cellular migration, this leads to the reversed oncogenic potential of lung adenocarcinoma cells. These results emphasize an essential role of chromatin architecture in the determination of oncogenic programs and illustrate a relationship between gene expression, epigenome, 3D genome and nuclear mechanics.


title: Chromatin accessibility combined with enhancer clusters activation mediates heterogeneous response to dexamethasone in myeloma cells

abstract:
Glucocorticoids (GC) effects occur through binding to the GC receptor (GR) which, once translocated to the nucleus, binds to GC response elements (GREs) to activate or repress target genes. Among GCs, dexamethasone (Dex) is widely used in treatment of multiple myeloma (MM), mainly in combination regimens. However, despite a definite benefit, all patients relapse. Moreover, while GC efficacy can be largely attributed to lymphocyte-specific apoptosis, its molecular basis remains elusive.

To determine the functional role of GR binding in myeloma cells, we generated bulk and single cell multi-omic data and high-resolution contact maps of active enhancers and target genes. We show that a minority (6%) of GR binding sites are associated with enhancer activity gains and increased interaction loops. We find that enhancers contribute to regulate gene activity through combinatorial assembly of large stretches of enhancers and/or enhancer cliques. Furthermore, one enhancer, proximal to GR-responsive genes, is predominantly associated with increased chromatin accessibility and higher H3K27ac occupancy. Finally, we show that Dex exposure leads to co-accessibility changes between predominant enhancer and other regulatory regions of the interaction network. Notably, these epigenomic changes are associated with cell-to-cell transcriptional heterogeneity. As consequences, BIM critical for GR-induced apoptosis and CXCR4 protective from chemotherapy-induced apoptosis are rather upregulated in different cells.

In summary, our work provides new insights into the molecular mechanisms involved in Dex escape.


title: The Sec61 translocon is a therapeutic vulnerability in Multiple Myeloma

abstract: Multiple Myeloma (MM) is an incurable malignancy characterized by the uncontrolled expansion of plasma cells in the bone marrow. While proteasome inhibitors like bortezomib efficiently halt MM progression, drug resistance or toxicity inevitably develop. Here, we used a recently discovered inhibitor, mycolactone, to assess the interest of targeting the Sec61 translocon in MM. In human cell lines and tumors from MM patients, mycolactone triggered pro-apoptotic endoplasmic reticulum stress responses synergizing with bortezomib for induction of MM cell death, irrespective of their resistance to proteasome inhibition. Notably, this synergy was selective of cancer cells and extended to B cell acute lymphoblastic leukemia. Sec61 blockade also caused collateral defects in MM secretion of immunoglobulins and expression of pro-survival interleukin (IL)-6 receptor and CD40, whose activation stimulates IL-6 production. Further, the mycolactone-bortezomib combination demonstrated superior therapeutic efficacy over single drug treatments in immunodeficient mice engrafted with MM cells, without inducing toxic side effects. Collectively, these findings establish Sec61 blockers as novel anti-MM agents and reveal the interest of targeting both the translocon and the proteasome in proteostasis-addicted tumors.


title: Comments on a small sabretooth cat in the Abismo Ponta de Flecha Cave, Vale do Ribeira, southeastern Brazil

abstract: The Vale do Ribeira, located in southeastern Brazil, is known for many caves with osteological material, including several extinct species. The saber-tooth cat Smilodon populator was a large predator that inhabited the Pleistocene and Holocene of South America. A specimen found in the Abismo Ponta de Flecha Cave based on small bones (metacarpals and phalanges) is commented here. The metacarpals have morphological characteristics of S. populator, but are smaller than that of S. fatalis and Panthera onca and larger than that of S. gracilis. The specimen is among the smallest ever found and is comparable in size to an adult lion.

category: paleontology


title: NR0B2 regulation during Primary Sclerosing Cholangitis defines a metabolic and pre-malignant reprogramming of Cholangiocyte

abstract:
Primary Sclerosing Cholangitis (PSC) is an idiopathic, cholestatic liver disease that is characterized by persistent, progressive, biliary inflammation leading to cirrhosis. These patients present higher risk for developing bile duct cancers.

Biomedical text-mining related to PSC symptoms like: biliary inflammation, biliary fibrosis, biliary cholestasis was initiated to collect gene associations with this pathophysiology. The text mining work was integrated in distinct omics data such as human transcriptome of PSC liver, Farnesoid X receptor (FXR) functional liver transcriptome and liver single cell transcriptome of the Abcb4-/- model of PSC. A molecular network implicated in abnormal hepatobiliary system physiology was built and confirming a major implication of Nr0b2 and its associated nuclear receptors like FXR in a metabolic cascade that could influence immune response. TNFRSF12A/TWEAK receptor, was found up regulated in PSC liver independently of FXR regulation and TWEAK signaling is known for its implication in pre-conditioning niche of cholangiocarcinoma. NR0B2 deregulation in PSC liver was found independent of gender, age and body mass index surrogates. At single cell level, Nr0b2 up regulation was found in cholangiocytes but not in hepatocytes. In affected cholangiocytes, the cell trajectory built on Nr0b2 expression, revealed implication of several metabolic pathways for detoxification like sulfur, glutathione derivative and monocarboxylic acid metabolisms. On this cell trajectory it was discovered some molecules potentially implicated in carcinogenesis like: GSTA3, ID2 and mainly TMEM45A a transmembrane molecule from golgi apparatus considered as oncogene in several cancers. All together, these observations found in humanPSC liver and in its murine models allowed to highlight an independent deregulation of NR0B2 with a metabolic and premalignant reprogramming of cholangiocytes.

category: pathology


title: Actions of camptothecin derivatives on larvae and adults of the arboviral vector Aedes aegypti

abstract:
Mosquito-borne viruses including dengue, Zika and Chikungunya viruses as well as parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti (New Orleans strain) were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility.

Of the 400 compounds screened, we identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that significantly reduced Ae. aegypti larval motility compared to DMSO controls. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC50s of 25.5 {+/-} 5.0 M and 22.3 {+/-} 5.4 M respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to Ae. aegypti adults at 100 M and 10 M and completely blocked egg laying when fed at 100 M.

Camptothecin and its derivatives, inhibitors of topoisomerase I, have known activity against several agricultural pests and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries. Both humans and mosquitoes express the highly conserved topoisomerase I target, however, the design of derivatives with differing pharmacokinetic properties may offer a promising route towards the development of insect-specificity of this chemistry.

category: pharmacology and toxicology


title: Rest/Stress Myocardial Perfusion Imaging by Positron Emission Tomography with 18F-Flurpiridaz: A Feasibility Study in Mice

abstract:
BackgroundMyocardial perfusion imaging by positron emission tomography (PET-MPI) is the current gold standard for quantification of myocardial blood flow. 18F-flurpiridaz was recently introduced as a valid alternative to currently used PET-MPI probes. Nonetheless, optimum scan duration and time interval for image analysis are currently unknown. Further, it is unclear whether rest/stress PET-MPI with 18F-flurpiridaz is feasible in mice.

MethodsRest/stress PET-MPI was performed with 18F-flurpiridaz (0.6-3.0 MBq) in 29 mice aged 7-8 months. Regadenoson (0.1 g/g) was used for induction of vasodilator stress. Kinetic modeling was performed using a metabolite-corrected arterial input function. Image-derived myocardial 18F-flurpiridaz uptake was assessed for different time intervals by placing a volume of interest in the left ventricular myocardium.

ResultsTracer kinetics were best described by a two-tissue compartment model. K1 ranged from 6.7-20.0 mL/cm3/min, while myocardial volumes of distribution (VT) were between 34.6 and 83.6 mL/cm3. Of note, myocardial 18F-flurpiridaz uptake (%ID/g) was significantly correlated with K1 at rest and following pharmacological stress testing for all time intervals assessed. However, while Spearmans coefficients (rs) ranged between 0.478 and 0.672, R2 values were generally low. In contrast, an excellent correlation of myocardial 18F-flurpiridaz uptake with VT was obtained, particularly when employing the averaged myocardial uptake from 20-40 min post tracer injection (R2 [≥]0.98). Notably, K1 and VT were similarly sensitive to pharmacological stress induction. Further, mean stress-to-rest ratios of K1, VT, and %ID/g 18F-flurpiridaz were virtually identical, suggesting that %ID/g 18F-flurpiridaz can be used to estimate CFR in mice.

ConclusionOur findings suggest that a simplified assessment of relative myocardial perfusion and coronary flow reserve (CFR), based on image-derived tracer uptake, is feasible with 18F-flurpiridaz in mice, enabling high-throughput mechanistic CFR studies in rodents.

category: physiology


title: Vulnerability to cavitation is linked to home climate precipitation across eight eucalypt species

abstract: Vulnerability to cavitation in leaves is the result of highly adaptive anatomical and physiological traits that can be linked to water availability in a species climate of origin. Despite similar gross leaf morphology, eucalypt species are often confined to specific climate envelopes across the variable rainfall environments of Australia. In this study, we investigate how the progression of cavitation differs among eucalypts and whether this is related to other hydraulic and physical leaf traits. We used the Optical Visualisation technique to capture cavitation progression across the leaves of eight eucalypt species (Angophora crassifolia, Corymbia tessellaris, Eucalyptus atrata, Eucalyptus grandis, Eucalyptus laevopinea, Eucalyptus longifolia, Eucalyptus macrandra, Eucalyptus tereticornis) from a wide range of climates and grown in a common garden setting. Vulnerability to cavitation, represented by the leaf water potential required for 50% cavitation of leaf vessels, varied significantly among species (-3.48 MPa to -8.25 MPa) and correlated linearly with home climate precipitation and leaf SLA (R2 of 0.64 and 0.75, respectively). P12-P88, the range of water potentials between which 12% to 88% of cavitation occurs, was decoupled from P50 but also correlated with leaf SLA (R2 of 0.72). We suggest the magnitude of P12-P88 may be representative of a species drought strategy - a large P12-P88 signifying leaves that exhibit drought tolerance (retention of leaves under drought conditions) and a small P12-P88 signifying drought avoidance (leaf shedding after a threshold of drought is reached). Our results agree with other studies that highlight these cavitation metrics as genetically fixed traits. Turgor loss point, on the other hand, may be more plastic, as evidenced by the low variability of this trait across these eucalypt species grown in a common garden environment. Further study will help to establish the SLA-related anatomical traits that impart cavitation resistance and to extend these conclusions to a greater number of species and home climates.

category: plant biology


title: An evolutionarily ancient Fatty Acid Desaturase is required for the synthesis of hexadecatrienoic acid, which is the main source of the bioactive jasmonate in Marchantia polymorpha

abstract: Jasmonates are fatty acid derived hormones that regulate multiple aspects of plant development, growth and stress responses. Bioactive jasmonates, defined as the ligands of the conserved COI1 receptor, differ between vascular plants and bryophytes (using jasmonoyl-L-isoleucine; JA-Ile and dinor-12-oxo-10,15(Z)-phytodienoic acid; dn-OPDA, respectively). Whilst the biosynthetic pathways of JA-Ile in the model vascular plant Arabidopsis thaliana have been elucidated, the details of dn-OPDA biosynthesis in bryophytes are still unclear. Here, we identify an ortholog of Arabidopsis Fatty Acid Desaturase 5 (AtFAD5) in the model liverwort Marchantia polymorpha and show that FAD5 function is ancient and conserved between species separated by more than 450 million years of independent evolution. Similar to AtFAD5, MpFAD5 is required for the synthesis of 7Z-hexadecenoic acid. Consequently, in Mpfad5 mutants the hexadecanoid pathway is blocked, dn-OPDA levels almost completely depleted and normal chloroplast development is impaired. Our results demonstrate that the main source of dn-OPDA in Marchantia is the hexadecanoid pathway and the contribution of the octadecanoid pathway, i.e. from OPDA, is minimal. Remarkably, despite extremely low levels of dn-OPDA, MpCOI1-mediated responses to wounding and insect feeding can still be activated in Mpfad5, suggesting that dn-OPDA is not the only bioactive jasmonate and COI1 ligand in Marchantia.


title: Continuous tracking of gravistimulated roots in a chambered coverslip by confocal microscopy allows first glimpse on mechanoadaptation of cell files during curvature initiation

abstract: Mechanical responses of individual cells to plant internal and external stimuli modulate organ movement and ensure plant survival as sessile organism in a constantly changing environment. The root is a complex, three-dimensional object, which continuously modifies its growth path. Autonomous and paratonic root movements are both orchestrated by different signaling pathways, whereby auxin modulated directional growth adaptations, including gravitropic response, were already subject of manifold studies. But we still know very little about how cells adapt upon gravitropic stimulus to initiate curvature establishment, which is required to align root tip growth again along the gravitropic vector. This manuscript shows first insights into cell file movements upon gravitropic stimulus of Arabidopsis thaliana roots that initiate curvature establishment. The roots were grown shaded from light and without exogenous sucrose supplementation, both growth conditions that are known to negatively interfere with directed root growth, which allowed a more uniform tracking of root bending by using a confocal microscope with vertical stage.


title: The potyviral protein 6K1 reduces plant protease activity during Turnip mosaic virus infection

abstract:
O_LIPotyviral genomes encode just 11 major proteins and multifunctionality is associated to most of these proteins at different stages of virus life cycle. The potyviral protein 6K1 is required for potyvirus replication at the early stages of viral infection and may mediate cell-to-cell movement at later stages.
C_LIO_LIOur study demonstrates that the 6K1 protein from Turnip mosaic virus (TuMV) reduces the abundance of transcripts related to jasmonic acid biosynthesis and transcripts that encode cysteine protease inhibitors when expressed in trans in Nicotiana benthamiana relative to controls. Furthermore, 6K1 stability increases when lipoxygenase and cysteine protease activity is inhibited chemically, linking a mechanism to the rapid turnover of 6K1 when expressed in trans.
C_LIO_LIUsing transient expression, we show 6K1 is degraded rapidly at early time points in the infection process, whereas at later stages of infection protease activity is reduced and 6K1 becomes more stable, resulting in higher TuMV accumulation in systemic leaves. There was no impact of 6K1 transient expression on TuMV accumulation in local leaves.
C_LIO_LITogether, these results suggest a novel function for the TuMV 6K1 protein which has not been reported previously and enhances our understanding of the complex interactions occurring between plants and potyviruses.
C_LI


title: Community science designed ribosomes with beneficial phenotypes

abstract: Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

category: synthetic biology


title: Transcriptional signatures of cell-cell interactions are dependent on cellular context

abstract: Cell-cell interactions are often predicted from single-cell transcriptomics data based on observing receptor and corresponding ligand transcripts in cells. These predictions could theoretically be improved by inspecting the transcriptome of the receptor cell for evidence of gene expression changes in response to the ligand. It is commonly expected that a given receptor, in response to ligand activation, will have a characteristic downstream gene expression signature. However, this assumption has not been well tested. We used ligand perturbation data from both the high-throughput Connectivity Map resource and published transcriptomic assays of cell lines and purified cell populations to determine whether ligand signals have unique and generalizable transcriptional signatures across biological conditions. Most of the receptors we analyzed did not have such characteristic gene expression signatures - instead these signatures were highly dependent on cell type. Cell context is thus important when considering transcriptomic evidence of ligand signaling, which makes it challenging to build generalizable ligand-receptor interaction signatures to improve cell-cell interaction predictions.

category: systems biology


title: The landscape of toxic intermediates in the metabolic networks of pathogenic fungi reveals targets for antifungal drugs

abstract: The burden of fungal infections for humans, animals and plants is widely underestimated and comprises deadly infections as well as great economic costs. Despite that, antifungal drugs are scarce and emergence of resistance in fungal strains contributes to a high mortality. To overcome this shortage, we propose toxic intermediates and their controlling enzymes in metabolic pathways as a resource for new targets and provide a web-service, FunTox-Networks to explore the landscape of toxic intermediates in the metabolic networks of fungal pathogens. The toxicity of metabolites is predicted by a new random forest regression model and is available for over one hundred fungal species. Further, for major fungal pathogens, metabolic networks from the KEGG database were enriched with data of toxicity and regulatory effort for each enzyme to support identification of targets. We determined several toxic intermediates in fungal-specific pathways like amino acid synthesis, nitrogen and sulfur assimilation, and the glyoxylate bypass. For the latter, we show experimentally that growth of the pathogen Candida albicans is inhibited when the detoxifying enzymes Mls1 and Hbr2 are deleted and toxic glyoxylate accumulates in the cell. Thus, toxic pathway intermediates and their controlling enzymes represent an untapped resource of antifungal targets.


title: Oxytocin neurons mediates the effect of social isolation via the VTA circuits

abstract: Social interaction during adolescence strongly influences brain function and behaviour, and the recent pandemic has emphasized the devastating effect of social distancing on mental health. While accumulating evidences have shown the importance of the reward system in encoding specific aspects of social interaction, the consequences of social isolation on the reward system and the development of social skills later in adulthood are still largely unknown. Here, we found that one week of social isolation during adolescence in mice increased social interaction at the expense of social habituation and social novelty preference. Behavioural changes were accompanied by the acute hyperexcitability of dopamine (DA) neurons in the ventral segmental area (VTA) and long-lasting expression of GluA2-lacking AMPARs at excitatory inputs onto DA neurons that project to the prefrontal cortex (PFC). Social isolation-dependent behavioural deficits and changes in neural activity and synaptic plasticity were reversed by chemogenetic inhibition of oxytocin neurons in the paraventricular nucleus (PVN) of the hypothalamus. These results demonstrate that social isolation has acute and long-lasting effects on social interaction and suggest that these effects are mediated by homeostatic adaptations within the reward circuit.

category: neuroscience


title: Expectations boost the reconstruction of auditory features from electrophysiological responses to noisy speech

abstract: Online speech processing imposes significant computational demands on the listening brain, the underlying mechanisms of which remain poorly understood. Here, we exploit the perceptual  pop-out phenomenon (i.e. the dramatic improvement of speech intelligibility after receiving information about speech content) to investigate the neurophysiological effects of prior expectations on degraded speech comprehension. We recorded electroencephalography and pupillometry from 21 adults while they rated the clarity of noise-vocoded and sine-wave synthesised sentences. Pop-out was reliably elicited following visual presentation of the corresponding written sentence, but not following incongruent or neutral text. Pop-out was associated with improved reconstruction of the acoustic stimulus envelope from low-frequency EEG activity, implying that improvements in perceptual clarity were mediated via top-down signals that enhance the quality of cortical speech representations. Spectral analysis further revealed that pop-out was accompanied by a reduction in theta-band power, consistent with predictive coding accounts of acoustic filling-in and incremental sentence processing. Moreover, delta-band power, alpha-band power, and pupil diameter were all increased following the provision of any written sentence information, irrespective of content. Together, these findings reveal distinctive profiles of neurophysiological activity that differentiate the content-specific processes associated with degraded speech comprehension from the context-specific processes invoked under adverse listening conditions.


title: Phase-amplitude coupling and phase synchronization between medial temporal, frontal and posterior brain regions support episodic autobiographical memory recall

abstract: Episodic autobiographical memory (EAM) is a complex cognitive function that emerges from the coordination of specific and distant brain regions. Specific brain rhythms, namely theta and gamma oscillations and their synchronization, are thought of as putative mechanisms enabling EAM. Yet, the mechanisms of inter-regional interaction in the EAM network remain unclear in humans at the whole brain level. To investigate this, we analyzed EEG recordings of participants instructed to retrieve autobiographical episodes. EEG recordings were projected in the source space, and time-courses of atlas-based brain regions-of-interest (ROIs) were derived. Directed phase synchrony in high theta (7-10 Hz) and gamma (30-80 Hz) bands and high theta-gamma phase-amplitude coupling were computed between each pair of ROIs. Using network-based statistics, a graph-theory method, we found statistically significant networks for each investigated mechanism. In the gamma band, two sub-networks were found, one between the posterior cingulate cortex (PCC) and the medial temporal lobe (MTL) and another within the medial frontal areas. In the high theta band, we found a PCC to ventromedial prefrontal cortex (vmPFC) network. In phase-amplitude coupling, we found the high theta phase of the left MTL biasing the gamma amplitude of posterior regions and the vmPFC. Other regions of the temporal lobe and the insula were also phase biasing the vmPFC. These findings suggest that EAM, rather than emerging from a single mechanism at a single frequency, involves precise spatio-temporal signatures mapping on distinct memory processes. We propose that the MTL orchestrates activity in vmPFC and PCC via precise phase-amplitude coupling, with vmPFC and PCC interaction via high theta phase synchrony and gamma synchronization contributing to bind information within the PCC-MTL sub-network or valuate the candidate memory within the medial frontal sub-network.


title: Speech categorization reveals the role of early-stage temporal-coherence processing in auditory scene analysis

abstract: Temporal coherence of sound fluctuations across spectral channels is thought to aid auditory grouping and scene segregation. Although prior studies on the neural bases of temporal-coherence processing focused mostly on cortical contributions, neurophysiological evidence suggests that temporal-coherence-based scene analysis may start as early as the cochlear nucleus (i.e., the first auditory region supporting cross-channel processing over a wide frequency range). Accordingly, we hypothesized that aspects of temporal-coherence processing that could be realized in early auditory areas may shape speech understanding in noise. We then explored whether physiologically plausible computational models could account for results from a behavioral experiment that measured consonant categorization in different masking conditions. We tested whether within-channel masking of target-speech modulations predicted consonant confusions across the different conditions, and whether predicted performance was improved by adding across-channel temporal-coherence processing mirroring the computations known to exist in the cochlear nucleus. Consonant confusions provide a rich characterization of error patterns in speech categorization, and are thus crucial for rigorously testing models of speech perception; however, to the best of our knowledge, they have not been utilized in prior studies of scene analysis. We find that within-channel modulation masking can reasonably account for category confusions, but that it fails when temporal fine structure (TFS) cues are unavailable. However, the addition of across-channel temporal-coherence processing significantly improves confusion predictions across all tested conditions. Our results suggest that temporal-coherence processing strongly shapes speech understanding in noise, and that physiological computations that exist early along the auditory pathway may contribute to this process.


title: Effective connectivity reveals distinctive patterns in response to others' genuine affective experience of disgust as compared to pain

abstract: Empathy is significantly influenced by the identification of others emotions. In a recent study, we have found increased activation in the anterior insular cortex (aIns) that could be attributed to affect sharing rather than perceptual saliency, when seeing another person genuinely experiencing pain as opposed to merely acting to be in pain. In that prior study, effective connectivity between aIns and the right supramarginal gyrus (rSMG) was revealed to track what another person really feels. In the present study, we used a similar paradigm to investigate the corresponding neural signatures in the domain of empathy for disgust - with participants seeing others genuinely sniffing unpleasant odors as compared to pretending to smell something disgusting. Consistent with the previous findings on pain, we found stronger activations in aIns associated with affect sharing for genuine disgust compared with pretended disgust. However, instead of rSMG we found engagement of the olfactory cortex. Using dynamic causal modeling (DCM), we estimated the neural dynamics of aIns and the olfactory cortex between the genuine and pretended conditions. This revealed an increased excitatory modulatory effect for genuine disgust compared to pretended disgust. For genuine disgust only, brain-to-behavior regression analyses highlighted a link between the observed modulatory effect and the perspective-taking empathic trait. Altogether, the current findings complement and expand our previous work, by showing that perceptual saliency alone does not explain responses in the insular cortex. Moreover, it reveals that different brain networks are implicated in a modality-specific way when sharing the affective experiences associated with pain vs. disgust.


title: Optimization of closed-loop electrical stimulation enables robust cerebellar-directed seizure control

abstract: Additional treatment options for temporal lobe epilepsy are needed, and potential interventions targeting the cerebellum are of interest. Previous animal work has shown strong inhibition of hippocampal seizures through on-demand optogenetic manipulation of the cerebellum. However, decades of work examining electrical stimulation - a more immediately translatable approach - targeting the cerebellum has produced very mixed results. We were therefore interested in exploring the impact that stimulation parameters may have on seizure outcomes. Using a mouse model of temporal lobe epilepsy, we conducted on-demand electrical stimulation of the cerebellar cortex, and varied stimulation charge, frequency, and pulse width, resulting in over a thousand different potential combinations of settings. To explore this parameter space in an efficient, data-driven, manner, we utilized Bayesian optimization with Gaussian process regression, implemented in Matlab with an Expected Improvement Plus acquisition function. We examined two different fitting conditions and two different electrode orientations. Following the optimization process, we conducted additional on-demand experiments to test the effectiveness of selected settings. Across all animals, we found that Bayesian optimization allowed identification of effective intervention settings. Additionally, generally similar optimal settings were identified across animals, suggesting that personalized optimization may not always be necessary. While optimal settings were consistently effective, stimulation with settings predicted from the Gaussian process regression to be ineffective failed to provide seizure control. Taken together, our results provide a blueprint for exploration of a large parameter space for seizure control, and illustrate that robust inhibition of seizures can be achieved with electrical stimulation of the cerebellum, but only if the correct stimulation parameters are used.


title: Behavioral state tunes mouse vision to ethological features through pupil dilation

abstract: Sensory processing changes with behavioral context to increase computational flexibility. In the visual system, active behavioral states enhance sensory responses but typically leave the preferred stimuli of neurons unchanged. Here we find that behavioral state does modulate stimulus selectivity in mouse visual cortex in the context of colored natural scenes. Using population imaging, behavior, pharmacology, and deep neural networks, we identified a shift of color selectivity towards ultraviolet stimuli exclusively caused by pupil dilation, resulting in a dynamic switch from rod to cone photoreceptors, extending their role beyond night and day vision. This facilitated the detection of ethological stimuli, such as aerial predators against the twilight sky. In contrast to previous studies that have used pupil dilation as an indirect measure of brain state, our results suggest that the brain uses pupil dilation itself to differentially recruit rods and cones on short timescales to tune visual representations to behavioral demands.


title: Analysis of Rod/Cone Gap Junctions from the Reconstruction of Mouse Photoreceptor Terminals

abstract: Using serial blockface-scanning electron microscopy (SBF-SEM) and focused ion beam-scanning electron microscopy (FIB-SEM), combined with confocal microscopy for the gap junction protein Cx36, we reconstructed mouse photoreceptor terminals and located the gap junctions between them. An exuberant spray of fine telodendria extends from each cone pedicle (including blue cones) to contact 40-50 nearby rod spherules where Cx36 clusters were located, close to the mouth of the synaptic opening. There were approximately 50 Cx36 clusters per cone pedicle and 2-3 per rod spherule. We were unable to detect rod/rod or cone/cone coupling. Thus, rod/cone coupling accounts for nearly all gap junctions between photoreceptors. Our calculations suggest a mean of 82 Cx36 channels between a rod/cone pair of which 25% are open at rest. Rod/cone gap junctions are modulated by dopamine. Comparing our morphological calculations of maximum coupling to previous physiological results suggests that dopamine antagonists can drive rod/cone gap junctions to a surprisingly high open probability, approaching 100%.


title: Inhibition of the SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

abstract: Natural Killer (NK) cells play a crucial role in immunity, killing virally infected and cancerous cells. The balance of signals initiated upon engagement of activating and inhibitory NK receptors with cognate ligands determines killing or tolerance. Nevertheless, the molecular mechanisms regulating rapid NK cell discrimination between healthy and malignant cells in a heterogeneous tissue environment are incompletely understood. The SHP-1 tyrosine phosphatase is the central negative NK cell regulator, which dephosphorylates key activating signaling proteins. Though the mechanism by which SHP-1 mediates NK cell inhibition has been partially elucidated, the pathways by which SHP-1 is itself regulated remain unclear. Here, we show that phosphorylation of SHP-1 in NK cells on the S591 residue by PKC-{theta} promotes the inhibited SHP-1 "folded" state. Silencing PKC-{theta} maintains SHP-1 in the active conformation, reduces NK cell activation and cytotoxicity, and promotes tumor progression in-vivo. This study reveals a molecular pathway that sustains the NK cell activation threshold through suppression of SHP-1 activity.

category: immunology


title: Viral tolerance in Aedes aegypti relies on the negative cooperativity between Toll5A and the cytokine ligand Spz1C

abstract: A. aegypti has evolved to become an efficient vector of Dengue viruses among other arboviruses despite Toll-regulated infection levels. Interestingly, both Toll and its ligand Spatzle (Spz) have undergone gene duplication in A. aegypti raising the possibility of neofunctionalization and mutualism to develop between arboviruses and mosquitoes. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A-Spz1C complexes that display transient but specific interactions. Binding of the first ligand alters receptor-receptor interactions and promotes asymmetric contacts in the vicinity of the Z-loop in Toll5A. This conformation then restricts binding of a second ligand, while bridging the C termini that promote signalling. In contrast, increased receptor concentrations promote inactivating head-to-head receptor assemblies. We also found that Spz1C differs from orthologous and paralogous cytokines in their transcriptional responses upon A. aegypti Aag2 cell stimulation. Interestingly, Spz1C uniquely controls genes involved in innate immunity, lipid metabolism and tissue regeneration. Given the remarkable DENV-induced expression patterns of these proteins, our data rationalises how Spz1C upregulation might promote innate immunity in the midgut, and Toll5A upregulation, viral tolerance in the salivary glands.


title: Mitochondrial sirtuin TcSir2rp3 affects TcSODA activity and oxidative stress response in Trypanosoma cruzi

abstract: Trypanosoma cruzi, the etiological agent of Chagas disease, faces a variety of environmental scenarios during its life cycle in both invertebrate and vertebrate hosts, which include changes in the redox environment that requires a fine regulation of a complex antioxidant arsenal of enzymes. Reversible post-translational modifications, as lysine acetylation, are a fast and economical way for cells to react to environmental conditions. Acetylation neutralizes the lysine positive charge conferring novel properties to the modified proteins, from changes in enzymatic activity to subcellular localization. Recently, we found that the main antioxidant enzymes, including the mitochondrial superoxide dismutase A (TcSODA) are acetylated in T. cruzi, suggesting that protein acetylation could participate in the oxidative stress response in T. cruzi. Therefore, we investigated whether mitochondrial lysine deacetylase sirtuin 3 (TcSir2rp3) was involved in the activity control of TcSODA. We observed an increased resistance to hydrogen peroxide and menadione two oxidant compounds in parasites overexpressing TcSir2rp3. Increased resistance was also found for benznidazole and nifurtimox, the two drugs available for treatment of Chagas disease, known to induce reactive oxidative and nitrosactive species in the parasite. In parallel, TcSir2rp3 overexpressing parasites showed parasites showed a reduction in the ROS levels after treatment with benznidazole and nifurtimox, suggesting a role of TcSir2rp3 in the oxidative stress response. To better understand the way TcSir2rp3 could contributes to oxidative stress response, we analyzed the expression of a key antioxidant enzyme, TcSODA, in the TcSir2rp3 overexpressing parasites and did not detect any increase in protein levels of this enzyme. However, we found that parasites overexpressing TcSir2rp3 presented higher levels of superoxide dismutase activity, and also that TcSir2rp3 and TcSODA interacts in vivo. Knowing that TcSODA is acetylated at lysine residues K44 and K97, and that K97 is located at similar region in the protein structure as K68 in human manganese superoxide dismutase (MnSOD), responsible to regulates MnSOD activity, we generated mutated versions of TcSODA at K44 and K97 and found that replacing K97 by glutamine, which mimics an acetylated lysine, negatively affects the enzyme activity in vitro. By using molecular dynamics approaches we revealed that acetylation of K97 induces specific conformational changes in TcSODA with respect of hydrogen bonding pattern to neighbor residues, specifically D94 and E96, suggesting a key participation of this residue to modulate the affinity to O2- by changing the charge availability on the surface of the enzyme. Taken together, our results showed for the first time the involvement of lysine acetylation in the maintenance of homeostatic redox state in trypanosomatids, contributing to the understanding of mechanisms used by T. cruzi to progress during the infection and opening the opportunity to explore protein acetylation as potential drug target in this parasite.

category: microbiology


title: High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

abstract: Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia have become to humans and agricultural animals, few reliable treatment options exist. To identify novel chemical inhibitors of microsporidia infection, we developed a high-throughput screening method using Caenorhabditis elegans and the microsporidia species Nematocida parisii. We screened the Spectrum Collection of 2,560 FDA-approved compounds and natural products to identify compounds that prevent C. elegans progeny inhibition caused by N. parisii infection. We developed a semi-automated method for quantifying C. elegans progeny number in liquid culture, confirming 11 candidate microsporidia inhibitors. We show that five compounds prevent microsporidia infection by inhibiting spore firing, and demonstrate that one compound, dexrazoxane, slows infection progression. Together, our results demonstrate the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens and provide a scalable high-throughput system for the identification and characterization of additional microsporidia inhibitors.


title: Chaos in small microbial communities

abstract: Predictability is a fundamental requirement in biological engineering. As we move to building coordinated multicellular systems, the potential for such systems to display chaotic behaviour becomes a concern. Therefore understanding which systems show chaos is an important design consideration. We developed a methodology to explore the potential for chaotic dynamics in small microbial communities governed by resource competition, intercellular communication and competitive bacteriocin interactions. We show that we can expect to find chaotic states in relatively small synthetic microbial systems, understand the governing dynamics and provide insights into how to control such systems. This work is the first to query the existence of chaotic behaviour in synthetic microbial communities and has important ramifications for the fields of biotechnology, bioprocessing and synthetic biology.


title: Mechanistic dissection of global proteomic changes in rats with heart failure and preserved ejection fraction

abstract:
IntroductionHeart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, pulmonary congestion and exercise intolerance. Previous preclinical studies show that treatment with cardiosphere-derived cells (CDCs) improves diastolic function, attenuates arrhythmias and prolongs survival in a rat HFpEF model. Here we characterize the myocardial proteome and diastolic function in HFpEF, with and without CDC therapy. As an initial strategy for identifying pathways worthy of further mechanistic dissection, we correlated CDC-responsive proteomic changes with functional improvements.

Methods and ResultsDahl salt-sensitive rats fed high-salt diet, with verified diastolic dysfunction, were randomly assigned to intracoronary CDCs or placebo. Dahl rats fed a low salt diet served as controls. Phenotyping was by echocardiography (E/A ratio) and invasive hemodynamic monitoring (time constant of relaxation Tau, and left ventricular end-diastolic pressure [LVEDP]). CDC treatment improved diastolic function as indicated by a normalized E/A ratio, a 33.3% reduction in Tau, and a 47% reduction of LVEDP. Mass spectrometry of left ventricular tissues (n=6/group) revealed changes in transcription and translation pathways in this rat HFpEF model and was also recapitulated in human HFpEF. These pathways were enhanced following CDC treatment in the animal model (205 proteins and 32 phosphorylated residues accounting for 37% and 19% of all changes, respectively). Among all CDC-sensitive pathways, 65% can be linked to at least 1 of 7 upstream regulators, among which several are of potential relevance for regulating protein expression. To probe newly-synthesized proteins AHA labeling was carried out in isolated rat cardiomyocytes obtained from HFpEF groups, with and without CDC therapy. Five of the initial upstream regulators (HNF4A, MTOR, MYC, TGF{beta}1, and TP53) were linked to proteins expressed exclusively (or increased) with CDC treatment. All 32 phosphorylated residues of proteins involved in transcription/translation altered specifically by CDC treatment had predicted kinases (Protein kinase C (PKC) being the most dominant) and known to be regulated by MYC, TGF{beta}1 and/or TP53. Western blot analysis of those 5 upstream regulators showed that TGF{beta}1, TP53, and Myc were significantly decreased in LV from CDC treated animals, whereas MTOR and HNF4A showed a significant increase compared to HFpEF alone. The cellular quantities of several upstream regulator correlated with indices of diastolic function (E/A ratio, Tau and/or LVEDP). Since CDCs act via the secretion of exosomes laden with signaling cargo, it is relevant that all 7 upstream regulators could, in principle, be regulated by proteins or miRNA that are present in CDC-derived exosomes.

ConclusionWe identified key cellular regulators of transcription and translation that underlie the therapeutic effects of CDCs in HFpEF, whose levels correlate quantitatively with measures of diastolic function. Among the multifarious proteomic changes associated with rat model of HFpEF which were also observed in human HFpEF samples, we propose that these regulators, and downstream effector kinases, be prioritized for further dedicated mechanistic dissection.

category: molecular biology


title: A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation

abstract: In plants, selfish genetic elements including retrotransposons and DNA viruses are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of [~]30 bp synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV initiating nucleotide choice, RDR2 initiation 1-2 nt internal to Pol IV transcript ends and RDR2 terminal transferase activity collectively yield a code that influences which end of the precursor is diced and whether 24 or 23 nt siRNAs are generated from the Pol IV or RDR2-transcribed strands. By diversifying the size, sequence, and strand polarity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow maximal siRNA coverage at methylated target loci.


title: Aging is associated with increased brain iron through brain-derived hepcidin expression

abstract: Iron is an essential molecule for biological processes, but its accumulation can lead to oxidative stress and cellular death. Due to its oxidative effects, iron accumulation is implicated in the process of aging and neurodegenerative diseases. However, the mechanism for this increase in iron with aging, and whether this increase is localized to specific cellular compartment(s), are not known. Here, we measured the levels of iron in different tissues of aged mice, and demonstrate that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain exhibits an increase in both the cytosolic and mitochondrial non-heme iron. This increase in brain iron is associated with elevated levels of local hepcidin mRNA and protein in the brain. We also demonstrate that the increase in hepcidin is associated with increased ubiquitination and reduced levels of the only iron exporter, feroportin-1 (FPN1). Overall, our studies provide a potential mechanism for iron accumulation in the brain through increased local expression of hepcidin, and subsequent iron accumulation due to decreased iron export. Additionally, our data support that aging is associated with mitochondrial and cytosolic iron accumulation only in the brain and not in other tissues.

category: cell biology


title: eIF6 rebinding dynamically couples ribosome maturation and translation

abstract: Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1 (both mutated in the leukaemia predisposition disorder Shwachman-Diamond syndrome) license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. Here, we show that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. Our data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukaemia predisposition disorder.


title: Wnt signaling rescues amyloid beta induced stem cell loss

abstract: Previously, we established an optogenetic model to induce Amyloid-{beta} intracellular oligomerization to model distinct disease etiologies (LO_SCPLOWIMC_SCPLOW et al. 2020). Here we examine the effect of Wnt signaling on Amyloid in this model. We observe that Wnt activation rescues the detrimental effects of Amyloid expression and oligomerization. We analyze the gene expression changes downstream of Wnt that contribute to this rescue and find changes in aging related genes, protein misfolding, metabolism and inflammation. We propose that Wnt expression reduces inflammation through repression of Toll activating factors and confirm that chronic Toll activation reduces lifespan. We propose that the protective effect observed for Lithium treatment functions at least in part through Wnt activation and inhibition of inflammation.


title: The second lineage differentiation of bovine embryos fails in the absence of OCT4/POU5F1

abstract: The mammalian blastocyst undergoes two lineage segregations, i.e., formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) the remaining pluripotent lineage. To clarify expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9 and 12 blastocysts completely derived ex vivo by staining for OCT4, NANOG, SOX2 (EPI) and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4-deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost of NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4-deficient cells. Our data show, that OCT4 is required cell-autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.

category: developmental biology


title: A cellular and molecular atlas reveals the basis of chytrid development.

abstract: The chytrids (phylum Chytridiomycota) are a major early-diverging fungal lineage of ecological and evolutionary importance. Despite their importance, many fundamental aspects of chytrid developmental and cell biology remain poorly understood. To address these knowledge gaps, we combined quantitative volume electron microscopy and comparative transcriptome profiling to create an  atlas of the cellular and molecular basis of the chytrid life cycle, using the model chytrid Rhizoclosmatium globosum. From our developmental atlas, we show that zoospores exhibit a specialised biological repertoire dominated by inactive ribosome aggregates, and that lipid processing is complex and dynamic throughout the cell cycle. We demonstrate that the chytrid apophysis is a distinct subcellular structure characterised by high intracellular trafficking, providing evidence for division of labour in the chytrid cell plan, and show that zoosporogenesis includes  animal like amoeboid cell morphologies resulting from endocytotic cargo transport from the interstitial maternal cytoplasm. Taken together, our results reveal insights into chytrid developmental biology and provide a basis for future investigations into early-diverging fungal cell biology.


title: Synthetic deconstruction of hunchback regulation by Bicoid

abstract:
For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as the model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation.

By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoids partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.


title: Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription


title: The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth

abstract:
Hedgehog (Hh) and bone morphogenetic proteins (BMPs) pattern the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. Here, we show that a previously unknown Hh-dependent mechanism fine-tunes the activity of BMPs. Through genome-wide expression profiling of the Drosophila wing imaginal discs, we identify nord as a novel target gene of the Hh signaling pathway. Nord is related to the vertebrate Neuron Derived Neurotrophic Factor (NDNF) involved in Congenital Hypogonadotropic Hypogonadism and several types of cancer. Loss- and gain-of-function analyses implicate Nord in the regulation of wing growth and proper crossvein patterning. At the molecular level, we present biochemical evidence that Nord is a secreted BMP-binding protein and localizes to the extracellular matrix. Nord binds to Decapentaplegic (Dpp) or the heterodimer Dpp-Glass bottom boat (Gbb) to modulate their release and activity. Furthermore, we demonstrate that Nord is a dosage-depend biphasic BMP modulator, where low levels of Nord promote and high levels inhibit BMP signaling. Taken together, we propose that Hh-induced Nord expression fine tunes both the range and strength of BMP signaling in the developing Drosophila wing.

One sentence summaryThe NDNF-like factor Nord is a Hedgehog-induced and extracellular-localized biphasic BMP modulator that regulates Drosophila wing patterning and growth


title: Missing the forest for the trees: A reappraisal of global seaweed carbon sequestration

abstract: Global seaweed carbon sequestration estimates are currently taken as the fraction of the net primary production (NPP) exported to the deep ocean. However, this perspective does not account for CO2 from the consumption of external subsidies. Here we clarify: i) the role of export relative to seaweed net ecosystem production (NEP) for a closed system and one more likely open to subsidies; ii) the importance of subsidies by compiling published estimates of NEP from seaweed-dominated ecosystems; and iii) discuss their impact on the global seaweed net carbon balance and other sequestration constraints as a mitigation service. Examples of seaweed NEP (n = 18) were sparse and variable. Nevertheless, the average NEP (-9.2mmol C m-2 day-1 SE {+/-} 11.6) suggested that seaweed ecosystems are a C source, becoming increasingly heterotrophic as their export is consumed. Critically, mitigation of greenhouse gas emissions was mixed relative to their replacement or baseline states, and where CO2 is supplied independently of organic metabolism and atmospheric exchange we caution a sole reliance on NEP or NPP. This will ensure a more accurate seaweed mitigation assessment, one that does exceed their capacity and is effective within a compliance and carbon trading scheme.

category: ecology


title: Missing the forest for the trees: Do seaweed ecosystems mitigate atmospheric CO2 emissions?


title: Sequence conservation and structural features that are common within TRP channels

abstract: TRP proteins are a large family of cation selective channels, surpassed in variety only by voltage-gated potassium channels. Detailed molecular mechanisms governing how membrane voltage, ligand binding, or temperature can induce conformational changes promoting the open state of the channel are still missing for TRP channels. Aiming to unveil distinctive structural features common to the transmembrane domains within the TRP family, we performed bioinformatic analyses over a large set of TRP channel genes. Here we report a discrete and exceptionally conserved set of residues. This fingerprint is composed of eleven residues localized at equivalent three-dimensional positions in TRP channels from the different subtypes. Moreover, these amino acids are arranged in three groups, connected by a set of aromatics located at the core of the transmembrane structure. We hypothesize that differences in the connectivity between these different groups of residues harbors the apparent differences in coupling strategies used by TRP subgroups.

category: evolutionary biology


title: Ecology dictates the value of memory for foraging bees

abstract: "Ecological intelligence" hypotheses posit that animal learning and memory evolves to meet the demands posed by foraging, and together with social intelligence and cognitive buffer hypotheses, provide a key framework for understanding cognitive evolution. However, identifying the critical environments where cognitive investment reaps significant benefits has proved challenging. Here, we capitalise upon seasonal variation in forage availability for a social insect model (Bombus terrestris) to establish how the benefits of short-term memory vary with resource availability. Through analysis of over 1700 foraging trips carried out over two years, we show that short-term memory predicts foraging efficiency - a key determinant of colony fitness - in plentiful spring foraging conditions, but that this relationship is reversed during the summer floral dearth. Our results suggest that selection for enhanced cognitive abilities is unlikely to be limited to harsh environments where food is hard to find or extract, highlighting instead that the complexity of rich and plentiful environments could be a broad driver in the evolution of certain cognitive traits.

category: animal behavior and cognition


title: Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins

abstract: Hemoglobin oxidation due to oxidative stress and disease conditions leads to generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the RBC membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2, 2-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-{beta}-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.

category: biochemistry


title: Clues to reaction specificity in PLP-dependent fold type I enzymes of monosaccharide biosynthesis

abstract: Novel functions can emerge in an enzyme family while conserving catalytic mechanism, motif or fold. PLP-dependent enzymes have evolved into seven fold-types and catalyse diverse reactions using the same mechanism for the formation of external aldimine. Nucleotide sugar aminotransferases (which will be henceforth referred to as aminotransferases) belong to fold type I and mediate the biosynthesis of several monosaccharides. They use diverse substrates but are highly selective to the C3 or C4 carbon to which amine group is transferred. Profile HMMs were able to identify aminotransferases but could not capture reaction specificity. A search for discriminating features led to the discovery of sequence motifs that are located near the pyranose binding site suggesting their role in imparting reaction specificity. Using a position weight matrix for this motif, we were able to assign reaction specificity to a large number of aminotransferases. Inferences from this analysis set way for future experiments that can shed light on mechanisms of functional diversification in nucleotide sugar aminotransferases of fold type I.

category: bioinformatics


title: Clues to reaction specificity in PLP-dependent fold type I aminotransferases of monosaccharide biosynthesis


title: tascCODA: Bayesian tree-aggregated analysis of compositional amplicon and single-cell data

abstract: Accurate generative statistical modeling of count data is of critical relevance for the analysis of biological datasets from high-throughput sequencing technologies. Important instances include the modeling of microbiome compositions from amplicon sequencing surveys and the analysis of cell type compositions derived from single-cell RNA sequencing. Microbial and cell type abundance data share remarkably similar statistical features, including their inherent compositionality and a natural hierarchical ordering of the individual components from taxonomic or cell lineage tree information, respectively. To this end, we introduce a Bayesian model for tree-aggregated amplicon and single-cell compositional data analysis (tascCODA) that seamlessly integrates hierarchical information and experimental covariate data into the generative modeling of compositional count data. By combining latent parameters based on the tree structure with spike-and-slab Lasso penalization, tascCODA can determine covariate effects across different levels of the population hierarchy in a data-driven parsimonious way. In the context of differential abundance testing, we validate tascCODAs excellent performance on a comprehensive set of synthetic benchmark scenarios. Our analyses on human single-cell RNA-seq data from ulcerative colitis patients and amplicon data from patients with irritable bowel syndrome, respectively, identified aggregated cell type and taxon compositional changes that were more predictive and parsimonious than those proposed by other schemes. We posit that tascCODA1 constitutes a valuable addition to the growing statistical toolbox for generative modeling and analysis of compositional changes in microbial or cell population data.


title: Glycoproteogenomics characterizes the CD44 splicing code driving bladder cancer invasion

abstract:
Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome of a large BC patient cohort for splicing signatures. Remarkable CD44 heterogeneity was observed, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. In parallel, immunoassays showed that targeting short O-glycoforms could hold the key to improve CD44 cancer specificity. This prompted the development of a glycoproteogenomics approach, building on the integration of transcriptomics-customized datasets and glycomics for protein annotation from nanoLC-ESI-MS/MS experiments. The concept was applied to invasive human BC cell lines, glycoengineered cells, and tumor tissues, enabling unequivocal CD44s identification. Finally, we confirmed the link between CD44s and invasion in vitro by siRNA knockdown, supporting findings from BC tissues. The key role played by short-chain O-glycans in CD44-mediated invasion was also demonstrated through glycoengineered cell models. Overall, CD44s emerged as biomarker of poor prognosis and CD44-Tn/STn as promising molecular signatures for targeted interventions. This study materializes the concept of glycoproteogenomics and provides a key vision to address the cancer splicing code at the protein level, which may now be expanded to better understand CD44 functional role in health and disease.

Significance StatementThe biological role of CD44, a cell membrane glycoprotein involved in most cancer hallmarks and widely explored in BC, is intimately linked to its protein isoforms. mRNA alternative splicing generates several closely related polypeptide sequences, which have so far been inferred from transcripts analysis, in the absence of workflows for unequivocal protein annotation. Dense O-glycosylation is also key for protein function and may exponentiate the number of proteoforms, rendering CD44 molecular characterization a daunting enterprise. Here, we integrated multiple molecular information (RNA, proteins, glycans) for definitive CD44 characterization by mass spectrometry, materializing the concept of glycoproteogenomics. BC specific glycoproteoforms linked to invasion have been identified, holding potential for precise cancer targeting. The approach may be transferable to other tumors, paving the way for precision oncology.

category: cancer biology


title: Simulations on the efficacy of radiotherapy with different time schemes of antiangiogenic therapy

abstract: The combination of radiotherapy and antiangiogenic agents has been suggested to be potent in tumor growth control compared to the application of antiangiogenic therapy or radiotherapy alone. Since radiotherapy is highly dependent on the oxygen level of the tumor area, antiangiogenic agents are utilized for the reoxygenation of tumor vasculature. We present a mathematical framework to investigate the efficacy of radiotherapy combined with antiangiogenic treatment. The framework consists of tumor cells, vasculature, and oxygenation levels evolving with time to mimic a tumor microenvironment. Non-linear partial differential equations (PDEs) are employed to simulate each component of the framework. Different treatment schemes are investigated to see the changes in tumor growth and oxygenation. To test combination schedules, radiation monotherapy, neoadjuvant, adjuvant, and concurrent cases are simulated. The efficiency of each therapy scheme on tumor growth control, the changes in tumor cell density, and oxygen levels shared by tumor cells are represented. The simulation results indicate that the application of radiotherapy after antiangiogenic treatment is more efficient in tumor growth control compared to other therapy schemes. The present study gives an insight into the possible interaction and timing of the combination of radiotherapy and antiangiogenic drug treatment.


title: Drug targeting to sites of oxidative stress using the Baeyer-Villiger reaction

abstract: The antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) is a desirable therapeutic target for a broad range of pathologies, including chronic diseases of the lung and liver, and autoimmune, neurodegenerative, and cardiovascular disorders. However, current Nrf2 activators are limited by unwanted effects due to non-specificity, and systemic distribution and action. Here we report that a 1,2-dicarbonyl moiety masks the electrophilic reactivity of the Nrf2 activator monomethyl fumarate (MMF), otherwise responsible for its non-specific effects. The 1,2-dicarbonyl compound is highly susceptible to Baeyer-Villiger oxidation, with generation of MMF specifically on exposure to pathological levels of hydrogen peroxide or peroxynitrite. Oral treatment with the MMF generating 1,2-dicarbonyl compound reversed chronic neuropathic and osteoarthritis pain in mice, and selectively activated Nrf2 at sites of oxidative stress. This 1,2-dicarbonyl platform may be used to treat additional disorders of oxidative stress, and to selectively target other therapeutics to sites of redox imbalance.

category: pharmacology and toxicology


title: Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation.

abstract:
Diatoms are amongst the most successful clades of oceanic phytoplankton, significantly contributing to photosynthesis on Earth. Their ecological success likely stems from their ability to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity of via Non Photochemical Quenching (NPQ). While the molecular effectors of this process, as well as the role of the Proton Motive Force (PMF) in its regulation are known, the putative regulators of the PET/PMF relationship in diatoms remain unidentified. Here, we demonstrate that the H+/K+ antiporter KEA3 is the main regulator of the coupling between PMF and PET in the model diatom Phaeodactylum tricornutum. By controlling the PMF, it modulates NPQ responses at the onset of illumination, during transients and in steady state conditions. Under intermittent light KEA3 absence results in reduced fitness. Using a parsimonious model including only two components, KEA3 and the diadinoxanthin de-epoxidase, we can describe most of the feedback loops observed between PET and NPQ. This two-components regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory Ca2+-binding domain in KEA3 that controls its activity. This circuit is likely finely tuned by the NPQ effector proteins LHCX, providing diatoms with the required flexibility to thrive in different ocean provinces.

One sentence summaryThe author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is Giovanni Finazzi.


title: Novel effector recognition capacity engineered into a paired NLR complex

abstract: The Arabidopsis RRS1-R Resistance gene confers recognition of the bacterial acetyltransferase PopP2 and another bacterial effector, AvrRps4. The RRS1-S allele recognizes AvrRps4 but not PopP2. RRS1- R/RRS1-S heterozygotes cannot recognize PopP2. RRS1-R and RRS1-S also suppress the constitutive RPS4-dependent autoactivity of RRS1-Rslh1. Phytoplasmas cause important plant diseases, and their effectors can cause degradation of specific host proteins. We tested whether attaching a pathogen effector-dependent degron to RRS1-R, enabling its degradation by phytoplasma effector SAP05, could derepress RRS1-Rslh1 autoactivity, resulting in SAP05-dependent resistance. In transient assays in tobacco, RRS1-R-derived constructs can confer a hypersensitive response (HR) to SAP05. However, phytoplasma infection assays in transgenic Arabidopsis resulted in delayed disease symptoms but not full resistance. We provide a proof-of-concept strategy utilizing the recessiveness of a plant immune receptor gene to engineer recognition of a pathogen effector that promotes degradation of a specific host protein.


title: Ascertaining cells' synaptic connections and RNA expression simultaneously with massively barcoded rabies virus libraries

abstract: Brain function depends on forming and maintaining connections between neurons of specific types, ensuring neural function while allowing the plasticity necessary for cellular and behavioral dynamics. However, systematic descriptions of how brain cell types organize into synaptic networks and which molecules instruct these relationships are not readily available. Here, we introduce SBARRO (Synaptic Barcode Analysis by Retrograde Rabies ReadOut), a method that uses single-cell RNA sequencing to reveal directional, monosynaptic relationships based on the paths of a barcoded rabies virus from its "starter" postsynaptic cell to that cells presynaptic partners1. Thousands of these partner relationships can be ascertained in a single experiment, alongside genome-wide RNA profiles - and thus cell identities and molecular states - of each host cell. We used SBARRO to describe synaptic networks formed by diverse mouse brain cell types in vitro, leveraging a system similar to those used to identify synaptogenic molecules. We found that the molecular identity (cell type/subtype) of the starter cell predicted the number and types of cells that had synapsed onto it. Rabies transmission tended to occur into cells with RNA-expression signatures related to developmental maturation and synaptic transmission. The estimated size of a cells presynaptic network, relative to that of other cells of the same type, associated with increased expression of Arpp21 and Cdh13. By tracking individual virions and their clonal progeny as they travel among host cells, single-cell, single-virion genomic technologies offer new opportunities to map the synaptic organization of neural circuits in health and disease.


title: Development of spontaneous firing of fusiform neurons from the dorsal cochlear nucleus of mice occurs after hearing onset.

abstract: The dorsal cochlear nucleus (DCN) in the auditory brainstem integrates auditory and somatosensory information. Mature fusiform neurons express two qualitative intrinsic states in equal proportions: quiet, with no spontaneous regular action potential firing, or active, with regular spontaneous action potential firing. However, how these firing states and other electrophysiological properties of fusiform neurons develop during early postnatal days to adulthood is not known. Thus, we recorded fusiform neurons from mice from P4 to P21 and analyzed their electrophysiological properties. In the pre-hearing phase (P4-P13), we found that fusiform neurons are mostly quiet, with the active state emerging after hearing onset at P14. Subthreshold properties present more variations before hearing onset, while action potential properties vary more after P14, developing bigger, shorter, and faster action potentials. Interestingly, the activity threshold is more depolarized in pre-hearing cells suggesting that persistent sodium current (INaP) increases its expression after hearing. In fact, INaP increases its expression after hearing, accordingly with the development of active neurons. Thus, we suggest that the post-hearing expression of INaP creates the active state of the fusiform neuron. At the same time, other changes refine the passive membrane properties and increase the speed of action potential firing of fusiform neurons.


title: A subset of nucleus accumbens neurons receiving dense and functional prelimbic cortical input are required for cocaine seeking

abstract:
BackgroundPrelimbic cortical projections to the nucleus accumbens core are critical for cue-induced cocaine seeking, but the identity of the accumbens neuron(s) targeted by this projection, and the transient neuroadaptations contributing to relapse within these cells, remain unknown.

MethodsMale Sprague-Dawley rats underwent cocaine or sucrose self-administration, extinction, and cue-induced reinstatement. Pathway-specific chemogenetics, patch-clamp electrophysiology, in vivo electrochemistry, and high-resolution confocal microscopy were used to identify and characterize a small population of nucleus accumbens core neurons that receive dense prelimbic cortical input to determine their role in regulating cue-induced cocaine and natural reward seeking.

ResultsChemogenetic inhibition of prelimbic cortical projections to the nucleus accumbens core suppressed cue-induced cocaine relapse and normalized real-time cue-evoked increases in accumbens glutamate release to that of sucrose seeking animals. Furthermore, chemogenetic inhibition of the population of nucleus accumbens core neurons receiving the densest prelimbic cortical input suppressed cocaine, but not sucrose seeking. These neurons also underwent morphological plasticity during the peak of cocaine seeking in the form of dendritic spine expansion and increased ensheathment by astroglial processes at large spines.

ConclusionsWe identified and characterized a unique subpopulation of nucleus accumbens neurons that receive dense prelimbic cortical input. The functional specificity of this subpopulation is underscored by their ability to mediate cue-induced cocaine relapse, but not sucrose seeking. This subset of cells represents a novel target for addiction therapeutics revealed by anterograde targeting to interrogate functional circuits imbedded within a known network.


title: A Novel Optical Design Enabling Lightweight and Large Field-Of-View Head-Mounted Microscopes

abstract: We present a novel fluorescence microscope light path that enables imaging of over a thousand neurons in freely-behaving mice and imaging in juvenile songbirds. The light path eliminates traditional illumination optics, allowing for head-mounted microscopes that have both a lower weight and a larger field-of-view (FOV) than previously possible. Using this light path, we designed two microscopes: one optimized for field-of-view ([~]4 mm FOV; 1.4 g), and the other optimized for weight (1.0 mm FOV; 1.0 g).


title: An unconventional GABAergic circuit differently controls pyramidal neuron activity in two visual cortical areas via endocannabinoids

abstract: Perisomatic inhibition of neocortical pyramidal neurons (PNs) coordinates cortical network activity during sensory processing, and it has been mainly attributed to parvalbumin-expressing basket cells (BCs). However, cannabinoid receptor type 1 (CB1)-expressing interneurons also inhibit the perisomatic region of PNs but the connectivity and function of these elusive - yet prominent - neocortical GABAergic cells is unknown. We found that the connectivity pattern of CB1-positive BCs strongly differs between primary and high-order cortical visual areas. Moreover, persistently active CB1 signaling suppresses GABA release from CB1 BCs in the medial secondary visual cortex (V2M), but not in the primary (V1) visual area. Accordingly, in vivo, tonic CB1 signaling is responsible for higher but less coordinated PN activity in V2M than in V1. Our results indicate a differential CB1-mediated mechanism controlling PN activity, and suggest an alternative connectivity schemes of a specific GABAergic circuit in different cortical areas


title: Synaptic and intrinsic mechanisms impair reticular thalamus and thalamocortical neuron function in a Dravet syndrome mouse model

abstract:
Thalamocortical network dysfunction contributes to seizures and sleep deficits in Dravet syndrome (DS), an infantile epileptic encephalopathy, but the underlying molecular and cellular mechanisms remain elusive. DS is primarily caused by mutations in the SCN1A gene encoding the voltage-gated sodium channel NaV1.1, which is highly expressed in GABAergic reticular thalamus (nRT) neurons as well as glutamatergic thalamocortical neurons. We hypothesized that NaV1.1 haploinsufficiency alters somatosensory corticothalamic circuit function through both intrinsic and synaptic mechanisms in nRT and thalamocortical neurons. Using Scn1a heterozygous mice of both sexes aged P25-P30, we discovered reduced intrinsic excitability in nRT neurons and thalamocortical neurons in the ventral posterolateral (VPL) thalamus, while thalamocortical ventral posteromedial (VPM) neurons exhibited enhanced excitability. NaV1.1 haploinsufficiency enhanced GABAergic synaptic input and reduced ascending glutamatergic sensory input to VPL neurons, but not VPM neurons. In addition, glutamatergic cortical input to nRT neurons was reduced in Scn1a heterozygous mice, whereas cortical input to VPL and VPM neurons remained unchanged. These findings introduce input-specific alterations in glutamatergic synapse function and aberrant glutamatergic neuron excitability in the thalamus as disease mechanisms in Dravet syndrome, which has been widely considered a disease of GABAergic neurons. This work reveals additional complexity that expands current models of thalamic dysfunction in Dravet syndrome and identifies new components of corticothalamic circuitry as potential therapeutic targets.

HIGHLIGHTSO_LIGABAergic reticular thalamus neurons have impaired tonic and burst firing properties in a NaV1.1 haploinsufficiency mouse model of Dravet syndrome.
C_LIO_LINaV1.1 haploinsufficiency has opposing effects on spike firing in two distinct glutamatergic thalamocortical neuron populations.
C_LIO_LINaV1.1 haploinsufficiency alters glutamatergic synaptic connectivity in an input-specific manner in the thalamus.
C_LIO_LIDysregulation of both intrinsic and synaptic mechanisms contribute to imbalanced thalamic excitation and inhibition in this Dravet syndrome mouse model.
C_LI


title: Therapeutic activation of virus-specific resident memory T cells within the glioblastoma microenvironment

abstract: Glioblastoma multiforme (GBM) is among the most aggressive, treatment resistant cancers, and despite standard of care surgery, radiation and chemotherapy, is invariably fatal. GBM is marked by local and systemic immunosuppression, contributing to resistance to existing immunotherapies that have had success in other tumor types. Memory T cells specific for previous infections reside in tissues throughout the host and these tissue resident memory T cells (TRM) are capable of rapid and potent immune activation. Here, we show that virus-specific memory CD8+ T cells expressing tissue resident markers populate the mouse and human glioblastoma microenvironment. Reactivating virus-specific TRM through intra-tumoral delivery of adjuvant-free virus-derived peptide triggered local immune activation. This delivery translated to anti-neoplastic effects, which improved survival in a murine glioblastoma model. Our results indicate that virus-specific TRM are a significant part of the glioblastoma immune microenvironment and can be leveraged to promote anti-tumoral immunity.