title: Quantifying the severity of adverse drug reactions using social media

abstract: Adverse drug reactions (ADRs) impact the health of 100,000s of individuals annually in the United States with associated costs in the hundreds of billions. The monitoring and analysis of the severity of adverse drug reactions is limited by the current qualitative and categorical system of severity classifications. Previous efforts have generated quantitative estimates for a subset of ADRs, but were limited in scope due to the time and costs associated with the efforts. We present a semi-supervised approach that estimates ADR severity by using a lexical network of ADR word embeddings and label propagation. We use this method to estimate the severity of 28,113 ADRs, representing 12,198 unique ADR concepts from MedDRA. Our Severity of Adverse Events Derived from Reddit (SO_SCPLOWAEDRC_SCPLOW) scores have good correlations with real-world outcomes. SO_SCPLOWAEDRC_SCPLOW scores had Spearman correlations with ADR case outcomes in FAERS of 0.595, 0.633, and -0.748 for death, serious outcome, and no outcome, respectively. We investigate different methods for defining initial seed term sets and evaluate their impact on severity estimates. We analyzed severity distributions for ADRs based on their appearance in Boxed Warning drug label sections, as well as ADRs with sex-specific associations. We find that ADRs discovered postmarket have significantly greater severity compared to those discovered in the clinical trial. We create quantitative Drug RIsk Profile (DO_SCPLOWRIPC_SCPLOW) scores for 968 drugs that have a Spearman correlation of 0.377 with drugs ranked by FAERS cases resulting in death, where the given drug was the primary suspect. We make the SO_SCPLOWAEDRC_SCPLOW and DO_SCPLOWRIPC_SCPLOW scores publicly available in order to enable more quantitative analysis of pharmacovigilance data.


title: Genotyping Copy Number Alterations from single-cell RNA sequencing

abstract:
MotivationCancers are composed by several heterogeneous subpopulations, each one harbouring different genetic and epigenetic somatic alterations that contribute to disease onset and therapy response. In recent years, copy number alterations leading to tumour aneuploidy have been identified as potential key drivers of such populations, but the definition of the precise makeup of cancer subclones from sequencing assays remains challenging. In the end, little is known about the mapping between complex copy number alterations and their effect on cancer phenotypes.

ResultsWe introduce CONGAS, a Bayesian probabilistic method to phase bulk DNA and single-cell RNA measurements from independent assays. CONGAS jointly identifies clusters of single cells with subclonal copy number alterations, and differences in RNA expression. The model builds statistical priors leveraging bulk DNA sequencing data, does not require a normal reference and scales fast thanks to a GPU backend and variational inference. We test CONGAS on both simulated and real data, and find that it can determine the tumour subclonal composition at the single-cell level together with clone-specific RNA phenotypes in tumour data generated from both 10x and Smart-Seq assays.

AvailabilityCONGAS is available as 2 packages: CONGAS (https://github.com/caravagnalab/congas), which implements the model in Python, and RCONGAS (https://caravagnalab.github.io/rcongas/), which provides R functions to process inputs, outputs, and run CONGAS fits. The analysis of real data and scripts to generate figures of this paper are available via RCONGAS; code associated to simulations is available at https://github.com/caravagnalab/rcongas_test.

Contactgcaravagna@units.it

Supplementary informationSupplementary data are available at Bioinformatics online.

category: bioinformatics


title: A Bayesian method to cluster single-cell RNA sequencing data using Copy Number Alterations


title: A variant selection framework for genome graphs

abstract:
MotivationVariation graph representations are projected to either replace or supplement conventional single genome references due to their ability to capture population genetic diversity and reduce reference bias. Vast catalogues of genetic variants for many species now exist, and it is natural to ask which among these are crucial to circumvent reference bias during read mapping.

ResultsIn this work, we propose a novel mathematical framework for variant selection, by casting it in terms of minimizing variation graph size subject to preserving paths of length  with at most{delta} differences. This framework leads to a rich set of problems based on the types of variants (SNPs, indels), and whether the goal is to minimize the number of positions at which variants are listed or to minimize the total number of variants listed. We classify the computational complexity of these problems and provide efficient algorithms along with their software implementation when feasible. We empirically evaluate the magnitude of graph reduction achieved in human chromosome variation graphs using multiple  and{delta} parameter values corresponding to short and long-read resequencing characteristics. When our algorithm is run with parameter settings amenable to long-read mapping ( = 10 kbp,{delta} = 1000), 99.99% SNPs and 73% indel structural variants can be safely excluded from human chromosome 1 variation graph. The graph size reduction can benefit downstream pan-genome analysis.

Implementationhttps://github.com/at-cg/VF

Contactchirag@iisc.ac.in, neda.tavakoli@gatech.edu, aluru@cc.gatech.edu


title: MicroPIPE: An end-to-end solution for high-quality complete bacterial genome construction

abstract: Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers; however, easy and automated construction of high-quality bacterial genomes remains challenging. Here we present MicroPIPE: a reproducible end-to-end bacterial genome assembly pipeline for ONT and Illumina sequencing. To construct MicroPIPE, we evaluated the performance of several tools for genome reconstruction and assessed overall genome accuracy using ONT both natively and with Illumina. Further validation of MicroPIPE was carried out using 11 sequence type (ST)131 Escherichia coli and eight publicly available Gram-negative and Gram-positive bacterial isolates. MicroPIPE uses Singularity containers and the workflow manager Nextflow and is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe.


title: A development guide for evaluating the maximum yield potential stage in barley

abstract:
Determining the grain yield potential contributed by grain number is a step towards advancing cereal crops yield. To achieve this aim, it is pivotal to recognize the maximum yield potential (MYP) of the crop. In barley (Hordeum vulgare L.), the MYP is defined as the maximum spikelet primordia number of a spike. Previous barley studies often assumed the awn primordium (AP) stage as the MYP stage regardless of genotypes and growth conditions. From our spikelet-tracking experiments using the two-rowed cultivar Bowman, we found that the MYP stage can be different from the AP stage. Importantly, we find that the occurrence of inflorescence meristem (IM) deformation and its loss of activity coincided with the MYP stage, indicating the end of further spikelet initiation. Thus, we recommend validating the barley MYP stage with the IMs shape and propose this approach (named Spikelet Stop) for MYP staging. Following this approach, we compared the MYP stage and the MYP in 27 two- and six-rowed barley accessions grown in the greenhouse and field. Our results reveal that the MYP stage can be reached at various developmental stages, which majorly depend on the genotype and growth conditions. Furthermore, we found that two-rowed barleys MYP and the duration reaching the MYP stage may determine their yield potential. Based on our findings, we suggest key steps for the identification of the MYP in barley that can also be applied in a related crop such as wheat.

HighlightWe show that the maximum yield potential stage in barley can be different from the awn primordium stage as proposed in earlier studies and it varies depending on the genotype and growth conditions. We suggest key steps to identify maximum yield potential in barley that might apply to related cereals.

category: plant biology


title: Marker-free imaging of α-Synuclein aggregates in a rat model of Parkinson' disease using Raman microspectroscopy

abstract: A hallmark of Parkinsons disease (PD) is the formation of Lewy bodies in the brain. Lewy bodies are rich in the aggregated form of misfolded -Synuclein (-Syn). The brain from PD patients can only be analysed after post-mortem, limiting the diagnosis of PD to the manifestation of motor symptoms. In PD patients and animal models phosphorylated -Syn was detected in the gut, thus, raising the hypothesizes that early-stage PD could be diagnosed based on colon tissues biopsies. Non-invasive marker-free technologies represent an ideal method to potentially detect aggregated -Syn in vivo. Raman microspectroscopy has been established for the detection of molecular changes such as alterations of protein structures. Here, the olfactory bulb in the brain and the muscularis mucosae of colon tissue sections of a human BAC-SNCA transgenic (TG) rat model was analysed using Raman imaging and microspectroscopy. Raman images from TG and WT rats were investigated using spectral, principal component and true component analysis. Spectral components indicated protein aggregates (spheroidal oligomers) in TG rat brain and colon tissues even at a young age but not in WT. In summary, we have demonstrated that Raman imaging is capable to detect -Syn aggregates in colon tissues of a PD rat model and making it a promising tool for future use in PD pathology.

category: neuroscience


title: A model of the appearance of the moving human eye

abstract: The entrance pupil and first Purkinje reflection ("glint") in an image of the human eye serve as important features in 3D, model-based eye tracking applications. Here I present a physically and biologically accurate, ray-traced model that supplies the appearance of the pupil and glint of a moving eye. Once biometrically calibrated for a subject under study, simultaneous fitting of the pupil and glint features by the model supplies eye rotation, radius of the aperture stop, and the translation of the eye relative to the initial camera position. The biometric parameters of the model, including corneal curvature and the depth of the centers of rotation, are obtained by model-based fitting of images of the eye posed at known gaze angles. This approach was applied to eye recordings from 30 people obtained during gaze calibration, and while head position was recorded simultaneously using echoplanar magnetic resonance imaging. The refractive and reflective effects of spectacle and contact lenses worn by some subjects were incorporated into the model. The fitted parameters reveal that the center of rotation for horizontal eye movements is deeper (13.8 mm) than that for vertical eye movements (12.1) mm, consistent with prior studies. Individual differences in the depth of the rotation centers, and in corneal curvature, were well related to biometric measures obtained from these subjects using clinical ophthalmologic instruments. Once biometrically calibrated for each subject, gaze position was modeled with a cross-validated, median (across subject) absolute error of 0.58{degrees}, and image-plane translation of the head was estimated with sub-millimeter accuracy. The open-source model described here produces biologically accurate simulations of the appearance of the eye in motion, and may be used in model-based search to derive eye pose and biometric properties from empirical data.


title: Defining a Connectome-Based Predictive Model of Attentional Control in Aging

abstract: With advancing age, declines in the executive control of attention are accompanied by shifts in the functional topology of brain networks. However, there is increasing recognition of the considerable individual variability in the extent and types of attentional deficits that older adults exhibit, with results from neuroimaging investigations paralleling behavioral heterogeneity. Emerging computational methods leverage whole-brain functional connectivity to predict individual-level behaviors. These approaches are well-suited to the cognitive aging context, as they may elucidate configurations of functional connections that best explain group- and individual-level differences across older adults. Two independent samples of neurologically and psychiatrically healthy older adults were used to separately derive a predictive model of attentional control and test the models external validity. Here we show that despite challenges posed by heterogeneity in these aging samples, select functional connections carried meaningful variance, allowing for successful prediction of attention in a novel sample of older individuals.


title: Year-round dynamics of amplicon sequence variant communities differ among eukaryotes, Mimiviridae, and prokaryotes in a coastal ecosystem

abstract:
Coastal seawater is the habitat of different microbial communities. These communities are affected by seasonal environmental changes and fluctuating nutrient availability, as well as competitive and cooperative interspecific interactions. In this work, we investigated the seasonal dynamics of communities of eukaryotes, a major group of double-stranded DNA viruses infecting eukaryotes (i.e. Mimiviridae), as well as prokaryotes in the Uranouchi Inlet, Kochi, Japan. We performed metabarcoding using ribosomal RNA genes and the Mimiviridae polB gene as marker genes in 43 seawater samples collected during 20 months. Communities characterized by the compositions of amplicon sequence variants (ASVs) showed synchronic seasonal cycles for eukaryotes, Mimiviridae, and prokaryotes. However, the community dynamics showed intriguing differences in several aspects such as the recovery rate after a year. We further show that the differences in the community dynamics can be explained by differences in the recurrence/persistence levels of individual ASVs among eukaryotes, Mimiviridae, and prokaryotes. Mimiviridae ASVs were less persistent than eukaryotic ASVs, and prokaryotic ASVs were the most persistent. We argue that the differences in the specificity of interactions (i.e. virus-eukaryote vs prokaryote-eukaryote) as well as the survival strategies are at the origin of the distinct community dynamics among eukaryotes, Mimiviridae, and prokaryotes.

One sentence summaryA one year observation of coastal microbial communities revealed similar but different community dynamics for eukaryotes, a group of large viruses, and prokaryotes.

category: microbiology


title: Transmission of pre-XDR and XDR-TB in the Mumbai Metropolitan Region, India

abstract:
Multidrug-resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis complex (MTBC) strains are a great challenge for tuberculosis (TB) control in India. Still, factors driving the MDR/XDR epidemic in India are not well defined.

To address this, whole genome sequencing (WGS) data from 1 852 MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai were used for phylogenetic strain classification, resistance prediction, and cluster analysis (12 allele distance threshold). Factors associated with pre-XDR/XDR-TB were defined by odds ratios and a multivariate logistic regression model.

Overall, 1 017 MTBC strains were MDR, out of which 57.8 % (n=591) were pre-XDR, and 17.9 % (n=183) were XDR. Lineage 2 (L2) strains represented 41.7 % of the MDR, 77.2 % of the pre-XDR, and 86.3 % of the XDR strains, and were significantly associated with pre-XDR/XDR-TB (P < 0.001). Cluster rates were high among MDR (78 %) and pre-XDR/XDR (85 %) strains with three dominant L2 strain clusters (Cl 1-3) representing half of the pre-XDR and two thirds of the XDR-TB cases. Cl 1 strains accounted for 52.5 % of the XDR MTBC strains. Transmission could be confirmed by identical mutation patterns of particular pre-XDR/XDR strains.

As a conclusion high rates of pre-XDR/XDR strains among MDR-TB patients require rapid changes in treatment and control strategies. Transmission of particular pre-XDR/XDR L2 strains is the main driver of the pre-XDR/XDR-TB epidemic. Accordingly, control of the epidemic in the region requires measures with stopping transmission especially of pre-XDR/XDR L2 strains.

category: molecular biology


title: Integration of APSIM and PROSAIL models to develop more precise radiometric estimation of crop traits using deep learning

abstract: A major challenge for the estimation of crop traits (biophysical variables) from canopy reflectance is the creation of a high-quality training dataset. This can be addressed by using radiative transfer models (RTMs) to generate training dataset representing  real-world data in situations with varying crop types and growth status as well as various observation configurations. However, this approach can lead to "ill-posed" problems related to assumptions in the sampling strategy and due to uncertainty in the model, resulting in unsatisfactory inversion results for retrieval of target variables. In order to address this problem, this research investigates a practical way to generate higher quality  synthetic training data by integrating a crop growth model (CGM, in this case APSIM) with an RTM (in this case PROSAIL). This allows control of uncertainties of the RTM by imposing biological constraints on distribution and co-distribution of related variables. Subsequently, the method was theoretically validated on two types of synthetic dataset generated by PROSAIL or the coupling of APSIM and PROSAIL through comparing estimation precision for leaf area index (LAI), leaf chlorophyll content (Cab), leaf dry matter (Cm) and leaf water content (Cw). Additionally, the capabilities of current deep learning techniques using high spectral resolution hyperspectral data were investigated. The main findings include: (1) Feedforward neural network (FFNN) provided with appropriate configuration is a promising technique to retrieve crop traits from input features consisting of 1 nm-wide hyperspectral bands across 400-2500 nm range and observation configuration (solar and viewing angles), leading to a precise joint estimation for LAI (RMSE=0.061 m2 m-2), Cab (RMSE=1.42 {micro}g cm-2), Cm (RMSE=0.000176 g cm-2) and Cw (RMSE=0.000319 g cm-2); (2) For the aim of model simplification, a narrower range in 400-1100 nm without observation configuration in input of FFNN model provided less precise estimation for LAI (RMSE=0.087 m2 m-2), Cab (RMSE=1.92 {micro}g cm-2), Cm (RMSE=0.000299 g cm-2) and Cw (RMSE=0.001271 g cm-2); (3) The introduction of biological constraints in training datasets improved FFNN model performance in both average precision and stability, resulting in a much accurate estimation for LAI (RMSE=0.006 m2 m-2), Cab (RMSE=0.45 {micro}g cm-2), Cm (RMSE=0.000039 g cm-2) and Cw (RMSE=0.000072 g cm-2), and this improvement could be further increased by enriching sample diversity in training dataset.

category: ecology


title: Evolutionary modelling of HCV subtypes provides rationale for their different disease outcomes

abstract: Hepatitis C virus (HCV) is a leading cause of liver-associated disease and liver cancer. Of the major HCV subtypes, patients infected with subtype 1b have been associated with having a higher risk of developing chronic infection, cirrhosis and hepatocellular carcinoma. However, underlying reasons for this increased disease severity remain unknown. Here, we provide an evolutionary rationale, based on a comparative study of fitness landscape and in-host evolutionary models of the envelope glycoprotein 2 (E2) of HCV subtypes 1a and 1b. Our analysis demonstrates that a higher chronicity rate of subtype 1b may be attributed to lower fitness constraints, enabling 1b viruses to more easily escape antibody responses. More generally, our results suggest that differences in evolutionary constraints between HCV subtypes may be an important factor in mediating distinct disease outcomes. Our analysis also identifies antibodies that appear to be escape-resistant against both subtypes 1a and 1b, providing directions for the design of HCV vaccines having cross-subtype protection.

category: evolutionary biology


title: Advancing admixture graph estimation via maximum likelihood network orientation

abstract:
MotivationAdmixture, the interbreeding between previously distinct populations, is a pervasive force in evolution. The evolutionary history of populations in the presence of admixture can be modeled by augmenting phylogenetic trees with additional nodes that represent admixture events. While enabling a more faithful representation of evolutionary history, admixture graphs present formidable inferential challenges, and there is an increasing need for methods that are accurate, fully automated, and computationally efficient. One key challenge arises from the size of the space of admixture graphs. Given that exhaustively evaluating all admixture graphs can be prohibitively expensive, heuristics have been developed to enable efficient search over this space. One heuristic, implemented in the popular method TreeMix, consists of adding edges to a starting tree while optimizing a suitable objective function.

ResultsHere, we present a demographic model (with one admixed population incident to a leaf) where TreeMix and any other starting-tree-based maximum likelihood heuristic using its likelihood function is guaranteed to get stuck in a local optimum and return an incorrect network topology. To address this issue, we propose a new search strategy that we term maximum likelihood network orientation (MLNO). We augment TreeMix with an exhaustive search for a MLNO, referring to this approach as OrientA-Graph. In evaluations including previously published admixture graphs, OrientAGraph outperformed TreeMix on 4/8 models (there are no differences in the other cases). Overall, OrientAGraph found graphs with higher likelihood scores and topological accuracy while remaining computationally efficient. Lastly, our study reveals several directions for improving maximum likelihood admixture graph estimation.

AvailabilityOrientAGraph is available under the GNU General Public License v3.0 on Github (https://github.com/sriramlab/OrientAGraph).


title: Autophagy as a mechanism for adaptive prediction-mediated emergence of drug resistance

abstract: Drug resistance is a major problem in treatment of microbial infections and cancers. There is growing evidence that a transient drug tolerant state may precede and potentiate the emergence of drug resistance. Therefore, understanding the mechanisms leading to tolerance is critical for combating drug resistance and for the development of effective therapeutic strategy. Through laboratory evolution of yeast, we recently demonstrated that adaptive prediction (AP), a strategy employed by organisms to anticipate and prepare for a future stressful environment, can emerge within 100 generations by linking the response triggered by a neutral cue (caffeine) to a mechanism of protection against a lethal agent (5-FOA). Here, we demonstrate that mutations selected across multiple laboratory evolved lines had linked the neutral cue response to core genes of autophagy. Across these evolved lines, conditional activation of autophagy through AP conferred tolerance, and potentiated subsequent selection of mutations in genes specific to overcoming the toxicity of 5-FOA. We propose a model to explain how extensive genome-wide genetic interactions of autophagy facilitates emergence of AP over short evolutionary timescales to potentiate selection of resistance-conferring mutations.


title: Characterization of Poldip2 knockout mice: avoiding incorrect gene targeting

abstract: POLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from two limitations: perinatal lethality in homozygotes and constitutive Poldip2 inactivation. To overcome these limitations, we developed a new conditional floxed Poldip2 model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication around Poldip2 with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxed Poldip2 mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects of Poldip2 ablation in the vasculature, an RNA-seq experiment was performed in constitutive knockout carotid arteries. Results support the involvement of POLDIP2 in multiple cellular processes and provide new opportunities for future in-depth study of its functions.

category: genetics


title: Chimpanzees use numerous flexible vocal sequences with more than two vocal units: A step towards language?

abstract: A major question in evolutionary science is how did language evolve? Syntax, as the core of language, combines meaning-bearing units (words) into hierarchical structures, thereby creating new meanings. Some other mammals and birds combine meaning-bearing vocalisations, but no documented examples exist of non-human animals combining more than two meaning-bearing vocalisations. Was the two-unit threshold only surpassed in the hominid lineage? Here, we examine the positional patterning of vocal sequences of chimpanzees. We analysed 4826 vocal utterances of 46 wild adult female and male chimpanzees. We found a flexible system with 390 multi-unit vocal sequences, some showing positional or transitional regularities. Two-unit pairs embedded in three-unit sequences predictably occurred either in head or tail positions, and co-occurred with specific other elements. The capacity to organise vocal output beyond the two-unit level may thus exist in species other than humans and could be viewed as an important evolutionary step towards language.

category: animal behavior and cognition


title: Specialization and selective social attention establishes the balance between individual and social learning

abstract: A key question individuals face in any social learning environment is when to innovate alone and when to imitate others. Previous simulation results have found that the best performing groups exhibit an intermediate balance, yet it is still largely unknown how individuals collectively negotiate this balance. We use an immersive collective foraging experiment, implemented in the Minecraft game engine, facilitating unprecedented access to spatial trajectories and visual field data. The virtual environment imposes a limited field of view, creating a natural trade-off between allocating visual attention towards individual search or to look towards peers for social imitation. By analyzing foraging patterns, social interactions (visual and spatial), and social influence, we shine new light on how groups collectively adapt to the fluctuating demands of the environment through specialization and selective imitation, rather than homogeneity and indiscriminate copying of others.


title: Lateral Undulation Aids Biological and Robotic Earthworm Anchoring and Locomotion

abstract: Earthworms (Lumbricus terrestris) are characterized by soft, highly flexible and extensible bodies, and are capable of locomoting in most terrestrial environments. Previous studies of earthworm movement have focused on the use of retrograde peristaltic gaits in which controlled contraction of longitudinal and circular muscles results in waves of shortening/thickening and thinning/lengthening of the hydrostatic skeleton. These waves can propel the animal across ground as well as into soil. However, worms can also benefit from axial body bends during locomotion. Such lateral undulation dynamics can aid locomotor function via hooking/anchoring (to provide propulsion), modify travel orientation (to avoid obstacles and generate turns) and even generate snake-like undulatory locomotion in environments where peristaltic locomotion results in poor performance. To the best of our knowledge, the important aspects of locomotion associated with the lateral undulation of an earthworm body are yet to be systematically investigated. In this study, we observed that within confined environments, the worm uses lateral undulation to anchor its body to the walls of their burrows and tip (nose) bending to search the environment. This relatively simple locomotion strategy drastically improved the performance of our soft bodied robophysical model of the earthworm both in a confined (in an acrylic tube) and above-ground heterogeneous environment (rigid pegs), where the peristaltic gait often fails. In summary, lateral undulation facilitates the mobility of earthworm locomotion in diverse environments and can play an important role in the creation of low cost soft robotic devices capable of traversing a variety of environments.


title: Enzyme kinetics of CRISPR molecular diagnostics

abstract: CRISPR diagnostic assays have gained significant interest in the last few years. This interest has grown rapidly during the current COVID-19 pandemic where CRISPR diagnostics have been frontline contenders for rapid testing solutions. This surge in CRISPR diagnostics research prompts the following question: What exactly are the achievable limits of detection and associated assay times enabled by the kinetics of Cas12 and Cas13 enzymes? To address this question, we here present a model based on Michaelis-Menten enzyme kinetics theory applied to Cas enzymes. We use the model to develop analytical solutions for reaction kinetics and develop back-of-the{-} envelope criteria to validate and check for consistency in reported enzyme kinetics parameters. We applied our analyses to all studies known to us which report Michaelis-Menten-type kinetics data for CRISPR associated enzymes. These studies include all subtypes of Cas12 and Cas13 and orthologs. We found all studies but one clearly violate at least two of our three rules of consistency. We further use our model to explore ranges of reaction time scales and degree of reaction completion for practically relevant target concentrations applicable to CRISPR-diagnostic assays.

category: bioengineering


title: Effect of air pockets in drug delivery in jet injections

abstract: Needle-free jet injections are actuated by a pressure impulse that can be delivered by different mechanisms, and the resultant jets are [Formula](102) m/s. Here, we report on the effect of entrapped air bubbles since filling procedures for pre-filled ampoules can induce bubbles, especially for viscous fluids. We use spring-piston devices as the principal actuation mechanism and vary both the location and size of the initial bubble. We find that the bubble location does have a statistically significant (p < 0.05) effect on the jet exit speed, based upon the volumetric flow rate. However, we reveal subtle features such as intermittent atomization when the gas pockets pass through the orifice and de-pressurize, which leads to spray formation and a temporary increase in jet dispersion, both of which can lead to product loss during an injection. These results have implications for the development of prefilled ampoules for jet injection applications.


title: Improving deep learning-based protein distance prediction in CASP14

abstract: Accurate prediction of residue-residue distances is important for protein structure prediction. We developed several protein distance predictors based on a deep learning distance prediction method and blindly tested them in the 14th Critical Assessment of Protein Structure Prediction (CASP14). The prediction method uses deep residual neural networks with the channel-wise attention mechanism to classify the distance between every two residues into multiple distance intervals. The input features for the deep learning method include co-evolutionary features as well as other sequence-based features derived from multiple sequence alignments (MSAs). Three alignment methods are used with multiple protein sequence/profile databases to generate MSAs for input feature generation. Based on different configurations and training strategies of the deep learning method, five MULTICOM distance predictors were created to participate in the CASP14 experiment. Benchmarked on 37 hard CASP14 domains, the best performing MULTICOM predictor is ranked 5th out of 30 automated CASP14 distance prediction servers in terms of precision of top L/5 long-range contact predictions (i.e. classifying distances between two residues into two categories: in contact (< 8 Angstrom) and not in contact otherwise) and performs better than the best CASP13 distance prediction method. The best performing MULTICOM predictor is also ranked 6th among automated server predictors in classifying inter-residue distances into 10 distance intervals defined by CASP14 according to the F1 measure. The results show that the quality and depth of MSAs depend on alignment methods and sequence databases and have a significant impact on the accuracy of distance prediction. Using larger training datasets and multiple complementary features improves prediction accuracy. However, the number of effective sequences in MSAs is only a weak indicator of the quality of MSAs and the accuracy of predicted distance maps. In contrast, there is a strong correlation between the accuracy of contact/distance predictions and the average probability of the predicted contacts, which can therefore be more effectively used to estimate the confidence of distance predictions and select predicted distance maps.


title: Sorghum pan-genome explores the functional utility to accelerate the genetic gain

abstract: Sorghum (Sorghum bicolor L.) is one of the most important food crops in the arid and rainfed production ecologies. It is a part of resilient farming and is projected as a smart crop to overcome the food and nutritional challenges in the developing world. The development and characterisation of the sorghum pan-genome will provide insight into genome diversity and functionality, supporting sorghum improvement. We built a sorghum pan-genome using reference genomes as well as 354 genetically diverse sorghum accessions belonging to different races. We explored the structural and functional characteristics of the pan-genome and explain its utility in supporting genetic gain. The newly-developed pan-genome has a total of 35,719 genes, a core genome of 16,821 genes and an average of 32,795 genes in each cultivar. The variable genes are enriched with environment responsive genes and classify the sorghum accessions according to their race. We show that 53% of genes display presence-absence variation, and some of these variable genes are predicted to be functionally associated with drought traits. Using more than two million SNPs from the pan-genome, association analysis identified 398 SNPs significantly associated with important agronomic traits, of which, 92 were in genes. Drought gene expression analysis identified 1,788 genes that are functionally linked to different conditions, of which 79 were absent from the reference genome assembly. This study provides comprehensive genomic diversity resources in sorghum which can be used in genome assisted crop improvement.


title: SpatialDWLS: accurate deconvolution of spatial transcriptomic data

abstract: Recent development of spatial transcriptomic technologies has made it possible to systematically characterize cellular heterogeneity while preserving spatial information, which greatly enables the investigation of structural organization of a tissue and its impact on modulating cellular behavior. On the other hand, the technology often does not have sufficient resolution to distinguish neighboring cells which may belong to different cell types, therefore it is difficult to identify cell-type distribution directly from the data. To overcome this challenge, we have developed a computational method, called spatialDWLS, to quantitatively estimate the cell-type composition at each spatial location. We benchmarked the performance of spatialDWLS by comparing with a number of existing deconvolution methods using both real and simulated datasets, and we found that spatialDWLS outperformed the other methods in terms of accuracy and speed. By applying spatialDWLS to analyze a human developmental heart dataset, we observed striking spatial-temporal changes of cell-type composition which becomes increasing spatially coherent during development. As such, spatialDWLS provides a valuable computational tool for faithfully extracting biological information from spatial transcriptomic data.


title: Three-dimensional interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis

abstract: Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art  HPV integrated site capture (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a  looping mechanism by which flanking host regions become amplified. Furthermore, using our  HPV16-specific Region Capture Hi-C technique, we have determined that three-dimensional (3D) interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive host gene dysregulation through the disruption of local host:host 3D interactions known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a  cancer-causing gene is not essential to influence such genes within an entire TAD and that these modifications to 3D interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

category: cancer biology


title: Voltage-Gated Potassium Channel Kv1.3 as a Therapeutic Target for Pancreatic Ductal Adenocarcinoma

abstract: The mitochondrial voltage-gated potassium channel, Kv1.3, has been emerged as an attractive oncologic target but its function in pancreas cancer (PDAC) is unknown. In this study we evaluated tissue expression of Kv1.3 in resected PDAC from 55 patients and tumor inhibition in orthotopic mouse models using the recently developed Kv1.3 inhibitors PCARBTP and PAPTP. Immunohistochemistry of 55 human PDAC specimens showed that all tumors expressed Kv1.3 with 60% of tumor specimens having high Kv1.3 expression. In pancreas tumor models (Pan02 cells injected into C57BL/6 mice), PCARBTP and PAPTP treatment resulted in tumor reductions of 87% and 70%, respectively. When combined with gemcitabine/abraxane, this increased to 95% and 80% without resultant organ toxicity. In vivo models indicated PCARBTP-mediated cell death occurred through the p38-MAPK pathway. In vitro-generated resistant clones to PCARBTP escaped cell death through upregulation of the anti-oxidant system as determined using SWATH-MS analysis. These data show Kv1.3 is highly expressed in resected human PDAC and the use of novel mitochondrial Kv1.3 inhibitors combined with cytotoxic chemotherapies might be novel, effective treatment for PDAC.


title: Redefining tumor classification and clinical stratification through a colorectal cancer single-cell atlas

abstract: Colorectal cancer (CRC), a disease of high incidence and mortality, has had few treatment advances owing to a large degree of inter- and intratumoral heterogeneity. Attempts to classify subtypes of colorectal cancer to develop treatment strategies has been attempted by Consensus Molecular Subtypes (CMS) classification. However, the cellular etiology of CMS classification is incompletely understood and controversial. Here, we generated and analyzed a single-cell transcriptome atlas of 49,859 CRC cells from 16 patients, validated with an additional 31,383 cells from an independent CRC patient cohort. We describe subclonal transcriptomic heterogeneity of CRC tumor epithelial cells, as well as discrete stromal populations of cancer-associated fibroblasts (CAFs). Within CRC CAFs, we identify the transcriptional signature of specific subtypes (CAF-S1 and CAF-S4) in more than 1,500 CRC patients using bulk transcriptomic data that significantly stratifies overall survival in multiple independent cohorts. We also uncovered two CAF-S1 subpopulations, ecm-myCAF and TGF{beta}-myCAF, known to be associated with primary resistance to immunotherapies. We demonstrate that scRNA analysis of malignant, stromal, and immune cells exhibit a more complex picture than portrayed by bulk transcriptomic-based Consensus Molecular Subtypes (CMS) classification. By demonstrating an abundant degree of heterogeneity amongst these cell types, our work shows that CRC is best represented in a transcriptomic continuum crossing traditional classification systems boundaries. Overall, this CRC cell map provides a framework to re-evaluate CRC tumor biology with implications for clinical trial design and therapeutic development.


title: Redefining colorectal cancer classification and clinical stratification through a single-cell atlas


title: Comparison of starch granule sizes of maize seeds between parental lines and derived hybrids

abstract: Maize starch is an important agricultural commodity that serves as food, feed, and a raw material for industrial purposes. It is organized into starch granules (SG) inside amyloplasts and is highly accumulated in endosperms. Maize hybrids exhibit heterosis over their parents. However, the parental effect on the size of SG in F1 hybrid seeds remains unclear. Here we compared the seed SG sizes among two parental inbred lines (Chang7-2 and Zheng58) as well as their reciprocal hybrids. SG was observed in situ and in vitro with SEM. The size of seed SG in hybrids was more like that of female parents, especially for large SG population. Thus, the control of SG size exhibits a maternal inheritance trend in the context of plastid (amyloplast) inheritance. Our results provide some insight on selecting parental inbred lines for breeding maize hybrids with different SG sizes.


title: Dynamic histone acetylation in floral volatile synthesis and emission in petunia flowers

abstract:
Biosynthesis of secondary metabolites relies on primary metabolic pathways to provide precursors, energy, and cofactors, thus requiring coordinated regulation of primary and secondary metabolic networks. However, to date it remains largely unknown how this coordination is achieved. Using Petunia hybrida flowers, which emit high levels of phenylpropanoid/benzenoid volatile organic compounds (VOCs), we uncovered genome-wide dynamic deposition of histone H3 lysine 9 acetylation (H3K9ac) during anthesis as an underlying mechanism to coordinate primary and secondary metabolic networks. The observed epigenome reprogramming is accompanied by transcriptional activation, at gene loci involved in primary metabolic pathways that provide precursor phenylalanine, as well as secondary metabolic pathways to produce volatile compounds. We also observed transcriptional repression among genes involved in alternative phenylpropanoid branches that compete for metabolic precursors. We show that GNAT family histone acetyltransferase(s) (HATs) are required for the expression of genes involved in VOC biosynthesis and emission, by using chemical inhibitors of HATs, and by knocking down a specific HAT, ELP3, through transient RNAi. Together, our study supports that chromatin level regulatory mechanisms may play an essential role in activating primary and secondary metabolic pathways to regulate VOC synthesis in petunia flowers.

HIGHLIGHTOur study shows that posttranslational modification of histones is essential for regulating the biosynthesis and emission of floral scent compounds, thus providing insights into chromatin level regulation of secondary metabolism.


title: The effect of mentee and mentor gender on scientific productivity of applicants for NIH training fellowships

abstract: Several studies have suggested that women in science are less productive than men, and that this gap contributes to their under-representation in the ranks of senior researchers. However, few studies have examined the role of mentoring, and in particular mentor gender, on the productivity of female scientists early in their careers. Such efforts are limited by the difficulties of unambiguously linking mentees to their mentors and measuring the research productivity resulting from those relationships. Here we use our novel author disambiguation solution to investigate the role of self-identified gender in mentorship of 12,932 trainees who either successfully or unsuccessfully applied to the National Institutes of Health for research fellowships between fiscal years 2011 and 2017, applying a multi-dimensional framework to assess productivity. We found that, after normalizing for the funding level of mentors, the productivity of female and male mentees is indistinguishable; it is also independent of the gender of the mentor, other than in measures of clinical impact, where women mentored by women outperform other mentee-mentor dyads.

category: scientific communication and education


title: High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

abstract: Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the {Delta}gacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.

category: systems biology


title: Effects of awareness and task relevance on neurocomputational models of mismatch negativity generation

abstract: Detection of regularities and their violations in sensory input is key to perception. Violations are indexed by an early EEG component called the mismatch negativity (MMN) - even if participants are distracted or unaware of the stimuli. On a mechanistic level, two dominant models have been suggested to contribute to the MMN: adaptation and prediction. Whether and how context conditions, such as awareness and task relevance, modulate the mechanisms of MMN generation is unknown. We conducted an EEG study disentangling influences of task relevance and awareness on the visual MMN. Then, we estimated different computational models for the generation of single-trial amplitudes in the MMN time window. Amplitudes were best explained by a prediction error model when stimuli were task-relevant but by an adaptation model when task-irrelevant and unaware. Thus, mismatch generation does not rely on one predominant mechanism but mechanisms vary with task relevance of stimuli.


title: Brain2Pix: Fully convolutional naturalistic video reconstruction from brain activity

abstract: Reconstructing complex and dynamic visual perception from brain activity remains a major challenge in machine learning applications to neuroscience. Here we present a new method for reconstructing naturalistic images and videos from very large single-participant functional magnetic resonance imaging data that leverages the recent success of image-to-image transformation networks. This is achieved by exploiting spatial information obtained from retinotopic mappings across the visual system. More specifically, we first determine what position each voxel in a particular region of interest would represent in the visual field based on its corresponding receptive field location. Then, the 2D image representation of the brain activity on the visual field is passed to a fully convolutional image-to-image network trained to recover the original stimuli using VGG feature loss with an adversarial regularizer. In our experiments, we show that our method offers a significant improvement over existing video reconstruction techniques.


title: Central inhibition of P2Y12R differentially regulates survival and neuronal loss in MPTP-induced Parkinsonism in mice

abstract: Parkinsons disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12 receptor (P2Y12R), which is exclusively expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that while pharmacological or genetic targeting of P2Y12R previous to disease onset augments acute mortality, these interventions protect against neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulate ROCK and p38 MAPK activity and control cytokine production. Understanding protective and detrimental P2Y12 receptor-mediated actions in the CNS may reveal novel approaches to control neuroinflammation and modify disease progression in PD.


title: Compensatory variability in network parameters enhances memory performance in the Drosophila mushroom body

abstract:
Neural circuits use homeostatic compensation to achieve consistent behaviour despite variability in underlying intrinsic and network parameters. However, it remains unclear how compensation regulates variability across a population of the same type of neurons within an individual, and what computational benefits might result from such compensation. We address these questions in the Drosophila mushroom body, the flys olfactory memory center. In a computational model, we show that memory performance is degraded when the mushroom bodys principal neurons, Kenyon cells (KCs), vary realistically in key parameters governing their excitability, because the resulting inter-KC variability in average activity levels makes odor representations less separable. However, memory performance is rescued while maintaining realistic variability if parameters compensate for each other to equalize KC average activity. Such compensation can be achieved through both activity-dependent and activity-independent mechanisms. Finally, we show that correlations predicted by our models compensatory mechanisms appear in the Drosophila hemibrain connectome. These findings reveal compensatory variability in the mushroom body and describe its computational benefits for associative memory.

Significance statementHow does variability between neurons affect neural circuit function? How might neurons behave similarly despite having different underlying features? We addressed these questions in neurons called Kenyon cells, which store olfactory memories in flies. Kenyon cells differ among themselves in key features that affect how active they are, and in a model of the flys memory circuit, adding this inter-neuronal variability made the model fly worse at learning the values of multiple odors. However, memory performance was rescued if compensation between the variable underlying features allowed Kenyon cells to be equally active on average, and we found the hypothesized compensatory variability in real Kenyon cells anatomy. This work reveals the existence and computational benefits of compensatory variability in neural networks.


title: Mechanoreceptor synapses in the brainstem shape the central representation of touch

abstract: Mammals use glabrous (hairless) skin of their hands and feet to navigate and manipulate their environment. Cortical maps of the body surface across species contain disproportionately large numbers of neurons dedicated to glabrous skin sensation, potentially reflecting a higher density of mechanoreceptors that innervate these skin regions. Here, we find that disproportionate representation of glabrous skin emerges over postnatal development at the first synapse between peripheral mechanoreceptors and their central targets in the brainstem. Mechanoreceptor synapses undergo developmental refinement that depends on proximity of their terminals to glabrous skin, such that those innervating glabrous skin make synaptic connections that expand their central representation. In mice that do not sense gentle touch, mechanoreceptors innervating glabrous skin still make more powerful synaptic connections in the brainstem. We propose that the skin region a mechanoreceptor innervates controls refinement of its central synapses over development to shape the representation of touch in the brain.


title: Down Regulation Of IL-1β Secretion By TGF-β1 In Macrophages Infected With Dengue Virus

abstract: Several pathogenic mechanisms have been linked to the severity of dengue virus infection, like viral cytotoxicity, underlying host genetics and comorbidities such as diabetes and dyslipidemia. It has been observed that patients with severe manifestations develop an uncontrolled immune response, with an increase in pro-inflammatory cytokines such as TNF, IL-1{beta}, IL-8, IL-6 and chemokines that damage the human microvascular endothelium, and also in anti-inflammatory cytokines IL-4, IL-10 and TGF-{beta}1. The role of TGF-{beta}1 on dengue is not clear; few studies have been published, and most of them from patient sera data, with both protective and pathological roles have described. The aim of this study was to evaluate the ability of TGF-{beta}1 to regulate the secretion of IL-1{beta} in macrophages infected by DENV using THP-1 cells treated with recombinant TGF-{beta}1 before or after DENV infection. By RT-PCR we did not observe a difference in IL-1{beta} expression between infected cells pretreated with TGF-{beta}1 and those that were not. However, secretion of IL-1{beta} was reduced only in cells stimulated with TGF-{beta}1 before infection, and not in those treated 2 hours post-infection. TGF-{beta}1 receptor blockage with SB505124 inhibitor, prior to the addition of TGF-{beta}1 and infection, abrogated the inhibitory effect of TGF-{beta}1. Our results suggest that DENV could regulate the function of TGF-{beta}1 on macrophages. This negative regulation of the TGF-{beta}1 pathway could be used by DENV to evade the immune response and could contribute to the immunopathology.

category: immunology


title: MTHFD2 is a Metabolic Checkpoint Controlling Effector and Regulatory T Cell Fate and Function

abstract: Antigenic stimulation promotes T cells metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme Methylenetetrahydrofolate Dehydrogenase-2 (MTHFD2) is highly expressed in inflammatory diseases and induced in activated T cells to promote proliferation and produce inflammatory cytokines. In pathogenic Th17 cells, MTHFD2 also prevented aberrant upregulation of FoxP3 and suppressive capacity. Conversely, MTHFD2-deficiency enhanced lineage stability of regulatory T (Treg) cells. Mechanistically, MTHFD2 maintained cellular 10-formyltetrahydrofolate for de novo purine synthesis and MTHFD2 inhibition led to accumulation of the intermediate 5-aminoimidazole carboxamide ribonucleotide that was associated with decreased mTORC1 signaling. MTHFD2 was also required for proper histone de-methylation in Th17 cells. Importantly, inhibiting MTHFD2 in vivo reduced disease severity in Experimental Autoimmune Encephalomyelitis and Delayed-Type Hypersensitivity. MTHFD2 induction is thus a metabolic checkpoint for pathogenic effector cells that suppresses anti-inflammatory Treg cells and is a potential therapeutic target within 1C metabolism.


title: Structural variability and concerted motions of the T cell receptor - CD3 complex

abstract: We investigate the structural and orientational variability of the membrane-embedded T cell receptor (TCR) - CD3 complex in extensive atomistic molecular dynamics simulations based on the recent cryo-EM structure determined by Dong et al. (2019). We find that the TCR extracellular (EC) domain is highly variable in its orientation by attaining tilt angles relative to the membrane normal that range from 15{degrees} to 55{degrees}. The tilt angle of the TCR EC domain is both coupled to a rotation of the domain and to characteristic changes throughout the TCR - CD3 complex, in particular in the EC interactions of the C{beta} FG loop of the TCR, as well as in the orientation of transmembrane helices. The concerted motions of the membrane-embedded TCR - CD3 complex revealed in our simulations provide atomistic insights for force-based models of TCR activation, which involve such structural changes in response to tilt-inducing forces on antigen-bound TCRs.


title: In vivo antigen expression regulates CD4 T cell differentiation and vaccine efficacy against Mycobacterium tuberculosis infection

abstract:
New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to IFN-{gamma} or nutrient/oxygen deprivation of in vitro infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analysed their corresponding CD4 T cell phenotype and vaccine-protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination and, against the overexpressing strain, vaccination with MPT70 conferred similar protection as ESAT-6. Together our data indicate that high in vivo antigen expression drives T cells towards terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less-differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune-balance in favor of the host.

ImportanceTuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current COVID-19 pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunisation with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.


title: A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

abstract: Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique KmAgo nuclease from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo preferentially binds 16-20 nt long 5'-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.


title: A biomechanical switch regulates the transition towards homeostasis in esophageal epithelium

abstract: Epithelial cells are highly dynamic and can rapidly adapt their behavior in response to tissue perturbations and increasing tissue demands. However, the processes that finely control these responses and, particularly, the mechanisms that ensure the correct switch to and from normal tissue homeostasis are largely unknown. Here we explore changes in cell behavior happening at the interface between postnatal development and homeostasis in the epithelium of the mouse esophagus, as a physiological model exemplifying a rapid but controlled tissue growth transition. Single cell RNA sequencing and histological analysis of the mouse esophagus reveal significant mechanical changes in the epithelium upon tissue maturation. Organ stretching experiments further indicate that tissue strain caused by the differential growth of the mouse esophagus relative to the entire body promotes the emergence of a defined committed population in the progenitor compartment as homeostasis is established. Our results point to a simple mechanism whereby the mechanical changes experienced at the whole tissue level are integrated with those "sensed" at the cellular level to control epithelial cell behavior and tissue maintenance.

category: cell biology


title: Frequent coexistence of closely related tree species on a small scale in temperate evergreen forests in Japan

abstract: Understanding how biotic interaction affects species composition and distribution is a major challenge in community ecology. In plants, negative reproductive interaction among closely related species (i.e., reproductive interference) is known to hamper the coexistence of congenic species. Since the magnitude of reproductive interference in plants depends on pollen flow distance, we hypothesized that the coexistence of congeners on a small spatial scale would be less likely to occur by chance but that such coexistence would be likely to occur on a scale larger than pollen flow distance. In the present study, we tested this hypothesis using spatially explicit woody plant survey data. Contrary to our prediction, congenic tree species often coexisted at the finest spatial scale and significant exclusive distribution was not detected. Our results suggest that cooccurrence of congenic tree species is not structured by reproductive interference, and they indicate the need for further research to explore the factors that mitigate the effects of reproductive interference.


title: Energy-harnessing problem solving of primordial life: modeling the emergence of catalytic host-parasite life cycles

abstract: All life forms on earth ultimately descended from a primordial population dubbed the last universal common ancestor or LUCA via Darwinian evolution. Extant living systems share two salient functional features, a metabolism extracting and transforming energy required for survival, and an evolvable, informational polymer - the genome - conferring heredity. Genome replication invariably generates essential and ubiquitous genetic parasites. Here we model the energetic, replicative conditions of LUCA-like organisms and their parasites, as well as adaptive problem solving of host-parasite pairs. We show using the Lotka-Volterra equations that three host-parasite pairs - individually a unit of a host and a parasite that is itself parasitized - are sufficient for robust and stable homeostasis, forming a life cycle. This catalytic life cycle efficiently captures, channels and transforms energy, enabling dynamic host survival and adaptation. We propose a Malthusian fitness model for an original quasispecies evolving through a host-parasite life cycle.


title: Mutational effects of ubiquitously present gamma radiation on Arabidopsis thaliana: insight into radiosensitivity in the reproductive stage

abstract: Earth has always been exposed to ionizing radiation from natural sources, and man-made sources have added to this radiation. In order to assess mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life-cycle of Arabidopsis thaliana under controlled conditions. Resequencing data from 12 M1 lines and 36 M2 progeny derived under gamma-irradiation conditions ranging from 0.0 to 2.0 Gy/d were obtained to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and a low-to-middling dose of radiation was assessed by statistical modeling. The increasing of de novo mutations in response to doses of irradiation fit the negative binomial model, accounting for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, with deletions being more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation are more intensive during the reproductive stage. These outcomes could provide valuable insights into practical strategies for environmental radioprotection of plants on Earth and in space.


title: APICURON: a database to credit and acknowledge the work of biocurators

abstract:
APICURON is an open and freely accessible resource that tracks and credits the work of biocurators across multiple participating knowledgebases. Biocuration is essential to extract knowledge from research data and make it available in a structured and standardized way to the scientific community. However, processing biological data - mainly from literature - requires a huge effort which is difficult to quantify and acknowledge. APICURON collects biocuration events from third party resources and aggregates this information, spotlighting biocurator contributions. APICURON promotes biocurator engagement implementing gamification concepts like badges, medals and leaderboards and at the same time provides a monitoring service for registered resources and for biocurators themselves. APICURON adopts a data model that is flexible enough to represent and track the majority of biocuration activities. Biocurators are identified through their ORCID. The definition of curation events, scoring systems and rules for assigning badges and medals are resource-specific and easily customizable. Registered resources can transfer curation activities on the fly through a secure and robust Application Programming Interface (API). Here we show how simple and effective it is to connect a resource to APICURON describing the DisProt database of intrinsically disordered proteins as a use case. We believe APICURON will provide biological knowledgebases with a service to recognize and credit the effort of their biocurators, monitor their activity and promote curators engagement.

Database URL: https://apicuron.org


title: DeepDRIM: a deep neural network to reconstruct cell-type-specific gene regulatory network using single-cell RNA-seq data

abstract: AO_SCPLOWBSTRACTC_SCPLOWSingle-cell RNA sequencing is used to capture cell-specific gene expression, thus allowing reconstruction of gene regulatory networks. The existing algorithms struggle to deal with dropouts and cellular heterogeneity, and commonly require pseudotime-ordered cells. Here, we describe DeepDRIM a supervised deep neural network that represents gene pair joint expression as images and considers the neighborhood context to eliminate the transitive interactions. Deep-DRIM yields significantly better performance than the other nine algorithms used on the eight cell lines tested, and can be used to successfully discriminate key functional modules between patients with mild and severe symptoms of coronavirus disease 2019 (COVID-19).


title: Co-linear chaining with overlaps and gap costs

abstract: Co-linear chaining has proven to be a powerful heuristic for finding near-optimal alignments of long DNA sequences (e.g., long reads or a genome assembly) to a reference. It is used as an intermediate step in several alignment tools that employ a seed-chain-extend strategy. Despite this popularity, efficient subquadratic-time algorithms for the general case where chains support anchor overlaps and gap costs are not currently known. We present algorithms to solve the co-linear chaining problem with anchor overlaps and gap costs in O(n) time, where n denotes the count of anchors. We also establish the first theoretical connection between co-linear chaining cost and edit distance. Specifically, we prove that for a fixed set of anchors under a carefully designed chaining cost function, the optimal  anchored edit distance equals the optimal co-linear chaining cost. Finally, we demonstrate experimentally that optimal co-linear chaining cost under the proposed cost function can be computed orders of magnitude faster than edit distance, and achieves correlation coefficient above 0.9 with edit distance for closely as well as distantly related sequences.


title: Diffusion of the disordered E-cadherin tail on β-catenin

abstract: Specific protein interactions typically require well-shaped binding interfaces. Here, we report a cunning exception. The disordered tail of the cell-adhesion protein E-cadherin dynamically samples a large surface area of the proto-oncogene {beta}-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without dissociation of the complex. A few persistent interactions provide specificity whereas unspecific contacts boost affinity. The energy landscape of this complex is rugged with many small barriers (3 - 4 kBT) and reconciles specificity, high affinity, and extreme disorder. Given the roles of {beta}-catenin in cell-adhesion, signalling, and cancer, this Velcro-like design has the potential to tune the stability of the complex without requiring dissociation.

category: biophysics


title: Multiscale three-dimensional imaging of intact human organs down to the cellular scale using hierarchical phase-contrast tomography

abstract: Human organs are complex, three-dimensional and multiscale systems. Spatially mapping the human body down through its hierarchy, from entire organs to their individual functional units and specialised cells, is a major obstacle to fully understanding health and disease. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique utilising the European Synchrotron Radiation Facilitys Extremely Brilliant Source: the worlds first high-energy 4th generation X-ray source. HiP-CT enabled three-dimensional and non-destructive imaging at near-micron resolution in soft tissues at one hundred thousand times the voxel size whilst maintaining the organs structure. We applied HiP-CT to image five intact human parenchymal organs: brain, lung, heart, kidney and spleen. These were hierarchically assessed with HiP-CT, providing a structural overview of the whole organ alongside detail of the organs individual functional units and cells. The potential applications of HiP-CT were demonstrated through quantification and morphometry of glomeruli in an intact human kidney, and identification of regional changes to the architecture of the air-tissue interface and alveolar morphology in the lung of a deceased COVID-19 patient. Overall, we show that HiP-CT is a powerful tool which can provide a comprehensive picture of structural information for whole intact human organs, encompassing precise details on functional units and their constituent cells to better understand human health and disease.

category: physiology


title: The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

abstract:
The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding transcription activator-like effector AvrBs3 from the bacterial pathogen Xanthomonas. Bs3 is homologous to flavin monooxygenases (FMOs), an enzyme class that has NADPH oxidase activity and can produce H2O2, a hallmark metabolite in plant immune reactions. Histochemical staining of infected pepper leaves and a translational fusion of Bs3 to the redox reporter roGFP2 both indicated that the Bs3-dependent HR induces a local increase in H2O2 levels in planta. Moreover, our in vitro studies with recombinant Bs3 protein confirmed its NADPH oxidase activity. To test if the NADPH oxidation of Bs3 induces HR, we adapted previous studies which have uncovered mutations in fungal FMOs that result in higher NADPH oxidase activity. We replicated one of these mutations and demonstrated that the generated recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3 in vitro. Translational fusions to roGFP2 showed that Bs3S211A also had increased NADPH oxidase activity in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta. Ultimately, this reveals that Bs3 produces H2O2 in planta, but that the H2O2 produced by Bs3 on its own is not sufficient to trigger HR. We also demonstrated that expression of Bs3 not only triggered HR in plants, but also inhibited proliferation of yeast, which lends this model system to be utilized for the genetic dissection of Bs3 function in future studies.

One sentence summaryThe executor-type resistance protein Bs3 from pepper (Capsicum annuum) acts as an NADPH oxidase but reactive oxygen species produced by Bs3 are not sufficient to trigger plant cell death


title: Improved alpha-beta power reduction via combined Electrical and Ultrasonic stimulation in a Parkinsonian Cortex-Basal Ganglia-Thalamus Computational Model

abstract:
ObjectiveTo investigate computationally the interaction of combined electrical and ultrasonic modulation of isolated neurons and of the Parkinsonian cortex-basal ganglia-thalamus loop.

MethodsContinuous-wave or pulsed electrical and ultrasonic neuromodulation is applied to isolated Otsuka plateau-potential generating subthalamic nucleus (STN) and Pospischil regular, fast and low-threshold spiking cortical cells in a temporally-alternating or simultaneous manner. Similar combinations of electrical/ultrasonic waveforms are applied to a Parkinsonian biophysical cortex-basal ganglia-thalamus neuronal network. Ultrasound-neuron interaction is modelled respectively for isolated neurons and the neuronal network with the NICE and SONIC implementations of the bilayer sonophore underlying mechanism. Reduction in --{beta} spectral energy is used as a proxy to express improvement in Parkinsons disease by insonication and electrostimulation.

ResultsSimultaneous electro-acoustic stimulation achieves a given level of neuronal activity at lower intensities compared to the separate stimulation modalities. Conversely, temporally alternating stimulation with 50 Hz electrical and ultrasound pulses is capable of eliciting 100 Hz STN firing rates. Furthermore, combination of ultrasound with hyperpolarizing currents can alter cortical cell relative spiking regimes. In the Parkinsonian neuronal network, high-frequency pulsed separated electrical and ultrasonic deep brain stimulation (DBS) reduce pathological  -- {beta} power by entraining STN-neurons. In contrast, continuous-wave ultrasound reduces pathological oscillations by silencing the STN. Compared to the separated stimulation modalities, temporally simultaneous or alternating electro-acoustic stimulation can achieve higher reductions in  -- {beta} power for the same contraints on electrical/ultrasonic intensity.

ConclusionContinuous-wave and pulsed ultrasound reduce pathological oscillations by different mechanisms. Electroacoustic stimulation further improves -- {beta} power for given safety limits and is capable of altering cortical relative spiking regimes.

Significancefocused ultrasound has the potential of becoming a non-invasive alternative of conventional DBS for the treatment of Parkinsons disease. Here, we elaborate on proposed benefits of combined electro-acoustic stimulation in terms of improved dynamic range, efficiency, resolution, and neuronal selectivity.


title: LIFE AND WORK OF RESEARCHERS TRAPPED IN THE COVID-19PANDEMIC VICIOUS CYCLE

abstract: AO_SCPLOWBSTRACTC_SCPLOWCOVID-19 has disrupted researchers work and posed challenges to their life routines. We have surveyed 740 researchers of which 66% experienced a decrease in productivity, 50% indicated increased workload, and 66% reported they have been feeling internal pressure to make progress. Those whose research required physical presence in a lab or the field experienced considerable disruption and productivity decrease. About 82% of this group will try to permanently reduce their work dependency on physical presence. Parents and those taking care of vulnerable dependents have been spending less time on research due to their role conflict. We further observed a gender gap in the overall disruption consequences; more female researchers have been experiencing a reduction in productivity and external pressure to make progress. The results of this study can help institution leaders and policymakers better understand the pandemics challenges for the research community and motivate appropriate measures to instill long-term solutions.


title: Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain

abstract: The COVID-19 pandemic caused by SARS-CoV-2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of the SARS-CoV-2 spike protein receptor binding domain using the CyDisCo system to create and maintain the correct disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce functional, active antigen in bacteria capable of recognizing and binding to the ACE2 (angiotensin-converting enzyme) receptor as well as antibodies in COVID-19 patient sera.

category: biochemistry


title: CovRadar: Continuously tracking and filtering SARS-CoV-2 mutations for molecular surveillance

abstract:
The SARS-CoV-2 pandemic underlined the importance of molecular surveillance to track the evolution of the virus and inform public health interventions. Fast analysis, easy visualization and convenient filtering of the latest virus sequences are essential for this purpose. However, access to computational resources, the lack of bioinformatics expertise, and the sheer volume of sequences in public databases complicate surveillance efforts. CovRadar combines an analytical pipeline and a web application designed for the molecular surveillance of the spike gene of SARS-CoV-2, an important vaccine target. The intuitive web front-end focuses on mutations rather than viral lineages and provides easy access to frequencies and spatio-temporal distributions from global sample collections. The data is regularly updated based on a scalable and reproducible analytical back-end. With this platform, we aim to give users, those with or without bioinformatics skills or sufficient computational resources, the possibility to track and explore mutational changes in the SARS-CoV-2 spike gene and to filter, download, and further analyze data that meet their questions and needs. Advanced computational users have the ability to apply the analytical pipeline and data visualization methods locally on their own data. CovRadar is freely accessible at https://covradar.net, source code is available at https://gitlab.com/dacs-hpi/covradar.

GRAPHICAL ABSTRACT

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category: bioinformatics


title: Revision of commonly accepted Warburg mechanism of cancer development. Redox-sensitive mitochondrial cytochromes in breast and brain cancers by Raman imaging

abstract:
BackgroundWe studied oncogenic processes that characterize human breast cancer (infiltrating ductal carcinoma (IDC)) and human brain tumors: glioma, astrocytoma and medulloblastoma based on the quantification of cytochrome redox status by exploiting the resonance-enhancement effect of Raman scattering.

MethodsWe used Raman imaging to monitor changes in the redox state of the mitochondrial cytochromes in ex vivo human brain and breast tissues surgically resected specimens of human tissues and in vitro human brain cells of normal astrocytes (NHA), astrocytoma (CRL-1718), glioblastoma (U87-MG) and medulloblastoma (Daoy), and human breast cells of normal cells (MCF 10A), slightly malignant cells (MCF7) and highly aggressive cells (MDA-MB-231) by means of Raman microspectroscopy at 532 nm.

ResultsWe visualized localization of cytochromes by Raman imaging in the major organelles in cancer cells. We demonstrated that the "redox state Raman marker" of the ferric low spin heme in cytochrome c at 1584 cm-1 can serve as a sensitive indicator of cancer aggressiveness. We compared concentration of reduced cytochrome c and the grade of cancer aggressiveness in cancer tissues and single cells and specific organelles in cells: nucleous, mitochondrium, lipid droplets, cytoplasm, and membrane.

ConclusionsWe found that the concentration of reduced cytochrome c becomes abnormally high in human brain tumors and breast cancers in human tissues. Our results suggest that the mechanisms controlling the electron transport chain are spectacularly deregulated in cancers and indicate that electron transport, organized in terms of electronegativity, is inhibited between complex III and cytochrome c for isolated cells in vitro and between cytochrome c and complex IV in brain and breast tissues. The results provide evidence that the extracellular matrix and interactions with cell microenvironment play an important role in the mechanisms controlling the electron transport chain by cytochrome c. Our results reveal the universality of Raman vibrational characteristics of mitochondrial cytochromes in metabolic regulation in cancers that arise from epithelial breast cells and brain glial cells.


title: Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain

abstract:
Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL ([~]0.043 nM) and 3.18 ng/mL ([~]0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19.

ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.


title: Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection

abstract:
The immune system of most SARS-CoV-2 infected individuals limits viral spread to the upper airways without pulmonary involvement. This prevents the development of pneumonic COVID-19. However, the protective immunological responses causative of successful viral containment in the upper airways remain unclear. Here, we combine longitudinal single-cell RNA sequencing, proteomic profiling, multidimensional flow cytometry, RNA-Seq of FACS-sorted leukocyte subsets and multiplex plasma interferon profiling to uncover temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients.

We compare host responses in a high-risk patient population infected with SARS-CoV-2 but without pulmonary involvement to patients with COVID-19 pneumonia. Our data reveal a distinct immunological signature of successful viral containment, characterized by an early prominent interferon stimulated gene (ISG) upregulation across immune cell subsets. In addition, reduced cytotoxic potential of Natural Killer (NK) and T cells, as well as a monocyte phenotype with immune-modulatory potential are hallmarks of protective immunity. Temporal resolution across disease trajectories highlights ISG upregulation as particularly prominent early in the disease and confirms increased expression also in comparison to healthy controls.

We validate this distinct temporal ISG signature by in-depth RNA-seq of FACS-sorted leukocyte subsets in a large prospective ambulatory SARS-CoV-2 infected cohort confirming early and robust ISG upregulation particularly in monocytes and T cells. In conclusion, our data demonstrate a protective ISG phenotype in patients with successful containment of SARS-CoV-2 infection without progression to COVID-19. This early protective interferon response might be exploited as a therapeutic approach and for disease course prediction.


title: Identification of the SHREK family of proteins as broad-spectrum host antiviral factors

abstract: Mucins and mucin-like molecules are highly glycosylated, high-molecular-weight cell surface proteins that possess a semi-rigid and highly extended extracellular domain. P-selectin glycoprotein ligand-1 (PSGL-1), a mucin-like glycoprotein, has recently been found to restrict HIV-1 infectivity through virion incorporation that sterically hinders virus particle attachment to target cells. Here, we report the identification of a family of antiviral cellular proteins, named the Surface-Hinged, Rigidly-Extended Killer (SHREK) family of virion inactivators (PSGL-1, CD43, TIM-1, CD34, PODXL1, PODXL2, CD164, MUC1, MUC4, and TMEM123), that share similar structural characteristics with PSGL-1. We demonstrate that SHREK proteins block HIV-1 infectivity by inhibiting virus particle attachment to target cells. In addition, we demonstrate that SHREK proteins are broad-spectrum host antiviral factors that block the infection of diverse viruses such as influenza A. Furthermore, we demonstrate that a subset of SHREKs also blocks the infectivity of a hybrid alphavirus-SARS-CoV-2 virus-like particle. These results suggest that SHREK proteins may be a part of host innate immunity against enveloped viruses.


title: Full Brain and Lung Prophylaxis against SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Mouse Model or Golden Hamsters

abstract: Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors are particularly suitable for mucosal vaccination and recently emerge as a promising strategy to elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models. Here, we demonstrate that a single intranasal administration of a lentiviral vector encoding a prefusion form of SARS-CoV-2 spike glycoprotein induces full protection of respiratory tracts and totally avoids pulmonary inflammation in the susceptible hamster model. More importantly, we generated a new transgenic mouse strain, expressing the human Angiotensin Converting Enzyme 2, with unprecedent brain permissibility to SARS-CoV-2 replication and developing a lethal disease in <4 days post infection. Even though the neurotropism of SARS-CoV-2 is now well established, so far other vaccine strategies under development have not taken into the account the protection of central nervous system. Using our highly stringent transgenic model, we demonstrated that an intranasal booster immunization with the developed lentiviral vaccine candidate achieves full protection of both respiratory tracts and brain against SARS-CoV-2.


title: Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

abstract: Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.


title: Structure of plant PSI-plastocyanin complex reveals strong hydrophobic interactions.

abstract:
Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 [A] resolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB / Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared to reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover.

One Sentence SummaryGenetic engineering, kinetics and cryo-EM structural data reveal a mechanism in a major step of oxygenic photosynthesis


title: Multiple indole glucosinolates and myrosinases defend Arabidopsis against Tetranychus urticae herbivory

abstract:
Arabidopsis defenses against herbivores are regulated by the jasmonate hormonal signaling pathway, which leads to the production of a plethora of defense compounds, including tryptophan-derived metabolites produced through CYP79B2/CYP79B3. Jasmonate signaling and CYP79B2/CYP79B3 limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, using Arabidopsis mutants that disrupt metabolic pathways downstream of CYP79B2/CYP79B3, and synthetic indole glucosinolates, we identified phytochemicals involved in the defense against T. urticae. We show that Trp-derived metabolites depending on CYP71A12 and CYP71A13 are not affecting mite herbivory. Instead, the supplementation of cyp79b2 cyp79b3 mutant leaves with the 3-indolylmethyl glucosinolate and its derived metabolites demonstrated that the indole glucosinolate pathway is sufficient to assure CYP79B2/CYP79B3-mediated defenses against T. urticae. We demonstrate that three indole glucosinolates can limit T. urticae herbivory, but that they have to be processed by the myrosinases to hinder T. urticae oviposition. Finally, the supplementation of the mutant myc2 myc3 myc4 with indole glucosinolates indicated that the transcription factors MYC2/MYC3/MYC4 induce additional indole glucosinolate-independent defenses that control T. urticae herbivory. Together, these results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple indole glucosinolates, specific myrosinases, and additional MYC2/MYC3/MYC4-dependent defenses.

One sentence summaryThree indole glucosinolates and the myrosinases TGG1/TGG2 help protect Arabidopsis thaliana against the herbivory of the two-spotted spider mite Tetranychus urticae.

category: plant biology


title: Touch-triggered bZIP translocation regulates elongation and secondary wall biosynthesis

abstract: Plant growth is mediated by the integration of internal and external cues, perceived by cells and transduced into a developmental program that gives rise to cell division, elongation, and wall thickening. Extra-, inter-, and intra- physical cellular forces contribute to this regulation. Across the plant kingdom thigmomorphogenesis is widely observed to alter plant morphology by reducing stem height and increasing stem diameter. The transcriptome is highly responsive to touch, including components of calcium signaling pathways and cell wall modification. One aspect of this cascade involves gibberellins (GA) and bZIP family transcription factors. Here, we present data connecting thigmomorphogenesis with secondary cell wall synthesis by GA inactivation and bZIP translocation into the nucleus. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus in response to mechanical stimulation. This translocation was mitigated by supplementing with exogenous bioactive GA, and induced by chemical inhibition of GA synthesis. Misregulation of SWIZ expression resulted in plants with reduced stem and root elongation, and following mechanical stimulation, increased secondary wall thickness. Classical touch responsive genes were upregulated in B. distachyon following touch, and we observe significant induction of the glycoside hydrolase 17 family, which may be indicative of a facet of grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. Thus, SWIZ is a transcriptional regulatory component of thigmomorphogenesis, which includes the thickening of secondary cell walls.