title: Functional wetland loss drives emerging risks to waterbird migration networks

abstract: Migratory waterbirds (i.e., shorebirds, wading birds, and waterfowl) rely on a diffuse continental network of wetland habitats to support annual life cycle needs. Emerging threats of climate and land-use change raise new concerns over the sustainability of these habitat networks as water scarcity triggers cascading ecological effects impacting wetland habitat availability. Here we use important waterbird regions in Oregon and California, USA, as a model system to examine patterns of landscape change impacting wetland habitat networks in western North America. Wetland hydrology and flooded agricultural habitats were monitored monthly from 1988 to 2020 using satellite imagery to quantify the timing and duration of inundation--a key delimiter of habitat niche values associated with waterbird use. Trends were binned by management practice and wetland hydroperiods (semi-permanent, seasonal, and temporary) to identify differences in their climate and land-use change sensitivity. Wetland results were assessed using 33 waterbird species to detect nonlinear effects of network change across a diversity of life cycle and habitat needs. Pervasive loss of semi-permanent wetlands was an indicator of systemic functional decline. Shortened hydroperiods caused by excessive drying transitioned semi-permanent wetlands to seasonal and temporary hydrologies--a process that in part counterbalanced concurrent seasonal and temporary wetland losses. Expansion of seasonal and temporary wetlands associated with closed-basin lakes offset wetland declines on other public and private lands, including wildlife refuges. Diving ducks, black terns, and grebes exhibited the most significant risk of habitat decline due to semi-permanent wetland loss that overlapped important migration, breeding, molting, and wintering periods. Shorebirds and dabbling ducks were beneficiaries of stable agricultural practices and top-down processes of functional wetland declines that operated collectively to maintain habitat needs. Outcomes from this work provide a novel perspective of wetland ecosystem change affecting waterbirds and their migration networks. Understanding the complexity of these relationships will become increasingly important as water scarcity continues to restructure the timing and availability of wetland resources.


title: Genetic regulation of human aortic smooth muscle cell gene expression and splicing predict causal coronary artery disease genes

abstract: Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies (GWAS) have identified over 175 loci associated with CAD. The majority of these loci are in non-coding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we hypothesized that a subset of the CAD GWAS risk loci are associated with the regulation of transcription in distinct SMC phenotypes. Here, we measured gene expression in SMCs isolated from the ascending aortas of 151 ethnically diverse heart transplant donors in quiescent or proliferative conditions and calculated the association of their expression and splicing with [~]6.3 million imputed single nucleotide polymorphism (SNP) markers across the genome. We identified 4,910 expression and 4,412 splice quantitative trait loci (sQTL) that represent regions of the genome associated with transcript abundance and splicing. 3,660 of the eQTLs had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 of the eQTLs were SMC- and sex-specific, respectively. To identify the effector transcript(s) regulated by CAD GWAS loci, we used four distinct colocalization approaches and identified 84 eQTL and 164 sQTLs that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. Two CAD loci colocalized with a SMC sex-specific eQTL (AL160313.1 and TERF2IP) and another locus colocalized with SMC-specific eQTL (ALKBH8). Also, 27% and 37% of the sQTLs were unique to quiescent or proliferative SMCs, respectively. The most significantly associated CAD locus, 9p21, was an sQTL for the long non-coding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. Collectively, these results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.

category: genetics


title: Transforming chemical proteomics enrichment into high throughput method using SP2E workflow.

abstract: Protein post-translational modifications (PTMs) play a critical role in the regulation of protein catalytic activity, localization and protein-protein interactions. Attachment of PTMs onto proteins significantly diversifies their structure and function resulting in so-called proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease specific, is still a highly challenging task. Sub-stoichiometric amounts and modification of low abundant proteins necessitate purification or enrichment of the modified proteins. Although the introduction of the mass spectrometry-based chemical proteomic strategies have enabled to screen protein PTMs with increased throughput, sample preparation has remained highly time consuming and tedious. Here, we report an optimized workflow for enrichment of PTM proteins in 96-well plate format which can be possible extended to robotic automatization. This platform allows to significantly lower the input of total protein, which opens up the opportunity to screen specialized and difficult to culture cell lines in high-throughput manner. The presented SP2E protocol is robust, time- and cost-effective as well as suitable for large-scale screening of proteoforms. Application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration and cancer and may contribute to an overall acceleration of the recently launched Human Proteoform Project.

category: biochemistry


title: Self-rectifying magnetoelectric metamaterials enable precisely timed remote neural stimulation and restoration of sensory motor functions

abstract: Magnetoelectric materials convert magnetic fields to electric fields and have applications in wireless data and power transmission, electronics, sensing, data storage, and biomedical technology. For example, magnetoelectrics could enable precisely timed remote stimulation of neural tissue, but the resonance frequencies where magnetoelectric effects are maximized are typically too high to stimulate neural activity. To overcome this challenge, we created the first self-rectifying magnetoelectric "metamaterial." This metamaterial relies on nonlinear charge transport across semiconductor layers that allow the material to generate a steady bias voltage in the presence of an alternating magnetic field. This "self-rectification" allows us to generate arbitrary electrical pulse sequences that have a time-averaged voltage in excess of 1 V. As a result, we can use magnetoelectric nonlinear metamaterials (MNMs) to remotely stimulate peripheral nerves with repeatable latencies of less than 5 ms, which is more than 120 times faster than previous neural stimulation approaches based on magnetic materials. These short latencies enable this metamaterial to be used in applications where fast neural signal transduction is necessary such as in sensory or motor neuroprosthetics. As a proof or principle, we show wireless stimulation to restore a sensory reflex in an anesthetized rat model as well as using the MNM to restore signal propagation in a severed nerve. The rational design of nonlinearities in the magnetic-to-electric transduction pathway as described here opens the door to many potential designs of MNMs tailored to applications spanning electronics, biotechnology, and sensing.

category: bioengineering


title: Genomics-based annotations help unveil the molecular composition of edible plants

abstract: Given the important role food plays in health and wellbeing, the past decades have seen considerable experimental efforts dedicated to mapping the chemical composition of food ingredients. As the composition of raw food is genetically predetermined, here we ask, to what degree can we rely on genomics to predict the chemical composition of natural ingredients. We therefore developed tools to unveil the chemical composition of 75 edible plants genomes, finding that genome-based annotations increase the number of compounds linked to specific plants by 42 to 100%. We rely on Gibbs free energy to identify compounds that accumulate in plants, i.e., those that are more likely to be detected experimentally. To quantify the accuracy of our predictions, we performed untargeted metabolomics on 13 plants, allowing us to experimentally confirm the detectability of the predicted compounds. For example, we find 59 novel compounds in corn, predicted by genomics annotations and supported by our experiments, but previously not assigned to the plant. Our study shows that genome-based annotations can lead to an integrated metabologenomics platform capable of unveiling the chemical composition of edible plants, and the biochemical pathways responsible for the observed compounds.

category: bioinformatics


title: FMSClusterFinder: A new tool for detection and identification of clusters of sequential motifs with varying characteristics inside genomic sequences

abstract:
This paper describes FMSClusterFinder, a new tool and algorithm for identification and detection of clusters of sequential blocks inside the DNA and RNA subject sequences. Gene expression and genomic groups performance is under the control of functional elements cooperating with each other as clusters. The functional motifs or blocks are often comparably short, degenerate and are located within varying distances from each other. Since functional motifs mostly act in relation to each other as clusters, finding such clusters of blocks is an effective approach to identify functional groups and their function and structure, which represents the need for development of new corresponding tools.

The presented web application finds clusters of sequential blocks, with even altering sequences and located in varying distances from each other inside the subject sequences, simultaneously. Additionally, the blocks could be searched with user defined constant or varying characteristics such as: a) different levels of similarity, b) varying minimum number of blocks required to build up the query cluster, c) different types of sequence (degenerate or standard) and d) one or multiple alternative sequences for each block. FMSClusterFinder is available freely at http://fmsclusterfinder.fmsbiog.com.


title: Dissecting Mutational Allosteric Effects in Alkaline Phosphatases Associated with Different Hypophosphatasia Phenotypes: An Integrative Computational Investigation

abstract:
Hypophosphatasia (HPP) is a rare inherited disorder characterized by defective bone mineralization, is highly variable in its clinical phenotype. The disease occurs due to various loss-of-function mutations in ALPL, the gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). In this work, a data-driven and biophysics-based approach for large-scale analysis of ALPL mutations - from nonpathogenic to severe HPPs is proposed. Allosteric molecular signatures of ALPL mutations were determined using an integrated pipeline of synergistic approaches including sequence and energetic-based analysis, structural topology network modeling and elastic network models. Statistical analysis of molecular features computed for the ALPL mutations showed a significant difference between the control and mild and severe HPP phenotypes, and the developed machine learning model suggested that the topological network parameters could serve as is a robust indicator for severe mutations. Molecular dynamics simulations coupled with protein structure network was employed to analyze the effect of single-residue variation on conformational dynamics of TNSALP dimers, and we found that severe disease-associated mutations have a significantly greater effect on allosteric communications. The results of this study suggested that ALPL mutations associated with mild and severe HPPs tend to have different effects on protein stability on local scale and long-range communications caused by network rewiring. By linking disease phenotypes with allosteric molecular signatures, the proposed integrative computational approach elucidates the complex sequence-structure-allostery relationships of ALPL mutations and dissects the role of allosteric effects in the pathogenesis of HPPs.

Author SummaryThe understanding of mutational genotype- disease phenotype relationship is a fundamental step for enabling precision medicine. It remains a challenging task to assess the molecular principle of the genotype- phenotype relationship. By focusing on Hypophosphatasia, a rare inherited disorder, as an example, we performed comprehensive analysis of single-amino-acid mutations in the encoded protein of Tissue Nonspecific Alkaline Phosphatase associated in terms of their sequence, structure, and dynamics properties. We further developed a machine learning -based method to classify different disease phenotypes, and the interpretability of the classification model was addressed by the structural-functional analysis of network topological important mutations. Our results highlighted the allosteric propensity of severe mutations, and show the potential allosteric principle of genotype- phenotype relationship.

category: biophysics


title: Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study

abstract: Gramicidin is a monomeric protein that is thought to non-selectively conduct cationic currents and water. Linear gramicidin is considered an antibiotic. This function is considered to be mediated by the formation of pores within the lipid membrane, thereby killing bacterial cells. The main non-psychoactive active constituent of the cannabis plant, cannabidiol (CBD), has recently gained interest, and is proposed to possess various potential therapeutic properties, including being an antibiotic. We previously determined that CBDs activity on ion channels could be, in part, mediated by altering membrane biophysical properties, including elasticity. In this study, our goal was to determine the empirical effects of CBD on gramicidin currents in human embryonic kidney (HEK) cells, seeking to infer potential direct compound-protein interactions. Our results indicate that gramicidin, when applied to the extracellular HEK cell membrane, followed by CBD perfusion, increases the gramicidin current.


title: The central clock suffices to drive the majority of circulatory metabolic rhythms

abstract: Life on Earth anticipates recurring 24-h environmental cycles via genetically-encoded molecular clocks active in all mammalian organs. Communication between these clocks is believed to control circadian homeostasis. Metabolism can be considered a form of inter- tissue communication language that results in temporal coordination of systemic metabolism between tissues. Here we characterize the extent to which clocks in different organs employ this means of communication, an area which remains largely unexplored. For this, we analysed the metabolome of serum from mice with tissue-specific expression of the clock gene Bmal1. Notably, having functional hepatic and muscle clocks can only drive a minority (13%) of the oscillating metabolites in circulation. Conversely, limiting Bmal1 expression to Syt10- expressing neurons (which are enriched in the suprachiasmatic nucleus [SCN], the master pacemaker that regulates circadian rhythms) restores rhythms to 57% of circulatory metabolites and 28% of liver transcripts, and rescues glucose intolerance. Importantly, these parameters were also restored in clock-less mice upon rhythmic feeding, indicating that the central clock mainly regulates metabolic rhythms via behavior. These findings explicate the circadian communication between tissues and highlight the importance of the central clock in governing those signals.

category: physiology


title: Increased parasite load is associated with reduced metabolic rates and escape responsiveness in pumpkinseed sunfish host

abstract: Wild animals have parasites that can compromise their physiological and/or behavioural performance. Yet, the extent to which parasite load is related to intraspecific variation in performance traits within wild populations remains relatively unexplored. We used pumpkinseed sunfish (Lepomis gibbosus) and their endoparasites as a model system to explore the effects of infection load on host aerobic metabolism and escape performance. Metabolic traits (standard and maximum metabolic rates, aerobic scope) and fast-start escape responses following a simulated aerial attack by a predator (responsiveness, response latency, and escape distance) were measured in fish from across a gradient of visible (i.e. trematodes causing black spot disease counted on fish surfaces) and non-visible (i.e. cestodes in fish abdominal cavity counted post-mortem) endoparasite infection. We found that a higher infection load of non-visible endoparasites was related to lower standard and maximum metabolic rates, but not aerobic scope in fish. Non-visible endoparasite infection load was also related to decreased responsiveness of the host to a simulated aerial attack. Visible endoparasites were not related to changes in metabolic traits nor fast-start escape responses. Our results suggest that infection with parasites that are inconspicuous to researchers can result in intraspecific variation in physiological and behavioral performance in wild populations, highlighting the need to more explicitly acknowledge and account for the role played by natural infections in studies of wild animal performance.


title: miniTurbo-based interactomics of two plasma membrane-localized SNARE proteins in Marchantia polymorpha

abstract:
O_LIMarchantia polymorpha is a model liverwort and its overall low genetic redundancy is advantageous for dissecting complex pathways. Proximity-dependent in vivo biotin-labelling methods have emerged as powerful interactomics tools in recent years. However, interactomics studies applying proximity labelling are currently limited to angiosperm species in plants.
C_LIO_LIHere, we established and evaluated a miniTurbo-based interactomics method in M. polymorpha using MpSYP12A and MpSYP13B, two plasma membrane- localized SNARE proteins, as baits.
C_LIO_LIWe show that our method yields a manifold of potential interactors of MpSYP12A and MpSYP13B compared to a co-immunoprecipitation approach. Our method could capture specific candidates for each SNARE.
C_LIO_LIWe conclude that a miniTurbo-based method is a feasible tool for interactomics in M. polymorpha and potentially applicable to other model bryophytes. Our interactome dataset on MpSYP12A and MpSYP13B will be a useful resource to elucidate the evolution of SNARE functions.
C_LI

category: plant biology


title: Rhythm of The Night (and Day): Predictive metabolic modeling of circadian growth in Chlamydomonas

abstract:
Algal cells experience strong circadian rhythms under diurnal light, with regular changes in both biomass composition and transcriptomic environment. However, most metabolic models - critical tools for bioengineering organisms - assume a steady state. The conflict between these assumptions and the reality of the cellular environment make such models inappropriate for algal cells, creating a significant obstacle in engineering cells that are viable under natural light. By transforming a set of discreet transcriptomic measurements from synchronized Chlamydomonas cells grown in a 12/12 diel light regime (1) into continuous curves, we produced a complete representation of the cells transcriptome that can be interrogated at any arbitrary timepoint. We clustered these curves, in order to find genes that were expressed in similar patterns, and then also used it to build a metabolic model that can accumulate and catabolize different biomass components over the course of a day. This model predicts qualitative phenotypical outcomes for the sta6 mutant, including excess lipid accumulation (2) and a failure to thrive when grown diurnally in minimal media (3), representing a qualitative prediction of phenotype from genotype even under dynamic conditions. We also extended this approach to simulate all single-knockout mutants with genes represented in the model and identified potential targets for rational engineering efforts.

SIGNIFICANCE STATEMENTWe have developed the first transient metabolic model for diurnal growth of algae based on experimental data and capable of predicting phenotype from genotype. This model enables us to evaluate the impact of genetic and environmental changes on the growth, biomass composition and intracellular fluxes of the model green alga, Chlamydomonas reinhardtii. The availability of this model will enable faster and more efficient design of cells for production of fuels, chemicals and pharmaceuticals.

category: systems biology


title: Theoretical Principles of Enhancer-Promoter Communication in Transcriptional Bursting

abstract:
Transcriptional regulation occurs through genomic contacts between enhancers and their cognate promoters, and most genes are transcribed in a bursty fashion. To understand the relationship between these two phenomena, we develop a general modeling framework in terms of the information transmission from upstream genomic organization to downstream transcriptional bursting. Importantly, we uncover fundamental theoretical principles of enhancer-promoter (E-P) spatial communication in the modulation of transcriptional burst size (BS) and burst frequency (BF). First, BS and BF obey their respective power-law dependences on the E-P communication strength and distinct scaling exponents. Second, the E-P spatial distance follows a Maxwell-Boltzmann distribution rather than the previously assumed Gauss distribution. Third, the E-P genomic distance affects transcriptional outcomes biphasically (i.e., in an exponential decay for small E-P genomic distances but insensitively to large E-P genomic distances). Fourth, the E-P communication mainly modulates BF rather than BS. Finally, the mutual information between BS (or BF) and E-P spatial distance further reveals essential characteristics of the information transfer from the upstream to the downstream. Our predictions are experimentally verifiable, e.g., confirmed by experimental data on Drosophila. The overall analysis provides insights into the role of the E-P communication in the control of transcriptional bursting.

SignificanceMeasurement technologies of chromatin conformations and genome-wide occupancy data of architectural proteins have revealed that genome topology is tightly intertwined with gene transcription. However, a long-standing question in transcriptional regulation is how the enhancer-promoter (E-P) spatial communication impacts transcriptional bursting kinetics. To address this issue, we develop a multiscale model that couples upstream chromatin dynamics to downstream transcriptional bursting. This model not only reveals fundamental principles of E-P communication in transcriptional bursting kinetics (e.g., burst size and frequency follow their own power-law behaviors) but also provides a general modeling framework toward the 4D nucleome project.


title: Impaired glutamate reuptake induces synaptic mistuning in rat hippocampal slices, that can be counteracted by ketamine

abstract: Mistuning of synaptic transmission has been proposed to underlie many psychiatric disorders, with decreased reuptake of the excitatory neurotransmitter glutamate as one contributing factor. Synaptic tuning occurs through several diverging and converging forms of plasticity. By recording evoked field postsynaptic potentials in the CA1 area in hippocampal slices, we found that inhibiting glutamate transporters using DL-TBOA causes retuning of synaptic transmission, resulting in a new steady state with reduced synaptic strength and a lower threshold for inducing long-term synaptic potentiation (LTP). Moreover, we also found reduced threshold for LTP in a rat model of depression that has decreased levels of glutamate transporters. Most importantly, we found that the antidepressant ketamine counteracts the effects of increased glutamate on the various steps involved in synaptic retuning. We therefore propose that ketamines mechanism of action as an antidepressant is to restore adequate synaptic tuning.

category: neuroscience


title: Two roles for choice selective inhibition in decision-making circuits

abstract: During perceptual decision-making, the firing rates of cortical neurons reflect upcoming choices. Recent work showed that excitatory and inhibitory neurons are equally selective for choice. However, functional consequences of inhibitory choice selectivity in decision-making circuits are unknown. We developed a circuit model of decision-making which accounts for the specificity of inputs to and outputs from inhibitory neurons. We found that selective inhibition expands the space of circuits supporting decision-making, allowing for weaker or stronger recurrent excitation when connected in a competitive or feedback motif. The specificity of inhibitory outputs sets the trade-off between speed and accuracy of decisions by altering the attractor dynamics in the circuit. Recurrent neural networks trained to make decisions display the same dependence on inhibitory specificity and the strength of recurrent excitation. Our results reveal two concurrent roles for selective inhibition in decision-making circuits: stabilizing strongly connected excitatory populations and maximizing competition between oppositely selective populations.


title: The Temporal and Contextual Stability of Activity Levels in Hippocampal CA1 Cells

abstract: Recent studies have shown that the activity levels of hippocampal neurons during exploration are not constant but change on a daily to weekly scale. However, a few other studies suggest that their activity levels are preserved over time. To resolve this contradiction, we repeatedly measured the neural activity of the mouse hippocampus in four different environments. The activity level of hippocampal neurons fluctuated greatly in one environment, but the mean activity level across all the environments was more stable. Each cells preferred environment frequently changed over time. The number of environments in which a cell showed activity varied across cells, ranging from zero to four, and the value also tended to be preserved. Cells that showed place cell activity in many environments had higher mean firing rates and higher spatial information content, whereas cells active only in a small number of environments were suitable for coding environmental identity. These results suggest that although the activity level in a single environment appears unstable, each cell has not only an inherent activity level but also play a characteristic role in the coding of space.


title: DciA helicase operators exhibit diversity across bacterial phyla

abstract:
A fundamental requirement for life is the replication of an organisms DNA. Studies in Escherichia coli and Bacillus subtilis have set the paradigm for DNA replication in bacteria. During replication initiation in E. coli and B. subtilis, the replicative helicase is loaded onto the DNA at the origin of replication by an ATPase helicase loader. However, most bacteria do not encode homologs to the helicase loaders in E. coli and B. subtilis. Recent work has identified the DciA protein as a predicted helicase operator that may perform a function analogous to the helicase loaders in E. coli and B. subtilis. DciA proteins, which are defined by the presence of a DUF721 domain (termed the DciA domain herein), are conserved in most bacteria but have only been studied in mycobacteria and {gamma}-proteobacteria (Pseudomonas aeruginosa and Vibrio cholerae). Sequences outside of the DciA domain in Mycobacterium tuberculosis DciA are essential for protein function but are not conserved in the P. aeruginosa and V. cholerae homologs, raising questions regarding the conservation and evolution of DciA proteins across bacterial phyla. To comprehensively define the DciA protein family, we took a computational evolutionary approach and analyzed domain architectures and sequence properties of DciA-domain containing proteins across the tree of life. These analyses identified lineage-specific domain architectures amongst DciA homologs as well as broadly conserved sequence-structural motifs. The diversity of DciA proteins represents the evolution of helicase operation in bacterial DNA replication and highlights the need for phylum-specific analyses of this fundamental biological process.

IMPORTANCEDespite the fundamental importance of DNA replication for life, this process remains understudied in bacteria outside of Escherichia coli and Bacillus subtilis. In particular, most bacteria do not encode the helicase loading proteins that are essential in E. coli and B. subtilis for DNA replication. Instead, most bacteria encode a DciA homolog that likely constitutes the predominant mechanism of helicase operation in bacteria. However, it is still unknown how DciA structure and function compare across diverse phyla that encode DciA proteins. In this study, we perform computational evolutionary analyses to uncover tremendous diversity amongst DciA homologs. These studies provide a significant advance in our understanding of an essential component of the bacterial DNA replication machinery.

category: microbiology


title: Environmental change alters nitrogen fixation rates and microbial parameters in a subarctic biological soil crust

abstract: Together, Biological Soil Crust (BSC) and other cryptogamic groundcovers can contribute up to half of the global nitrogen (N) fixation. BSC also stabilizes the soil (reducing erosion and dust emissions), fixes carbon (C), retains moisture, and acts as a hotspot of microbial diversity and activity. Much of the knowledge about how climate change is affecting the composition and functioning of BSC comes from hot arid and semiarid regions. The comparatively smaller body of research on BSC from cold and mesic environments has been primarily observational, for example along chronosequences after a glacier retreat. Few studies have experimentally investigated the effects of the environment on BSC from high latitudes. Such experiments allow unraveling of relationships at a resolution that can only be achieved by controlling for confounding factors. We measured short-term (2-4 days) responses of a liverwort-based (Anthelia juratzkana) BSC from the south of Iceland to a range of temperature, moisture and light conditions. Warming increased N fixation rates, especially when moisture was at a saturation level, and only when light was not limiting. A correlation analysis suggests that increases in N fixation rates were linked to cyanobacterial abundance on the BSC surface and to the rates of their metabolic activity. Warming and moisture changes also induced compositional and structural modification of the bacterial community, with consequences at the functional level. In contrast to many observations on BSC from hot drylands, the BSC from our cold and mesic study site is more limited by low temperature and light than by moisture. Our findings show possible ways in which BSC from cold and mesic ecosystems can respond to short-term manifestations of climate change, such as increasingly frequent heat waves.


title: Light-controlled phosphorylation in the TrkA-Y785 site by photosensitive UAAs activates the MAPK/ERK signaling pathway

abstract: TrkA is a membrane receptor that upon ligand binding, induces autophosphorylation of tyrosine residues in the intracellular domain, and this process includes sites in the kinase activation loop (Y670, Y674, Y675) and two direct sites involved in downstream signaling pathways (Y490, Y785). At present, researchers cannot fully elucidate the regulatory mechanism of TrkA phosphorylation because TrkA signaling is a highly dynamic process, and a strategy with high temporal and spatial resolution will be beneficial to the mechanism research. Our previous study proposed a design scheme for photosensitive TrkA, which utilizes a new molecular light control technology to target TrkA-Y490 and three kinase domain sites (Y670, Y674, and Y675) through Genetic Code Expansion (GCE) technology combined with site-directed mutagenesis. We chose two light-controllable unnatural amino acids (UAAs) to introduce at the specific phosphorylation sites of the target protein TrkA. We focused on the regulation mechanism of these sites on the MAPK/ERK pathway downstream of TrkA. However, this method has not yet been validated for the TrkA-Y785 site. Therefore, this paper will continue to test the light-controlled method we established earlier in the Y785 site. We aim to improve further the experimental model of light-controlled phosphorylation of TrkA that we have established and finally lay the foundation for the comprehensive analysis of kinase-related pathways.

category: molecular biology


title: Decoding the IGF1 Signaling Gene Regulatory Network Behind Alveologenesis from A Mouse Model of Bronchopulmonary Dysplasia

abstract:
Lung development is precisely controlled by underlying Gene Regulatory Networks (GRN). Disruption of genes in the network can interrupt normal development and cause diseases such as bronchopulmonary dysplasia (BPD), a chronic lung disease in preterm infants with morbid and sometimes lethal consequences that is characterized by lung immaturity and reduced alveolarization.

Here, we generated a transgenic mouse exhibiting a moderate severity BPD phenotype by blocking IGF1 signaling in secondary crest myofibroblasts (SCMF) at the onset of alveologenesis. Using approaches mirroring the construction of the model GRN in sea urchins development, we constructed the IGF1 signaling network underlying alveologenesis using this mouse model that phenocopies BPD. The constructed GRN, consisting of 43 genes, provides a birds-eye view of how the genes downstream of IGF1 are regulatorily connected. The GRN also reveals a mechanistic interpretation of how the effects of IGF1 signaling are transduced within SCMF from its specification genes to its effector genes and then from SCMF to its neighboring alveolar epithelial cells with WNT5A and FGF10 signaling as the bridge. Consistently, blocking of WNT5A signaling in mice phenocopies BPD as inferred by the network. A comparative study on human samples suggests that a GRN of similar components and wiring underlies human BPD.

Our network view of alveologenesis is transforming our perspective to understand and treat BPD. The new perspective calls for the construction of the full signaling GRN underlying alveologenesis, upon which targeted therapies for this neonatal chronic lung disease can be viably developed.

category: developmental biology


title: Types of cis- and trans-gene regulation of expression quantitative trait loci across human tissues

abstract: AO_SCPLOWBSTRACTC_SCPLOWExpression quantitative trait loci (eQTLs) have been identified for most genes in the human genome across nearly 50 tissues or cell types. While most of the eQTLs are near the associated genes, some can be far away or on different chromosomes, with the regulatory mechanisms largely unknown. Here, we study cis- and trans-regulation of eQTLs across multiple tissues and cell types. Specifically, we constructed trios consisting of an eQTL, its cis-gene and trans-gene and inferred the regulatory relationships with causal network inference. We identify multiple types of regulatory networks for trios: across all the tissues, more than half of the trios are inferred to be conditionally independent, where the two genes are conditionally independent given the genotype of the eQTL (cis-gene [<-] eQTL [->] trans-gene). Around 1.5% of the trios are inferred to be mediation (eQTL [->] mediator [->] target), around 1.3% fully connected among the three nodes, and just a handful v-structures (eQTL [->] gene 1 [<-] gene 2). Unexpectedly, across the tissues, on average more than half of the mediation trios have the trans-gene as the mediator. Most of the mediators (cis and trans) are tissue specific. Furthermore, cis-gene mediators are significantly enriched for protein-coding genes compared with the genome average, whereas trans-gene mediators are significantly enriched for pseudogenes and depleted for long noncoding RNAs (lncRNAs).

category: genomics


title: High throughput RNA sequencing discovers symptomatic and latent viruses: an example from ornamental Hibiscus

abstract: Hibiscus rosa-sinensis L. (Hibiscus, Malvaceae) is an ornamental species grown widely in amenity plantings. We collected leaves on an urban roadside pavement (sidewalk) near a market in Guangzhou which showed multiple symptoms of leaf rolling, deformation and chlorosis. Initial evaluation by electron microscopy using negative staining of drip preparations revealed the presence of tobamovirus-like particles. Total RNA was extracted, and, unusually, without any RNA selection based on sequence, was used for cDNA library construction and high-throughput survey sequencing. From the 814 Mb of clean sequence data (from 2,712,161 paired reads of 150 bp) reads representing chloroplast, ribosomal, and mitochondrial genes were filtered out, eliminating 79.1% of reads. 1,135,848 x 150 bp of the sequence was retained and screened for viral sequences. Assembly of these sequences detected nine virus species from seven virus genera comprising three tobamoviruses, namely, Tobacco mosaic virus, Tobacco mild green mosaic virus and Hibiscus latent Singapore virus, Turnip mosaic virus (Potyvirus), Potato virus M (Carlavirus), Hibiscus chlorotic ringspot virus (Betacarmovirus), Fabavirus sp (Fabavirus), Cotton leaf curl Multan virus (Begomovirus) and a putative mitoviruses replicating in mitochondria, Chenopodium quinoa mitovirus 1. Mapping the reads to complete virus reference sequences showed high and uniform coverage of the genomes from 3,729 x coverage for Turnip mosaic virus to 22 x for Cotton leaf curl Multan virus. By comparison, nuclear reference genes actin showed 14 x coverage and polyubiquitin 27 x. Notable variants from reference sequences (SNPs) were identified. With the low cost of sequencing and potential for semi-automated bioinformatic pipelines, the whole-RNA approach has huge potential for identifying multiple undiagnosed viruses in ornamental plants, resulting in the ability to take preventive measures in production facilities against spread and to product quality for the mutual benefit of producers and consumers.


title: DisGUVery: a versatile open-source software for high-throughput image analysis of Giant Unilamellar Vesicles

abstract: Giant Unilamellar Vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVerys three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple pre-defined analysis modules allow the user to extract properties such as membrane fluorescence, vesicle shape and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of non-spherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in GUV research.


title: Single cell sequencing reveals microglia induced angiogenesis by specific subsets of endothelial cells following spinal cord injury

abstract: Spinal cord injury (SCI) results in dynamic alterations of the microenvironment at the lesion site, which inevitably leads to neuron degeneration and functional deficits. The prominent deterioration of the milieu, derived from the destruction of spinal vascular system, not only activates innate immunity but also makes cells in the lesion lose nutrient supports. Limited endogenous angiogenesis happens after SCI, but the cell events at the lesion site underpinning this process have not been delineated so far. Here, we conducted single-cell RNA sequencing (scRNA-seq) of tissues in the spinal lesion at different time points after rat SCI. After performing clustering and cell-type identification, we focused on the vascular endothelial cells (ECs), which play a pivot role in angiogenesis, and drew a comprehensive cellular and molecular atlas for endogenous angiogenesis after SCI. We found that microglia and macrophage promote endogenous angiogenesis by regulating EC subsets through SPP1 and IGF1 signal pathways. Our results indicated that immune cells promotes angiogenesis by the regulation of specific cell subsets of vascular ECs, which provides new clues for the development of interventions for SCI.


title: Defects in DNA double-strand break repair re-sensitise antibiotic-resistant Escherichia coli tomultiple bactericidal antibiotics

abstract: Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance-breaker compounds, which inhibit resistance mechanisms and thus re-sensitise bacteria to existing antibiotics. In the current study, we identify bacterial DNA double-strand break repair as a promising target for the development of resistance-breaking co-therapies. We examined genetic variants of Escherichia coli that combined antibiotic-resistance determinants with DNA repair defects. We observed that defects in the double-strand break repair pathway led to significant re-sensitisation towards five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full re-sensitisation. For ciprofloxacin and nitrofurantoin, sensitisation manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the pro-mutagenic SOS response. Disruption of double-strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break-repair inhibitors can be developed they could re-sensitise antibiotic-resistant bacteria to multiple classes of existing antibiotics and may supress the development of de novo antibiotic-resistance mutations.


title: The scotopic band: primate detailed scotopic vision and perceptual uncertainty

abstract: Primates perceive detailed images in photopic (daylight) vision via a preferred-processing, high-acuity pathway, made up of a small photopic center (the fovea), and dedicated eye movements, whose function is to shift images of target objects to the fovea and keep them while foveal cones sense the image. Thus, the preferred processing pathway processes images serially, not in parallel. No such pathway is known for scotopic (night-light) vision or mesopic (twilight) vision - though details are informative in dim light, even vital; it remains unclear whether and how scotopic vision of details is accomplished. Here we show that primates do have a scotopic preferred-processing pathway. It consists of a  scotopic center, located on a  scotopic band, and eye movements that shift target images not to the fovea but directly to the scotopic center. In contrast to the stationary fovea, the scotopic center can relocate over the scotopic band. The scotopic center relocates to match changing visual conditions and contextual needs. Ambient light intensity is mapped monotonically onto the scotopic band, with mesopic vision at one end. The dimmer the light is, the more dorsal the scotopic center relocates on the scotopic band. Importantly, the scotopic center relocates (or  set) not only in passive response to ambient light but also actively, driven by internal factors. In near-threshold conditions the scotopic center relocates to a more dorsal band location than in salient conditions. That the same relocation of the scotopic center can be instigated both by dimming the ambient light and by reducing the detectability of targets indicates that, at the core, the longitudinal axis of the scotopic band encodes the level of perceptual uncertainty.


title: Design of immunogens for eliciting antibody responses that may protect against SARS-CoV-2 variants

abstract:
The rise of SARS-CoV-2 variants and the history of outbreaks caused by zoonotic coronaviruses point to the need for next-generation vaccines that confer protection against variant strains. Here, we combined analyses of diverse sequences and structures of coronavirus spikes with data from deep mutational scanning to design SARS-CoV-2 variant antigens containing the most significant mutations that may emerge. We trained a neural network to predict RBD expression and ACE2 binding from sequence, which allowed us to determine that these antigens are stable and bind to ACE2. Thus, they represent viable variants. We then used a computational model of affinity maturation (AM) to study the antibody response to immunization with different combinations of the designed antigens. The results suggest that immunization with a cocktail of the antigens is likely to promote evolution of higher titers of antibodies that target SARS-CoV-2 variants than immunization or infection with the wildtype virus alone. Finally, our analysis of 12 coronaviruses from different genera identified the S2 cleavage site and fusion peptide as potential pan-coronavirus vaccine targets.

Author SummarySARS-CoV-2 variants have already emerged and future variants may pose greater threats to the efficacy of current vaccines. Rather than using a reactive approach to vaccine development that would lag behind the evolution of the virus, such as updating the sequence in the vaccine with a current variant, we sought to use a proactive approach that predicts some of the mutations that could arise that could evade current immune responses. Then, by including these mutations in a new vaccine antigen, we might be able to protect against those potential variants before they appear. Toward this end, we used various computational methods including sequence analysis and machine learning to design such antigens. We then used simulations of antibody development, and the results suggest that immunization with our designed antigens is likely to result in an antibody response that is better able to target SARS-CoV-2 variants than current vaccines. We also leveraged our sequence analysis to suggest that a particular site on the spike protein could serve as a useful target for a pan-coronavirus vaccine.

category: immunology


title: Mutations of SARS-CoV-2 variants of concern escaping Spike-specific T cells

abstract:
The amino acid (AA) mutations that characterise the different variants of concern (VOCs), which replaced the ancestral SARS-CoV-2 Wuhan-Hu-1 isolate worldwide, provide biological advantages such as increased infectivity and partial escape from humoral immunity. Here we analysed the impact of these mutations on vaccination- and infection-induced Spike-specific T cells. We confirmed that, in the majority of infected or vaccinated individuals, different mutations present in a single VOC (Delta) or a combined mosaic of more than 30 AA substitutions and deletions found in Alpha, Beta, Gamma, Delta and Omicron VOCs cause modest alteration in the global Spike-specific T cell response. However, distinct numerically dominant Spike-specific CD4 and CD8 T cells preferentially targeted regions affected by AA mutations and do not recognise the mutated peptides. Importantly, some of these mutations, such as N501Y (present in Alpha, Beta, Gamma, and Omicron) and L452R (present in Delta), known to provide biological advantage to SARS-CoV-2 in terms of infectivity also abolished CD8 T cell recognition.

Taken together, our data show that while global mRNA vaccine- and infection-induced Spike-specific T cells largely tolerate the diverse mutations present in VOCs, single Spike-specific T cells might contribute to the natural selection of SARS-CoV-2 variants.


title: Close relatives of MERS-CoV in bats use ACE2 as their functional receptors

abstract: Middle East Respiratory Syndrome coronavirus (MERS-CoV) and several bat coronaviruses employ Dipeptidyl peptidase-4 (DPP4) as their functional receptors1-4. However, the receptor for NeoCoV, the closest MERS-CoV relative yet discovered in bats, remains enigmatic5. In this study, we unexpectedly found that NeoCoV and its close relative, PDF-2180-CoV, can efficiently use some types of bat Angiotensin-converting enzyme 2 (ACE2) and, less favorably, human ACE2 for entry. The two viruses use their spikes S1 subunit carboxyl-terminal domains (S1-CTD) for high-affinity and species-specific ACE2 binding. Cryo-electron microscopy analysis revealed a novel coronavirus-ACE2 binding interface and a protein-glycan interaction, distinct from other known ACE2-using viruses. We identified a molecular determinant close to the viral binding interface that restricts human ACE2 from supporting NeoCoV infection, especially around residue Asp338. Conversely, NeoCoV efficiently infects human ACE2 expressing cells after a T510F mutation on the receptor-binding motif (RBM). Notably, the infection could not be cross-neutralized by antibodies targeting SARS-CoV-2 or MERS-CoV. Our study demonstrates the first case of ACE2 usage in MERS-related viruses, shedding light on a potential bio-safety threat of the human emergence of an ACE2 using "MERS-CoV-2" with both high fatality and transmission rate.


title: Substantial immune response in Omicron infected breakthrough and unvaccinated individuals against SARS-CoV-2 variants of concerns

abstract: The recent emergence of highly mutated SARS-CoV-2 Omicron variant has debilitating effect on public health system of the affected countries worldwide. Currently India is facing third wave of COVID-19 pandemic and going through a severe crisis. Within short span of time, the variant has shown high transmissibility and capability of evading the immune response generated against natural infection and vaccination. The immune escape potential of Omicron is a serious concern and further needs to be explored. In the present study, we have assessed the IgG and neutralizing antibody (NAb) response in breakthrough individuals vaccinated with two doses ChAdOx1 nCoV-19 vaccine (n=25), breakthrough individuals vaccinated with two doses of BNT162b2 mRNA vaccine (n=8) and unvaccinated individuals (n=6). All these individuals were infected with Omicron variant. The IgG antibody activity in the sera of the ChAdOx1 nCoV-19 and BNT162b2 mRNA breakthrough individuals was comparable with S1-RBD, while it was lesser in BNT162b2 mRNA breakthrough individuals with N protein and inactivated whole antigen IgG ELISA. BNT162b2 mRNA breakthrough individuals showed moderate reduction in NAb GMTs compared to ChAdOx1 nCoV-19 against Alpha, Beta and Delta. However, 3-fold higher reduction was observed with omicron variant in BNT162b2 mRNA than ChAdOx1 nCoV-19. Apparently, Alpha variant was modestly resistant to the sera of unvaccinated individuals than Beta, Delta and Omicron. Our study demonstrated substantial immune response in the individuals infected with Omicron. The neutralizing antibodies could effectively neutralize the Omicron and other VOCs including the most prevalent Delta variant.


title: Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail

abstract: The SARS-CoV-2 receptor-binding domain (RBD) E406W mutation abrogates neutralization mediated by the REGEN-CoV therapeutic monoclonal antibody (mAb) COVID-19 cocktail and the cilgavimab (AZD1061) mAb. Here, we show that this residue substitution remodels the ACE2-binding site allosterically, thereby dampening receptor recognition severely and altering the epitopes recognized by these three mAbs. Although vaccine-elicited neutralizing antibody titers are decreased similarly against the E406 mutant and the Delta or Epsilon variants, broadly neutralizing sarbecovirus mAbs, including a clinical mAb, inhibit the E406W spike mutant.


title: Similarity bias from averaging signatures from the Connectivity Map

abstract: The Next Generation L1000 Connectivity Map (L1000) is a massive, high-throughput dataset measuring transcriptional changes - or signatures - in cancer cell lines due to chemical and genetic perturbation[1]. Zhu et al [2] recently presented Deep Learning-Based Efficacy Prediction System (DLEPS), a method that models averages of many signatures (CTPs or meta-signatures) of the same compound across conditions. However, our analysis shows that averaging increasing numbers of signatures from L1000 results in a substantial positive increase in signature similarities even among entirely unrelated perturbations, drastically shifting the baseline threshold for similarity. Consequently, DLEPS overstates how informative the model is about each compound, highlighting the need to account for this similarity bias in any L1000 Connectivity Map analysis.


title: Template-based design of peptides to inhibit SARS-CoV-2 RNA-dependent RNA polymerase complexation.

abstract: The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between the subunits of the RdRp complex are highly conserved, facilitating the design of inhibitors with high affinity for the interaction hotspots of the complex. Here, we report development and application of a structural bioinformatics protocol to design peptides that can inhibit RdRp complex formation by targeting the interface of its core subunit nonstructural protein (nsp) 12 with accesory factor nsp7. We adopt a top-down approach for protein design by using interaction hotspots of the nsp7-nsp12 complex obtained from a long molecular dynamics trajectory as template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in silico to determine peptide sequences with highest shape and interaction complementarity for the nsp7-nsp12 interface. Two lead designed peptide are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation and anti-viral activity. Their binding affinity to nsp7 (target), as determined from surface plasmon resonance (SPR) assay, is found to be comparable to that of the nsp7-nsp12 complex. Further, one of the designed peptides gives 46 % inhibition of nsp7-nsp12 complex at 10:1 peptide:nsp7 molar concentration (from ELISA assay). Further optimization of cell penetrability and target affinity of these designed peptides is expected to provide lead candidates with high anti-viral activity against SARS-CoV-2.


title: Visual uncertainty unveils the distinct role of haptic cues in multisensory grasping

abstract: Multisensory grasping movements (i.e., seeing and feeling a handheld object while grasping it with the contralateral hand) are superior to movements guided by each separate modality. This multisensory advantage might be driven by the integration of vision with either the haptic position only or with both position and size cues. To contrast these two hypotheses, we manipulated visual uncertainty (central vs. peripheral vision) and the availability of haptic cues during multisensory grasping. We showed a multisensory benefit irrespective of the degree of visual uncertainty suggesting that the integration process involved in multisensory grasping can be flexibly modulated by the contribution of each modality. Increasing visual uncertainty revealed the role of the distinct haptic cues. The haptic position cue was sufficient to promote multisensory benefits evidenced by faster actions with smaller grip apertures, whereas the haptic size was fundamental in fine-tuning the grip aperture scaling. These results support the hypothesis that, in multisensory grasping, vision is integrated with all haptic cues, with the haptic position cue playing the key part. Our findings highlight the important role of non-visual sensory inputs in sensorimotor control and hint at the potential contributions of the haptic modality in developing and maintaining visuomotor functions.


title: Towards the identification and optimization of the "dose-response" relationship of transcranial direct current stimulation

abstract:
Transcranial direct current stimulation (tDCS) is being investigated as a therapeutic tool in several neuropsychiatric disorders, but its mechanisms of action remain incompletely understood and its effectiveness is highly variable from subject to subject. Some of this inter-subject variance is thought to stem from individual differences in head anatomy, which result in different electric field distributions across individuals for the same stimulation settings. In a recent block-randomized, sham-controlled pilot study, 18 older adults with mild-to-moderate motor and cognitive impairments underwent a 2-week, 10-session intervention of 2 mA bipolar tDCS with the anode over the left dorsolateral prefrontal cortex (DLPFC) and cathode over the right supraorbital region (anodal F3 vs. FP2 using sponge electrodes in the 10-20 EEG system). tDCS, compared to sham stimulation, improved average cognitive-motor performance over a two-week follow-up. For a subset of 12 participants from this cohort who underwent structural brain MRIs at baseline, we derived tDCS-induced electric field properties using MRI-derived realistic, individualized finite element modeling. We hypothesized that the electric field component orthogonal (En) to the cortex over the left DLPFC would correlate with observed cognitive-motor improvements. Across the 12 subjects the average tDCS-induced electric field over the target area was highly variable with a standard deviation of the surface average En over the left DLPFC of 21% of the mean. For those subjects who were randomized to tDCS intervention (n=6), those who exhibited greater improvement in cognitive-motor performance were exposed to greater normal component of the tDCS electric field averaged over the left DLPFC (r95 % [isin] [-0.99,-0.59]). In contrast, averaged over the right DLPFC, En was a poor predictor of outcome (r95 % [isin] [-0.08,0.97]), as was the amplitude of the current injected (a widely adopted dosing parameter). In light of these results, we conducted a retrospective modeling experiment using personalized montage optimization algorithms to achieve a desired En amplitude over the left DLPFC. The optimized montages induced an average En on the left DLPFC comparable to the bipolar montages, but with half the variability (standard deviation was 11% compared to 21% with bipolar montages). In addition, these results suggest that optimized multichannel montages afford more focal targeting, with lower "spill-over" of electric field onto other cortical areas. These proof-of-concept results, though based upon a small sample size, indicate that assessment of the E-field distribution is critical when evaluating the effects of tDCS intervention. They also reinforce the importance of controlled cortical electric field dosing in tDCS and the potential advantages of optimized multichannel stimulation.

Main findings: O_LIThe lDPFC averaged normal component of the electric field correlated with cognitive-motor improvements following tDCS intervention.
C_LIO_LIIn older adults, head and brain anatomical differences resulted in high inter-subject variability of tDCS-induced electric fields.
C_LIO_LItDCS montages optimized from modeling and delivered via multichannel electrode arrays can control variance and maximize electric field on target, while minimizing effects elsewhere.
C_LI


title: Biodiversity of the Genus Trichoderma in the Rhizosphere of Coffee (Coffea arabica) Plants in Ethiopia and their Potential Use in Biocontrol of Coffee Wilt Disease

abstract: The present study investigated the distribution status and biodiversity of Trichoderma species surveyed from coffee rhizosphere soil samples from Ethiopia and their potential for biocontrol of coffee wilt disease (CWD) caused by Fusarium xylarioides. Trichoderma isolates were identified based on molecular approaches and morphological characteristics followed by biodiversity analysis using different biodiversity indices. The antagonistic potential of Trichoderma isolates was evaluated against F. xylarioides using the dual confrontation technique and agar diffusion bioassays. A relatively high diversity of species was observed including 16 taxa and 11 undescribed isolates. Trichoderma asperellum, T. asperelloides and T. longibrachiatum were classified as abundant species, with dominance (Y) values of 0.062, 0.056 and 0.034, respectively. Trichoderma asperellum was the most abundant species (comprising 39.6% of all isolates) in all investigated coffee ecosystems. Shannons biodiversity index (H), the evenness (E), Simpsons biodiversity index (D), and the abundance index (J) were calculated for each coffee ecosystem, revealing that species diversity and evenness were highest in the Jimma zone (H =1.97, E = 0.76, D = 0.91, J = 2.73). The average diversity values for Trichoderma species originating from the coffee ecosystem were H = 1.77, D = 0.7, E = 0.75 and J = 2.4. In vitro confrontation experiments revealed that T. asperellum AU131 and T. longibrachiatum AU158 reduced the mycelial growth of F. xylarioides by over 80%. The potential use of these Trichoderma species for disease management of F. xylarioides and to reduce its impact on coffee cultivation is discussed in relation to Ethiopias ongoing coffee wilt disease crisis.


title: Characterization and identification of a novel daidzein reductase involved in (S)-equol biosynthesis in Clostridium C1

abstract:
(S)-equol is an isoflavone with high estrogen-like activity and no toxic effects in the human body, and is only produced by some gut bacteria in vivo. It plays an important role in maintaining individual health, however, the dearth of resources associated with (S)-equol-producing bacteria has seriously restricted the production and application of (S)-equol. We report here a novel functional gene C1-07020 that was identified from a chick (S)-equol-producing bacterium (Clostridium C1). We found that recombinant protein of C1-07020 possessed similar function to daidzein reductase (DZNR), which can convert daidzein (DZN) into R/S-dihydrodaidzein (R/S-DHD). Interestingly, C1-07020 can reverse convert (R/S)-DHD (DHD oxidases) into DZN even without cofactors or anaerobic conditions. Additionally, high concentrations of (S)-equol can directly promote DHD oxidase but inhibit DZNR activity. Molecular docking and site-directed mutagenesis revealed that the amino acid Arg 75 was the active site of DHD oxidases. Subsequently, an engineered E. coli strain based on C1-07020 was constructed and showed higher yield of (S)-equol than the engineered bacteria from our previous work. Metagenomics analysis and PCR detection surprisingly revealed that C1-07020 and related bacteria may be prevalent in the gut of humans and animals and their (S)-equol production state may cause differed between (S)-equol producer and non-producer. Overall, a novel DZNR from Clostridium C1 was found and identified in this study, and its bidirectional enzyme activities and wide distribution in the gut of humans and animals provide alternative strategies for revealing the individual regulatory mechanisms of (S)-equol-producing bacteria.

Importance(S)-equol is a final product of DZN that metabolized by some enteric bacteria. Although (S)-equol played very important roles in maintaining human health, larger differences in equol production varied between different populations. Here, a novel DZNR gene C1-07020, which related to (S)-equol production, was reported. The bidirectional enzyme functions and wide distribution of C1-07020 in human and animal gut provided additional insights into the metabolic regulation of (S)-equol. Additionally, C1-07020 can be used for improving the production of (S)-equol in vitro.


title: Computationally efficient assembly of a Pseudomonas aeruginosa gene expression compendium

abstract:
Over the past two decades, thousands of RNA sequencing (RNA-seq) gene expression profiles of Pseudomonas aeruginosa have been made publicly available via the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). In the work we present here, we draw on over 2,300 P. aeruginosa transcriptomes from hundreds of studies performed by over seventy-five different research groups. We first developed a pipeline, using the Salmon pseudo-aligner and two different P. aeruginosa reference genomes (strains PAO1 and PA14), that transformed raw sequence data into a uniformly processed data in the form of sample-wise normalized counts. In this workflow, P. aeruginosa RNA-seq data are filtered using technically and biologically driven criteria with characteristics tailored to bacterial gene expression and that account for the effects of alignment to different reference genomes. The filtered data are then normalized to enable cross experiment comparisons. Finally, annotations are programmatically collected for those samples with sufficient meta-data and expression-based metrics are used to further enhance strain assignment for each sample. Our processing and quality control methods provide a scalable framework for taking full advantage of the troves of biological information hibernating in the depths of microbial gene expression data. The re-analysis of these data in aggregate is a powerful approach for hypothesis generation and testing, and this approach can be applied to transcriptome datasets in other species.

SignificancePseudomonas aeruginosa causes a wide range of infections including chronic infections associated with cystic fibrosis. P. aeruginosa infections are difficult to treat and people with CF-associated P. aeruginosa infections often have poor clinical outcomes. To aid the study of this important pathogen, we developed a methodology that facilitates analyses across experiments, strains, and conditions. We aligned, filtered for quality and normalized thousands of P. aeruginosa RNA-seq gene expression profiles that were publicly available via the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). The workflow that we present can be efficiently scaled to incorporate new data and applied to the analysis of other species.


title: Choice of 16S ribosomal RNA primers impacts urinary microbiota profiling

abstract: Accessibility to next-generation sequencing (NGS) technologies has enabled the profiling of microbial communities living in distinct habitats. 16S ribosomal RNA (rRNA) gene sequencing is widely used for microbiota profiling with NGS technologies. Since most used NGS platforms generate short reads, sequencing the full-length 16S rRNA gene is impractical. Therefore, choosing which 16S rRNA hypervariable region to sequence is critical in microbiota profiling studies. All nine 16S rRNA hypervariable regions are taxonomically informative, but due to variability in profiling performance for specific clades, choosing the ideal 16S rRNA hypervariable region will depend on the bacterial composition of the habitat under study. Recently, NGS allowed the identification of microbes in the urinary tract, and urinary microbiota has become an active research area. However, there is no current study evaluating the performance of different 16S rRNA hypervariable regions for urinary microbiota profiling. We collected urine samples from male volunteers and profiled their urinary microbiota by sequencing a panel of six amplicons encompassing all nine 16S rRNA hypervariable regions. After systematically comparing their performance, we show that V1V2 hypervariable regions better assess the taxa commonly present in urine samples and V1V2 amplicon sequencing is more suitable for urinary microbiota profiling. We believe our results will be helpful to guide this crucial methodological choice in future urinary microbiota studies.


title: Nonsynonymous mutations in fepR are associated with Listeria adaptation to low concentrations of benzalkonium chloride, but not increased survival in use level concentrations of benzalkonium chloride.

abstract:
Presence of and selection for Listeria spp. and Listeria monocytogenes that are less effectively inactivated by quaternary ammonium compounds (such as benzalkonium chloride [BC]) is a food safety concern, including in fresh produce environments. An initial MIC assay on 67 produce-associated Listeria strains showed that strains carrying BC resistance genes bcrABC (n=10) and qacH (n=1) showed higher MIC (4-6 mg/L BC) compared to strains lacking these genes (MIC of 1-2 mg/L BC). Serial passaging experiments that exposed the 67 strains to increasing BC concentrations revealed that 62/67 strains showed growth in BC concentrations above their parent MIC (range of 4-20 mg/L). Two serially passaged isolates were obtained for each parent strain and substreaked onto BHI agar in the absence of BC for seven rounds; 105/134 substreaked isolates showed higher substreaked MIC (range of 4 - 6 mg/L) compared to parent MIC. These results suggested isolates acquired genetic adaptations that confer BC resistance. Substreaked isolates were characterized by a combination of whole genome sequencing and Sanger sequencing of fepR, a local repressor of the MATE family efflux pump FepA. These data identified nonsynonymous fepR mutations in 48/67 isolates including 24 missense, 16 nonsense, and 8 frameshift mutations. The mean inactivation of substreaked isolates after exposure to use level concentrations of BC (300 mg/L) was 4.48 log, which was not significantly different from inactivation observed in parent strains. Serial passage experiments performed on cocultures of Listeria strains containing bcrABC or qacH did not yield growth at higher BC concentrations than monoculture experiments.

IMPORTANCEListeria resistance to quaternary ammonium compounds has been raised as a concern with regards to Listeria persistence in food environments, which can increase risk of product contamination, food recalls and foodborne illness outbreaks. Findings from our study show that individual Listeria strains can acquire genetic adaptations that confer resistance to low concentrations of benzalkonium chloride, but these genetic adaptations dont increase Listeria survival when exposed to concentrations of benzalkonium chloride used for food contact surface sanitation (300 mg/L). Our study also suggests that there is limited risk of benzalkonium chloride resistance genes bcrABC and qacH spreading through horizontal gene transfer and conferring enhanced resistance of L. monocytogenes to benzalkonium chloride. Overall, our study suggests that emergence of benzalkonium chloride resistant Listeria strains in food processing environments is of limited concern, as even strains adapted to gain higher MIC in vitro maintain full sensitivity to use level concentrations of benzalkonium chloride.


title: L-threonine mediated DAF-16/HSF-1 activation inhibits ferroptosis and increases healthspan

abstract: The pathways that impact longevity in the wake of dietary restriction (DR) pathway remain still ill-defined. Most studies have focused on nutrient limitation and perturbations of energy metabolism to explain the beneficial effects of DR. We showed that the essential amino acid L-threonine was elevated in Caenorhabditis elegans under DR, and that L-threonine supplementation increased its healthspan. To elucidate the underlying mechanism, we conducted metabolic and transcriptomic profiling using LC-MS/MS and RNA-seq analyses in worms that were fed with RNAi to induce loss of function of key candidate mediators and evaluated healthspan. L-threonine supplementation and loss-of-threonine dehydrogenase, which govern L-threonine metabolism, increased the healthspan of C. elegans by attenuating ferroptosis in a ferritin-dependent manner. Tran-scriptomic analysis of C. elegans supplemented with L-threonine showed that FTN-1 encoding ferritin was elevated, implying FTN-1 is an essential mediator of longevity promotion through L-threonine. Organismal ferritin levels were positively correlated with chronological aging and L-thre-onine supplementation further increased ferritin levels, which protected against age-associated ferroptosis through the DAF-16 and HSF-1 pathways. Our investigation uncovered the role of a distinct and universal metabolite, L-threonine, in DR-mediated improvement in organismal healthspan, suggesting it could be an effective intervention for preventing senescence progression and age-induced ferroptosis.

category: cell biology


title: Historical records of plant-insect interactions in subarctic Finland

abstract: Historical ecological records document the diversity and composition of communities decades or centuries ago and provide a valuable benchmark for modern comparisons. Historical datasets on plant-animal interactions allow for modern comparisons that examine the stability of species and interaction networks over long periods of time and in response to anthropogenic change. Here we present a curated dataset of interactions between plants and insects in subarctic Finland, generated from digitizing a historical document from the late 19th century and updating the taxonomy using currently accepted nomenclature. The resulting dataset contains 654 records of plant-insect interactions observed during the years 1895-1900, and includes 498 unique interactions between 86 plant species and 173 insect taxa. Syrphidae, Apidae and Muscidae were the insect families involved in most interactions, and interactions were most observed with the plant species Angelica archangelica, Salix caprea, and Chaerophyllum prescottii. Interaction data are available as csv-file and provide a valuable resource on plant-insect interactions over 120 years ago in a high latitude ecosystem that is undergoing rapid climate change.

category: ecology


title: The chromosome-scale genome assembly of Jasminum sambac var. unifoliatum provide insights into the formation of floral fragrance

abstract: The high-quality genome assembly of the JSU-FSP provides valuable information on how jasmine produces the abundant VTs and VPBs that synergistically contribute to the pleasant aroma of jasmine flower.


title: Exploring repeats in rice genomes: Identification, Characterization and its Applications

abstract: Biodiversity is a fundamental property of all natural systems existing in the field biology. It refers to the underlying heterogeneity at different levels of ecology, genetics and evolution. In case of plant systems, dramatic variability has been observed during the Anthropocene at different spatial scales. Environmental stress is one of the major influencing factors behind this plant biodiversity. Huge genetic diversity has been also demonstrated across varieties of important crop species like rice. Repetitive sequences which are a major contributor of genomic diversity in polyploidy plants have been found to occur ubiquitously in their genomes. To date diverse repeat types have been characterized in the plant genomes performing various functions starting from qualitative trait markers to genome evolution and stress management. With an objective to identify of plant stress associated genes using DNA repeat probes, a robust method has been developed. The method has been modularized into three distinct sections. First part is dedicated for identification of different types of repeats. Earlier review has suggested building a pipeline of multiples tools for capturing different types of repeats from the genome sequences. Specialized tools like TROLL, Tandem repeat finder (TRF), PHOBOS and database like REPBASE have been selected for performing this job. Second module is intended for screening of stress related genes from the published articles and databases and the last module has been designed for the association mining between genomic repetitive patterns with stress phenotypes. The method has been used to explore stress associated repeats from 9 Oryza species from different continents and other plants like Arabidopsis and Brachypodium. In case of Oryza species distribution repeats has been found to be significantly different between stress associated and housekeeping genes. More than 55% of the repeats are found to be in positive association (nPMI > 0) whereas 26% of the repeats are false-positives. These repetitive probes have been utilized in several applications. Firstly, using as molecular markers to identify stress related genes in different Oryza species where availability is limited. Secondly, using as a probe to reanalyzing the evolutionary lineage of Oryza species etc.


title: LPS-TLR4 pathway stimulating in acute-on-chronic liver failure by promote NETs formation

abstract: Backgrounds: Intrahepatic infiltration of neutrophils is a character of alcoholic acute-on-chronic liver failure (AACLF) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. Intestinal bacteria and the gut-liver axis have been thought to play a key role in many liver diseases also including AACLF. However, whether NETs appear in AACLF and play a role in AACLF is still unsure. Methods: WT, NE KO, and TLR4 KO mice were used to build the AACLF model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AACLF related markers were detected. Results: The serum MPO-DNA and LPS concentration was increased in AACLF patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AACLF mice by testing MPO-DNA, Cit H3, and NE. These markers decreased with gut detergent and restored markers with gut detergent plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AACLF related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with combined treatment with antibiotics plus LPS. But the liver injury, intrahepatic fat, fibro deposition, and liver inflammation-related markers did show a significant difference in TLR4 KO mice when they received the same treatment. Conclusion: Intestinal-derived LPS promotes NETs formation in AACLF through the TLR4 pathway and further accelerates the AACLF process by NETs.

category: pathology


title: LPS-TLR4 pathway exaggerates alcohol-induced acute-on-chronic liver failure via provoking NETs formation


title: Inference and Uncertainty Quantification of Stochastic Gene Expression via Synthetic Models

abstract: AO_SCPLOWBSTRACTC_SCPLOWEstimating uncertainty in model predictions is a central task in quantitative biology. Biological models at the single-cell level are intrinsically stochastic and nonlinear, creating formidable challenges for their statistical estimation which inevitably has to rely on approximations that trade accuracy for tractability. Despite intensive interest, a sweet spot in this trade off has not been found yet. We propose a flexible procedure for uncertainty quantification in a wide class of reaction networks describing stochastic gene expression including those with feedback. The method is based on creating a tractable coarse-graining of the model that is learned from simulations, a synthetic model, to approximate the likelihood function. We demonstrate that synthetic models can substantially outperform state-of-the-art approaches on a number of nontrivial systems and datasets, yielding an accurate and computationally viable solution to uncertainty quantification in stochastic models of gene expression.


title: Impacts of development and adult sex on brain cell numbers in the Black Soldier Fly, Hermetia illucens L. (Diptera: Stratiomyidae)

abstract: The Black Soldier Fly (Hermetia illucens, Diptera: Stratiomyidae) has been introduced across the globe, with numerous industry applications predicated on its tremendous growth during the larval stage. However, basic research on H. illucens biology (for example, studies of their central nervous system) are lacking. Despite their small brain volumes, insects are capable of complex behaviors; understanding how these behaviors are completed with such a small amount of neural tissue requires understanding processing power (e.g. number of cells) within the brain. Brain cell counts have been completed in only a few insect species (mostly Hymenoptera), and almost exclusively in adults. This limits the taxonomic breadth of comparative analyses, as well as any conclusions about how development and body size growth may impact brain cell populations. Here, we present the first images and cell counts of the H. illucens brain at four time points across development (early, mid, and late larval stages, and both male and female adults) using immunohistochemistry and isotropic fractionation. To assess sexual dimorphism in adults, we quantified the number of cells in the central brain vs. optic lobes of males and females separately. To assess if increases in body size during development might independently affect different regions of the CNS, we quantified the larval ventral nerve cord and central brain separately at all three stages. Together, these data provide the first description of the nervous system of a popular, farmed invertebrate and the first study of brain cell numbers using IF across developmental stages in any insect.


title: The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2

abstract: Cell differentiation involves profound changes in global gene expression that often have to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. Contrasting gene expression with chromatin accessibility, we find that p27 is coincidentally necessary to globally repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2, and ASCL1. Our data reveal both direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of the Sox2 gene leads to reduced levels of SOX2-downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.


title: Prior exposure to speech rapidly modulates cortical processing of high-level linguistic structure

abstract: Neural activity has been shown to track hierarchical linguistic units in connected speech and these responses can be directly modulated by changes in speech intelligibility caused by spectral degradation. In the current study, we manipulate prior knowledge to increase the intelligibility of physically identical speech sentences and test the hypothesis that the tracking responses can be enhanced by this intelligibility improvement. Cortical magnetoencephalography (MEG) responses to intelligible speech followed by either the same (matched) or different (unmatched) unintelligible speech were measured in twenty-three normal hearing participants. Driven by prior knowledge, cortical coherence to "abstract" linguistic units with no accompanying acoustic cues (phrases and sentences) was enhanced relative to the unmatched condition, and was lateralized to the left hemisphere. In contrast, cortical responses coherent to word units, aligned with acoustic onsets, were bilateral and insensitive to contextual information changes. No such coherence changes were observed when prior experience was not available (unintelligible speech before intelligible speech). This dissociation suggests that cerebral responses to linguistic information are directly affected by intelligibility, which in turn are powerfully shaped by physical cues in speech. These results provide an objective and sensitive neural index of speech intelligibility, and explain why previous studies have reported no effect of prior knowledge on cortical entrainment.


title: Norepinephrine and dopamine contribute to distinct repetitive behaviors induced by novel odorant stress in male and female mice

abstract: Exposure to unfamiliar odorants induces an array of repetitive defensive and non-defensive behaviors in rodents which likely reflect adaptive stress responses to the uncertain valence of novel stimuli. Mice genetically deficient for dopamine {beta}-hydroxylase (Dbh-/-) lack the enzyme required to convert dopamine (DA) into norepinephrine (NE), resulting in globally undetectable NE and supranormal DA levels. Because catecholamines modulate novelty detection and reactivity, we investigated the effects of novel plant-derived odorants on repetitive behaviors in Dbh-/- mice and Dbh+/- littermate controls, which have catecholamine levels comparable to wild-type mice. Unlike Dbh+/- controls, which exhibited vigorous digging in response to novel odorants, Dbh-/- mice displayed excessive grooming. Drugs that block NE synthesis or neurotransmission suppressed odorant-induced digging in Dbh+/- mice, while a DA receptor antagonist attenuated grooming in Dbh-/- mice. The testing paradigm elicited high circulating levels of corticosterone regardless of Dbh genotype, indicating that NE is dispensable for this systemic stress response. Odorant exposure increased NE and DA abundance in the prefrontal cortex (PFC) of Dbh+/- mice, while Dbh-/- animals lacked NE and had elevated PFC DA levels that were unaffected by novel smells. Together, these findings suggest that novel odorant-induced increases in central NE tone contribute to repetitive digging and reflect psychological stress, while central DA signaling contributes to repetitive grooming. Further, we have established a simple method for repeated assessment of stress-induced repetitive behaviors in mice, which may be relevant for modeling neuropsychiatric disorders like Tourette syndrome or obsessive-compulsive disorder that are characterized by stress-induced exacerbation of compulsive symptoms.


title: The tyrosine phosphatase PRL regulates attachment of Toxoplasma gondii to host cells and is essential for virulence

abstract:
The pathogenesis of Toxoplasma gondii is mainly due to tissue damage caused by the repeating lytic cycles of the parasite. Many proteins localized to the pellicle of the parasite, particularly kinases, have been identified as critical regulators of the Toxoplasma lytic cycle. However, little is known about the associated protein phosphatases. Phosphatase of regenerating liver (PRL), a highly conserved tyrosine phosphatase, is an oncogene that plays pivotal roles in mammalian cells and typically associates with membranes via a conserved prenylation site. PRL in Toxoplasma has a predicted prenylation motif in the C-terminus like other homologs. We have determined that TgPRL localizes to the plasma membrane and that disruption of TgPRL results in a defect in the parasites ability to attach to host cells. This function is dependent both on TgPRLs membrane localization and phosphatase activity. Importantly, in vivo experiments have shown that while mice infected with parental strain parasites die within days of infection, those infected with parasites lacking TgPRL not only survive but also develop immunity that confers protection against subsequent infection with wild-type parasites. Immunoprecipitation experiments revealed that the PRL-CNNM (Cyclin M) complex, which regulates intracellular Mg2+ homeostasis in mammalian cells, is also present in Toxoplasma. Consistent with this interaction, parasites lacking TgPRL had higher intracellular Mg2+ levels than the parental or complemented strains, suggesting TgPRL is involved in regulating intracellular Mg2+ homeostasis. Thus, TgPRL is a vital regulator of the Toxoplasma lytic cycle and virulence, showing its potential as a target of therapeutic intervention.

IMPORTANCEInfection with Toxoplasma gondii can lead to severe and even life-threatening diseases in people with compromised or suppressed immune systems. Unfortunately, drugs to combat the parasite are limited, highly toxic, and ineffective against the chronic stage of the parasite. Consequently, there is a strong demand for the discovery of new treatments. A comprehensive understanding of how the parasite propagates in the host cells and which proteins contribute to the parasites virulence will facilitate the discovery of new drug targets. Our study meets this objective and adds new insights to understanding the lytic cycle regulation and virulence of Toxoplasma by determining that the protein phosphatase TgPRL plays a vital role in the parasites ability to attach to host cells and that it is essential for parasite virulence.


title: Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD

abstract: Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. There are currently four FDA-approved drugs for DMD that aim to restore expression of dystrophin by exon skipping using splice switching oligonucleotides. While these therapies require lifelong repeat administration, recent advancements in gene editing technologies have raised the possibility of achieving  permanent exon skipping, and thereby curing the disease with a single treatment. Here we have investigated the use of the Staphylococcus aureus CRISPR/Cas9 system and a double-cut strategy, delivered using a pair of AAV9 vectors, for dystrophin restoration in the severely-affected dystrophin/utrophin double knock-out (dKO) mouse. Single guide RNAs were designed to induce double-strand DNA breaks on either side of Dmd exon 23, such that the intervening exon 23 sequence is excised when the flanking intronic regions are joined via the non-homologous end joining repair pathway. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD.


title: PPMO-mediated exon skipping induces uniform sarcolemmal dystrophin rescue with dose-dependent restoration of circulating microRNA biomarkers and muscle biophysical properties

abstract: Duchenne muscular dystrophy (DMD) is a paediatric muscle-wasting disorder caused by genetic loss of the gene encoding the dystrophin protein. Therapies that restore dystrophin expression are presumed to correct the disease, with antisense-mediated exon skipping being the leading approach. In this study, we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. To this end, dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping and dystrophin rescue at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed two weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed. Importantly, dystrophin expression was uniformly distributed at the sarcolemma of corrected myofibers at all doses. The abundance of serum microRNA biomarkers (i.e. miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase were restored towards wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e. Youngs modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression, and that both serum miRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in the context of DMD.


title: The effect of COVID-19 mRNA vaccine on respiratory system: human lung carcinoma cells by means of Raman spectroscopy and imaging.

abstract: The effect of COVID-19 mRNA vaccine on human lung carcinoma epithelial cells (A549) in vitro has been studied by means of Raman spectroscopy and imaging. The paper focuses on Raman imaging as a tool to explore apoptosis and oxidative phosphorylation in mitochondrial dysfunctions. The Raman results demonstrate alterations in the oxidation-reduction pathways associated with cytochrome c. We found that the COVID-19 mRNA vaccine downregulates the concentration of cytochrome c upon incubation with tumorous lung cells. Concentration of oxidized form of cytochrome c in mitochondria of lung cells decreases upon incubation the COVID-19 mRNA vaccine. Lower concentration of oxidized cytochrome c in mitochondria illustrates lower effectiveness of oxidative phosphorylation (respiration), reduced apoptosis and lessened ATP production. Moreover, mRNA vaccine increases significantly de novo lipids synthesis in lipid droplets up to 96 hours and alterations in biochemical composition. It seems that lipid composition of cells returns to the normal level for longer incubation time (14 days). In cell nucleus the mRNA vaccine does not produce statistically significant changes. The observed alterations in biochemical profiles upon incubation with mRNA vaccine in the specific organelles of the tumorous lung cells are similar to those we observe for other types of cancers, particularly brain glial cells.


title: Emergence of glycogen synthase kinase-3 interaction domain enhances phosphorylation of SARS-CoV-2 nucleocapsid protein

abstract: A structural protein of SARS-CoV-2, nucleocapsid (N) protein is abundantly expressed during viral replication. The N protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3{beta} constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), commonly found in many endogenous GSK-3{beta} binding proteins, such as Axins, FRATs, WWOX and GSKIP. Indeed, N interacts with GSK-3{beta} similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. Unlike with endogenous GID proteins, the N interaction neither disturbs endogenous GSK-3 activity nor regulates subsequent canonical Wnt activity and the Snail-EMT program. Notably, N abundance in SARS-CoV-2 is incomparably high compared to other coronaviruses, such as 229E, OC43 and HKU1. Compared to other coronaviruses, N harbors a CDK1 primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3{beta}. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allows increased abundance and hyper-phosphorylation of N. Our observations suggest that the emergence of GID and mutations for increased phosphorylation in N may have contributed to the emergence of SARS-CoV-2 and evolution of variants, respectively. Further study, especially in a BSL3-equipped facility, is required to elucidate the functional importance of GID and N phosphorylation in SARS-CoV-2 and variants.