title: Cardiac dysfunction affects eye development and vision by reducing supply of lipids in fish

abstract: Developing organisms are especially vulnerable to environmental stressors. We aimed to understand the underlying mechanisms of phenanthrene (Phe) and crude oil induced eye malformations. We exposed Atlantic haddock (Melanogrammus aeglefinus) embryos to a known L-type calcium channel blocker, nicardipine hydrochloride (Nic), and compared to early embryonic crude oil (Oil) and late embryonic Phe toxicity. All treatments lead to severe, eye, jaw and spinal deformities at early larval stages. At 3 days post hatching, larvae from all treatments and corresponding controls were dissected. Eyes, trunk, head and yolk sac were subjected to lipid profiling, and eyes were also subjected to transcriptomic profiling. Changes in lipid profiles and the transcriptome suggested that the dysfunctional and abnormal eyes in our treatments were due to both disruption of signaling pathways and insufficient supply of essential fatty acids and other nutrients form the yolk.


title: Non-coding RNAs Associated with Prader-Willi Syndrome Regulate Transcription of Neurodevelopmental Genes in                   Human Induced Pluripotent Stem Cells

abstract: Mutations and aberrant gene expression during cellular differentiation lead to neurodevelopmental disorders such as Prader-Willi syndrome (PWS) which results from the deletion of an imprinted locus on chromosome 15. We analysed chromatin-associated RNA in human induced pluripotent cells (iPSCs) upon depletion of hybrid small nucleolar long non-coding RNAs (sno-lncRNAs) and 5 snoRNA capped and polyadenylated long non-coding RNAs (SPA-lncRNAs) transcribed from the locus deleted in PWS. We found that rapid ablation of these lncRNAs affects transcription of specific gene classes. Downregulated genes contribute to neurodevelopment and neuronal maintenance while genes that are upregulated are predominantly involved in the negative regulation of cellular metabolism and apoptotic processes. Our data revealed the importance of SPA-lncRNAs and sno-lncRNAs in controlling gene expression in iPSCs and provided a platform for synthetic experimental approaches in PWS studies. We conclude that ncRNAs transcribed from the PWS locus are critical regulators of a transcriptional signature important for neuronal differentiation and development.

category: molecular biology


title: The Aspartyl Protease Ddi1 Is Essential for Erythrocyte Invasion by the Malaria Parasite

abstract: Malaria pathology is caused by the exponential replication of Plasmodium parasites in the blood stream. The bottleneck of the parasite life cycle is the invasion of erythrocytes immediately after parasites egress from infected red blood cells. DNA damage-inducible protein 1 (Ddi1) is a conserved eukaryotic proteasome shuttle protein containing an internal retroviral-like protease domain. Using conditional genetics, we now show that the proteolytic activity of the P. falciparum homologue, PfDdi1, is critically required for invasion of red blood cells. Furthermore, PfDdi1 disruption results in the accumulation of highly polyubiquitinated proteins that can be processed by purified PfDdi1 or distant eukaryotic homologues. We also show that PfDdi1 interacts with multiple components of the ubiquitin-proteasome system and that parasites lacking PfDdi1 are more sensitive to proteasome inhibition. Overall, this study establishes PfDdi1 as a key component of the eukaryotic ubiquitin-proteasome system and as a promising antimalarial target.

category: cell biology


title: Myeloid-mediated IL-1R signaling in immuno-responsive Thy-1 negative fibroblasts is critical for pulmonary fibrosis

abstract: Idiopathic pulmonary fibrosis (IPF) is a fatal disease with poorly defined pathogenic mechanism and no cure. It is characterized by chronic inflammation, myofibroblast accumulation, and aberrant extracellular matrix (ECM) remodeling. Fibrosis progression is considered to occur due to sustained aberrant fibroblast mechanotransduction: sensing "normal" soft tissue as stiff scarred tissue leading to the overproduction of ECM that then stiffens the microenvironment, thus reinforcing a progressive, stiffness-dependent fibrotic program. How chronic inflammation leads to aberrant mechanotransduction is not well understood. Thy-1 is a regulator of mechanotransduction in fibroblasts. Thy-1 expression is lost in fibroblastic foci, the active sites of fibrosis, although the mechanism of this loss is unknown. We demonstrate that in IPF tissue, the SMA+ fibroproliferative foci express the Type 1 IL-1 receptor (IL-1RI) and IL-1RI-deficient mice did not develop bleomycin-induced pulmonary fibrosis. Using ASC speck formation during inflammasome activation as a marker of mature IL-1{beta} release, we identified the immune compartment as the source of active IL-1{beta} during bleomycin-induced fibrosis. Furthermore, incubating mouse lung fibroblasts on soft (2kPa) hydrogels with IL-1{beta} was sufficient to reduce Thy-1 surface expression and induce v{beta}3 integrin activation. As expected, Thy-1 negative fibroblasts exhibited elevated v{beta}3 integrin activation but surprisingly, Thy-1 negative fibroblasts also expressed higher levels of IL-1RI, potentially linking the immuno-responsive and mechanosensitivity of this fibroblast subpopulation. Leveraging the non-resolving fibrosis that occurs in Thy-1-/- mice, we observed that crossing Thy-1-/- mice onto the IL-1RI-/- background was sufficient to reduce fibrosis. Together, these data indicate that Thy-1 negative fibroblasts are an immuno-responsive subpopulation that also display altered mechanotransduction, potentially serving as the link between the noted inflammation and aberrant mechanotransduction observed in IPF.


title: Designing single-cell experiments to harvest fluctuation noise while rejecting measurement noise

abstract: Measurement error is a complicating factor that reduces or distorts the information contained in any experiment. This problem becomes even more serious in the context of experiments that are specifically designed to quantify single-cell gene expression heterogeneity, where important RNA and protein copy numbers are themselves subject to the inherently random fluctuations of biochemical reactions. It is not clear how measurement noise should be managed, in addition to other experiment design variables such as sampling size and frequency, to ensure that collected data will provide useful insights on gene expression mechanisms of interest. To address these fundamental single-cell experiment design challenges, we propose a computational framework that makes explicit use of measurement error modeling and Fisher Information Matrix-based criteria to decide between experimental methods. This unified approach not only quantifies how different noise characteristics affect uncertainty in parameter estimation, but also enables a systematic approach to design hybrid experiments that combine different measurement modalities.

category: systems biology


title: Designing single-cell experiments to harvest fluctuation information while rejecting measurement noise


title: Mitogen-activated protein kinases contribute to temperature induced cardiac remodelling in rainbow trout (Oncorhynchus mykiss) heart

abstract: Rainbow trout (Oncorhynchus mykiss) live in temperate environments and experience seasonal changes in temperature that range between 4{degrees}C and 20{degrees}C. Laboratory studies demonstrate that cold and warm acclimation of male trout can have oppositional effects on cardiac hypertrophy and the collagen content of the heart. The cellular mechanisms behind temperature induced cardiac remodelling are unclear, as is why this response differs between male and female fish. Recent work utilizing cultured trout cardiac fibroblasts suggests that collagen deposition is regulated, at least in part, by mitogen-activated protein kinase (MAPK) cell signalling pathways. We therefore hypothesized that temperature-dependent cardiac remodelling is regulated by these same cell signalling pathways. To test this, male and female trout were acclimated to 18{degrees}C (warm) in the summer and to 4{degrees}C (cold) in the winter and the activation of MAPK pathways in the hearts were characterized and compared to that of control fish maintained at 12{degrees}C. Animals, maintained under a natural photoperiod matched to time of year, were sampled throughout each acclimation. p38 MAPK phosphorylation increased in the hearts of female fish during the cold acclimation protocol and the phosphorylation of extracellular signal-regulated kinase (ERK) increased in the hearts of male fish with warm acclimation. These results indicate that thermal acclimation has transient and sex-specific effects on the phosphorylation of MAPKs.

category: zoology


title: The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

abstract:
Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila homologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and spermatocytes, the only two Drosophila cell types bearing motile cilia/flagella. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

SUMMARY STATEMENTDNAAF3 function as a dynein assembly factor for motile cilia is conserved in Drosophila.


title: Genetic analysis argues for a coactivator function for the Saccharomyces cerevisiae Tup1 corepressor

abstract: The Tup1-Cyc8 corepressor complex of Saccharomyces cerevisiae is recruited to promoters by DNA-binding proteins to repress transcription of genes, including the a-specific mating type genes. We report here a tup1(S649F) mutant that displays mating irregularities similar to a tup1 null and an -predominant growth defect. RNA-Seq and ChIP-Seq were used to analyze gene expression and Tup1 occupancy changes in mutant vs. wild-type in both a and  cells. Increased Tup1(S649F) occupancy tended to occur upstream of upregulated genes, whereas locations with decreased occupancy usually did not show changes in gene expression, suggesting this mutant not only loses corepressor function but also behaves as a coactivator. Based upon studies demonstrating a dual role of Tup1 in both repression and activation, we postulate that the coactivator function of Tup1(S649F) results from diminished interaction with repressor proteins, including 2. We also found that large changes in mating type-specific gene expression between a and  or between mutant and wild-type were not easily explained by the range of Tup1 occupancy levels within their promoters, as predicted by the classic model of a-specific gene repression by Tup1. Most surprisingly, we observed Tup1 occupancy upstream of the a-specific gene MFA2 and the -specific gene MF(ALPHA)1 in cells in which each gene was expressed rather than repressed. These results, combined with identification of additional mating related genes upregulated in the tup1(S649F)  strain, illustrate that the role of Tup1 in distinguishing mating types in yeast appears to be both more comprehensive and more nuanced than previously appreciated.

category: genetics


title: Performance of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study

abstract: The use of blood-based extracellular RNA (exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated 10 blood collection tubes, 3 time points between blood draw and downstream processing, and 8 RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics is assessed by deep transcriptome profiling of both small and messenger RNA from healthy donors plasma or serum. Experiments are conducted in triplicate (for a total of 276 transcriptomes) using 189 synthetic spike-in RNAs as processing controls. When comparing blood tubes, so-called blood preservation tubes do not stabilize RNA very well, as is reflected by increasing RNA concentration and number of detected genes over time, and by compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow for your own experiments. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated standard operating procedures (SOPs) for processing, representing paramount groundwork for future exRNA-based precision medicine applications.

category: genomics


title: Convergent loss of chemoreceptors across independent origins of slave-making in ants

abstract: The evolution of an obligate parasitic lifestyle often leads to the reduction of morphological and physiological traits, which may be accompanied by loss of genes and functions. Slave-maker ants are social parasites that exploit the work force of closely related ant species for social behaviours such as brood care and foraging. Recent divergence between these social parasites and their hosts enables comparative studies of gene family evolution. We sequenced the genomes of eight ant species, representing three independent origins of ant slavery. During the evolution of eusociality, chemoreceptor genes multiplied due to the importance of chemical communication in societies. We investigated evolutionary patterns of chemoreceptors in relation to slave-making in ants. We found that slave-maker ant genomes harboured only half as many gustatory receptors as their hosts, potentially mirroring the outsourcing of foraging tasks to host workers. In addition, parasites had fewer odorant receptors and their loss shows patterns of convergence across origins of parasitism, representing a rare case of convergent molecular evolution. This convergent loss of specific odorant receptors suggests that selective deprivation of receptors is adaptive. The 9-exon odorant receptor subfamily, previously linked to social evolution in insects, was significantly enriched for convergent loss across the three origins of slavery in our study, indicating that the transition to social parasitism in ants is accompanied by the loss of receptors that are likely important for mediating eusocial behaviour. Overall, gene loss in slave-maker ants suggests that a switch to a parasitic lifestyle accompanies relaxed selection on chemical perception.


title: Integrated phospho-proteogenomic and single-cell transcriptomic analysis of meningiomas establishes robust subtyping and reveals subtype-specific immune invasion

abstract: Meningiomas are the most frequent primary intracranial tumors. They can follow a wide clinical spectrum from benign to highly aggressive clinical course. No specific therapy exists for refractory cases or cases not amenable to resection and radiotherapy. Identification of risk of recurrence and malignant transformation for the individual patients is challenging. However, promising molecular markers and prognostic subgrouping by DNA methylation are emerging. Still, the biological underpinnings of these diagnostic subgroups are elusive, and, consequently, no novel therapeutic options arise thereof. Here we establish robust subgroups across the full landscape of meningiomas, consistent through DNA methylation, mutations, the transcriptomic, proteomic and phospho-proteomic level. Pronounced proliferative stress and DNA damage repair signals in malignant cells and in clusters exclusive to recurrent tumors are in line with their higher mitotic activity, but also provide an explanation for the accumulation of genomic instability in anaplastic meningiomas. Although homozygous deletion of CDKN2A/B is a diagnostic marker of high-grade meningioma, the expression of its gene product increased from low to non-deleted high-grade cases. Differences between subgroups in lymphocyte and myeloid cell infiltration, representing a majority of tumor mass in low-grade NF2 tumors, could be assigned to cluster-specific interaction with tumor cells. Activation to a more proinflammatory phenotype and decreased infiltration of myeloid cells in high-grade cases correlated with lower expression of CSF1, located on chromosome arm 1p, whose deletion is known as prognostic marker, with no proposed mechanism before. Our results demonstrate a robust molecular subclassification of a tumor type across multiple layers, provide insight into heterogeneous growth dynamics despite shared pathognomonic mutations, and highlight immune infiltration modulation as a novel target for meningioma therapy.


title: O-GlcNAcylation reduces phase separation and aggregation of the EWS N-terminal low complexity region

abstract:
Many membraneless organelles are thought to be biomolecular condensates formed by phase separation of proteins and other biopolymers. Post-translational modifications (PTMs) can impact protein phase separation behavior, although for many PTMs this aspect of their function is unknown. O-linked {beta}-D-N-acetylglucosaminylation (O-GlcNAcylation) is an abundant form of intracellular glycosylation whose roles in regulating biomolecular condensate assembly and dynamics have not been delineated. Using an in vitro approach, we found that O-GlcNAcylation reduces the phase separation propensity of the EWS N-terminal low complexity region (LCRN) under different conditions, including in the presence of the arginine-and glycine-rich RNA-binding domains (RBD). O-GlcNAcylation enhances fluorescence recovery after photobleaching (FRAP) within EWS LCRN condensates and causes the droplets to exhibit more liquid-like relaxation following fusion. Following extended incubation times, EWS LCRN+RBD condensates exhibit diminished FRAP, indicating a loss of fluidity, while condensates containing the O-GlcNAcylated LCRN do not. In HeLa cells, EWS is less O-GlcNAcylated following OGT knockdown and more prone to aggregation based on a filter retardation assay. Relative to the human proteome, O-GlcNAcylated proteins are enriched with regions that are predicted to phase separate, suggesting a general role of O-GlcNAcylation in regulation of biomolecular condensates.

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category: biochemistry


title: Atmospheric plasma jet device for versatile electron microscope grid treatment

abstract: Atmospheric pressure plasmas have been widely applied in surface modification and biomedical treatment due to its ability to generate highly reactive radicals and charged particles. In negative-stain electron microscopy (Neg-EM) and cryogenic electron microscopy (cryo-EM), plasmas have been used in eliminating the surface contaminants as well as generating the hydrophilic surface to embed the specimen on grids. Plasma treatment is a prerequisite for negative stain and quantifoil grids, which are coated with hydrophobic carbon on the grid surface. Here we introduce a non-thermal atmospheric plasma jet system as an alternative new tool for surface treatment. Unlike the conventional glow discharger, we found that the plasma jet system successfully cleans the grid surface and introduces hydrophilicity on grids in the ambient environment without introducing a vacuum. Therefore, we anticipate the plasma jet system will be beneficial in many aspects, such as cost-effective, convenient, versatile, and potential applications in surface modification for both negative stain and cryo-EM grid treatment.


title: Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

abstract: CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3 end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5 end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co-expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

category: bioengineering


title: Probing the Structural Basis of Citrus Phytochrome B using Computational Modelling and Molecular Dynamics Simulation Approaches

abstract:
Phytochromes (Phys) are known as red/far-red light photoreceptors and are responsible for directing the photosensory responses across the species, majorly from fungal, bacterial and plant kingdoms. Such responses majorly include photosynthetic potential and pigmentation in bacteria, whereas in a plant, they are involved in chloroplast development and photomorphogenesis. Many prokaryotic Phys have been modelled for their structural and functional analysis, but their plant counterparts have not been explored yet. To date, only the crystal structures of the photo-sensing module (PSM) of PhyB isoform from Arabidopsis thaliana and Glycine max have been resolved experimentally. Thus, in this study, we elucidated the complete 3D structure of Citrus PhyB. Initially, the structure and organisation of the Citrus PhyB have been predicted computationally, which were found to have the same domain organisation as A. thaliana and G. max PhyBs, yet their considerable distinct structural difference indicated potential divergence in signaling and functioning. Therefore, to evaluate the structural and functional implications of Citrus PhyB, we compared its structure with A. thaliana and G. max PhyBs using molecular dynamics (MD) simulation approaches. The modeling studies revealed that the region of Citrus PhyB-GAF domain possibly contributes to the variations between Citrus, A. thaliana and G. max PhyBs structures/functions. Hence, structural and molecular insights into Citrus PhyB can help to discover the Phys signaling and thus, an essential framework can be designed for optogenetic reagents and various agricultural/horticulture benefits.

One sentence summaryA complete Citrus PhyB structure together with photo-sensory and out-put modules provides significant information to evaluate its biological activities for agricultural benefits.

category: biophysics


title: PIP2-dependent coupling of voltage sensor and pore domains in Kv7.2

abstract: Phosphatidylinositol-4,5-bisphosphate (PIP2) is a signaling lipid which regulates voltage-gated Kv7/KCNQ potassium channels. Altered PIP2 sensitivity of neuronal Kv7.2 channel is involved in KCNQ2 epileptic encephalopathy. However, the molecular action of PIP2 on Kv7.2 gating remains largely elusive. Here, we use molecular dynamics simulations and electrophysiology to characterize PIP2 binding sites in a human Kv7.2 channel. In the closed state, PIP2 localizes to the periphery of the voltage-sensing domain (VSD). In the open state, PIP2 binds to 4 distinct interfaces formed by the cytoplasmic ends of the VSD, the gate, intracellular helices A and B and their linkers. PIP2 binding induces bilayer-interacting conformation of helices A and B and the correlated motion of the VSD and the pore domain, whereas charge-neutralizing mutations block this coupling and reduce PIP2 sensitivity of Kv7.2 channels by disrupting PIP2 binding. These findings reveal the allosteric role of PIP2 in Kv7.2 channel activation.


title: Effect of plant tubulin kinetic diversification on microtubule lengths

abstract: Microtubules (MTs) are dynamic polymers vital for cellular physiology. Bulk tubulin polymerization is nucleation dependent, while individual filaments exhibit  dynamic instability driven by GTP hydrolysis rates. Although MTs assembled from well-studied animal brain tubulins have very comparable nucleation and GTP-hydrolysis rates, the kinetic rates of evolutionarily more distant species could diverge. Here we focus on a plant tubulin, the legume Vigna sp. (mung bean) to test the effect of kinetic diversification on MT polymerization. We activity purify tubulin from seedlings and find MT filaments are fewer and shorter than animal brain tubulin. We find mung tubulin polymerization kinetics is nucleation dependent with a high rate of GTP hydrolysis and a critical concentration lower than previously reported for tubulins. A computational model of the kinetics based on the relative influence of rates of nucleation and hydrolysis demonstrates increased rates of hydrolysis can affect MT filament numbers and their lengths, as compared to increasing nucleation rates. Our approach provides a framework to compare the effect of evolutionary diversification of MT nucleation and elongation.


title: Molecular basis and functional consequences of the interaction between the Base Excision Repair DNA glycosylase NEIL1 and RPA

abstract: NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein replication protein A (RPA) modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd [~] 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against ionizing radiation-induced DNA lesions in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed that RPA interaction is not required for NEIL1 activity against oxidative damage in duplex DNA, and furthermore revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.


title: Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global crisis, urgently necessitating the development of safe, efficacious, convenient-to-store, and low-cost vaccine options. A major challenge is that the receptor-binding domain (RBD)-only vaccine fails to trigger long-lasting protective immunity if used solely for vaccination. To enhance antigen processing and cross-presentation in draining lymph nodes (DLNs), we developed an interferon (IFN)-armed RBD dimerized by immunoglobulin fragment (I-R-F). I-R-F efficiently directs immunity against RBD to DLN. A low dose of I-R-F induces not only high titer long-lasting neutralizing antibodies but also comprehensive T cell responses than RBD, and even provides comprehensive protection in one dose without adjuvant. This study shows that the I-R-F vaccine provides rapid and complete protection throughout upper and lower respiratory tracts against high dose SARS-CoV-2 challenge in rhesus macaques. Due to its potency and safety, this engineered vaccine may become one of the next-generation vaccine candidates in the global race to defeat COVID-19.

category: immunology


title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

abstract: The pathways that regulate cytokine responses in T cells are disrupted in autoimmunity, immune deficiencies, and cancer, and include immunotherapy targets. Systematic discovery of cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now have accomplished genome-wide pooled CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) screens in primary human T cells to map gene networks controlling Interleukin-2 and Interferon-{gamma} production. Arrayed CRISPRa confirmed key hits and enabled multiplexed T cell secretome characterization, revealing reshaped cytokine responses driven by individual regulators. CRISPRa uncovered genes not canonically expressed in T cells, including the transcription factor FOXQ1, whose overexpression promoted the expression of most cytokines, while selectively dampening T helper 2 (Th2) cytokines. Paired CRISPRa and CRISPRi screens reveal signaling components that tune critical immune cell functions, which could inform design of future immunotherapies.


title: Neutralization potential of Covishield vaccinated individuals against B.1.617.1

abstract: Covishield comprises the larger proportion in the vaccination program in India. Hence, it is of utmost importance to understand neutralizing capability of vaccine against the B.1.617.1 variant which is considered to responsible for surge of the cases in India. The neutralizing-antibody (NAb) titer against B.1.167.1 and prototype B.1 variant (D614G) was determined of the vaccine sera (4 weeks after second dose) of COVID-19 naive subjects (n=43) and COVID-19 recovered subjects (n=18). The results demonstrated that sera of COVID-19 recovered subjects (n=18) who received two doses of Covishield have higher NAb response compared to the COVID-19 naive with a significant difference (p<0.0001) in NAb titer against B.1 and B.1.617.1 In-spite of reduction in the neutralizing titer against B.1.617.1 variant; Covishield vaccine-induced antibodies are likely to be protective to limit the severity and mortality of the disease in the vaccinated individuals.


title: Neutralization potential of Covishield vaccinated individuals sera against B.1.617.1


title: Acquired Drug Resistance Enhances Imidazoquinoline Efflux by P-glycoprotein

abstract: Multidrug-Resistant (MDR) cancers mitigate the action of chemotherapeutics through drug efflux that occurs via ABC (ATP-Binding Cassette) transporters, including P-glycoprotein 1 (P-gp or ABCB1/MDR1). Because Toll-Like Receptor (TLR) agonist immunotherapies elicit abscopal anti-tumoral effects by modulating the activity of bystander tumor infiltrating immune cells, they not only circumvent the neutralizing effects of drug efflux, but could also work in synergy with this process. However, the effect of drug resistance on TLR agonist efflux is largely unknown. We begin to address this by investigating P-gp mediated efflux of model TLR agonists in cancer cell lines before and after acquired drug resistance. First, we used functionalized liposomes to determine that imidazoquinoline TLR agonists Imiquimod, Resiquimod, and Gardiquimod are substrates for P-gp. Next, we created Doxorubicin-resistant cancer cell lines from B16 melanoma, TRAMP prostate, and 4T1 breast cancer and observed that each cell line increased P-gp expression in response to Doxorubicin. Comparing imidazoquinoline efflux in Doxorubicin-resistant cell lines, relative to parent cancer cell lines, we used P-gp competitive substrates and inhibitors to demonstrate that imidazoquinoline efflux occurs through P-gp and is enhanced as a consequence of acquired drug resistance. We found that the most hydrophobic, yet least potent imidazoquinoline (Imiquimod), was the best substrate for efflux. This suggests a new parameter, susceptibility to drug efflux, could be an important consideration in the rationale design of the next generation of TLR agonist immunotherapies that are targeted to cancer cells, yet effect their mechanisms of action by modulating the activity of tumor infiltrating immune cells.

category: cancer biology


title: KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in P. falciparum-infected erythrocytes

abstract: The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually co-localizes with remnant actin junctions. We further present a 35[A] map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected [~]60 KAHRP molecules per knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.

category: microbiology


title: A novel lineage of polyomaviruses identified in bark scorpions

abstract: Polyomaviruses are non-enveloped viruses with circular double-stranded DNA genomes that range in size from [~]4-7 kilobasepairs. Initially identified in mammals, polyomaviruses have now been identified in birds and a few fish species. Although fragmentary polyomavirus-like sequences have been detected as apparent  hitchhikers in shotgun genomics datasets for various arthropods, the possible diversity of these viruses in invertebrates remains unclear. In general, polyomaviruses are host-specific, showing strong evidence of host-virus co-evolution. Identification of polyomaviruses in a broader range of animals could shed useful light on the evolutionary history of this medically important group of viruses. Scorpions are predatory arachnids that are among the oldest terrestrial animals. Thus far, viromes of arachnids have been under-sampled and understudied. Here, high-throughput sequencing and traditional molecular techniques were used to explore the diversity of circular DNA viruses associated with bark scorpions (Centruroides sculpturatus) from the greater Phoenix area, Arizona, USA. The complete genomes of eight novel polyomaviruses were identified. Analysis of Centruroides transcriptomic datasets elucidated the splicing of the viral late gene array, which is more complex than that of vertebrate polyomaviruses. Phylogenetic analysis provides further evidence of co-divergence of polyomaviruses with their hosts, suggesting that at least one ancestral species of polyomaviruses was circulating amongst the primitive common ancestors of arthropods and chordates.


title: A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli

abstract:
As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation as well as virulence. sRNAs can alter target mRNA translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify new regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in E. coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX and McaS, previously found to act through Hfq competition, RNase T, a 3'-5' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two new specific regulators were identified. AspX, a novel 3'end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.

SignificanceThe ability to promptly switch genes on and off allows bacteria to adapt rapidly to changing environments for better survival. Many sRNAs, including RyhB, a sRNA made in response to iron starvation, are important switches in bacteria. We discovered new factors that can keep the sRNA switch off by using a facile genetic screen platform. These new factors include a new RNA sponge and an adaptor protein for a ribonuclease, providing new perspectives on controlling sRNA signaling in bacteria.


title: OsbZIP47 an integrator for meristem regulators during rice plant growth and development

abstract:
Stem cell homeostasis by the WUS-CLV negative feedback loop is generally conserved across species; however, its links with other meristem regulators may have species-specific distinctions, rice being an example. We characterize rice OsbZIP47 for vegetative and inflorescence phenotypes in knockdown (OsbZIP47KD) transgenics and uncover its role in meristem maintenance and developmental progression. The shoot apical meristem (SAM) size in five day old OsbZIP47KD seedlings, was reduced as compared to the wild-type (WT). Whereas SAM in older twenty-five-day OsbZIP47KD plants was larger with increased size for L1 and underlying cells. We tested protein interactions of OsbZIP47 with other transcription factors and found partnerships with OsMADS1, RFL, and OSH1. Results from meta-analysis of deregulated panicle transcriptome datasets, in OsbZIP47KD, OsMADS1KD and RFLKD knockdown transgenics, and OSH1 genome-wide binding sites divulge potential targets coregulated by OsbZIP47, OsMADS1, OSH1 and RFL. Transcript analysis in OsbZIP47KD SAM and panicles showed abnormal gene expression for CLAVATA peptide-like signaling FON2-LIKE CLE PROTEIN1 (FCP1), FLORAL ORGAN NUMBER 2 (FON2), and hormone pathway: cytokinin (CK) Isopenteyltransferase2 (OsIPT2), Isopenteyltransferase8 (OsIPT8); auxin biosynthesis OsYUCCA6, OsYUCCA7; gibberellic acid (GA) biosynthesis GA20Ox1, GA20Ox4 and brassinosteriod biosynthesis CYP734A4 genes. The effects on ABBERANT PANICLE ORGANIZATION1 (APO1), OsMADS16, and DROOPING LEAF relate to second and third whorl organ phenotypes in OsbZIP47KD florets. Further, we demonstrate that OsbZIP47 redox status affects its DNA binding to cis elements in the FCP1 locus. Taken together, we provide insights on unique functional roles for OsbZIP47 in rice shoot meristem maintenance, its progression through inflorescence branching and floret development.

One sentence summaryOsbZIP47 regulates rice shoot meristem size, panicle and floret development in concert with other meristem regulators such as OsMADS1, RFL and OSH1.

category: plant biology


title: Modeling SARS-CoV-2 and Influenza Infections and Antiviral Treatments in Human Lung Epithelial Tissue Equivalents

abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.


title: The repeat region of the circumsporozoite protein is an elastic linear spring with a functional role in Plasmodium sporozoite motility

abstract:
The circumsporozoite protein (CSP) forms a dense coat on the surface of the sporozoite, the infective stage of the malaria parasite. The central repeat region of CSP is a critical component of the only licensed malaria vaccine yet little is known about its structure or function. We found that sporozoite mutants with severely truncated or scrambled repeats have impaired motility due to altered adhesion site formation and dynamics, suggesting that the CSP repeats provide a cohesive environment in which adhesion sites can form. We hypothesized that biophysical properties of the repeats are important in this role and interrogated this using single-molecule fluorescence-force spectroscopy. We show that the repeats are a stiff, linear spring with elastic properties, dependent upon length and lost when the repeats are scrambled. These data are the first evidence that the CSP repeat region serves a functional role during infection and motility, likely mediated through its biophysical properties.

SummaryNo clear function of the central repeat region of the malaria circumsporozoite protein has been described to date, despite its central role in the only licensed malaria vaccine. Here we use mutational analysis and single-molecule fluorescence-force spectroscopy to describe the structural properties and determine the function of this conserved region and important vaccine target.

HighlightsThe CSP repeats have properties of a linear spring

Scrambling or large truncations of the repeats leads to defects in sporozoite motility

Motility defects are attributed to abnormal formation of adhesion sites


title: Unravelling consensus genomic regions conferring leaf rust resistance in wheat via meta-QTL analysis

abstract: Leaf rust, caused by the fungus Puccinia triticina Erikss (Pt), is a destructive disease affecting wheat and a threat to food security. Developing resistant varieties represents a useful method of disease control, and thus, understanding the genetic basis for leaf rust resistance is required. To this end, a comprehensive bibliographic search for leaf rust resistance quantitative trait loci (QTLs) was performed, and 393 QTLs were collected from 50 QTL mapping studies. Afterwards, a consensus map with a total length of 4567 cM consisting of different types of markers (SSR, DArT, Chip-based SNP markers and SNP markers from GBS) was used for QTL projection, and meta-QTL analysis was performed on 320 QTLs. A total of 75 genetic map positions (gmQTLs) were discovered and refined to 15 high confidence mQTLs (hcmQTLs). The candidate genes discovered within the hcmQTL interval were then checked for differential expression using data from three transcriptome studies, resulting in 92 differentially expressed genes (DEGs). The expression of these genes in various leaf tissues during wheat development was explored. This study provides insight into leaf rust resistance in wheat and thereby provides an avenue for developing resistant varieties by incorporating the most important hcmQTLs.


title: Conservation of isolate substrate preferences in mixed communities revealed through ribosomal marker protein profiling

abstract: Assessment of structure-function relationships is a central theme in microbial ecology. However, the degree that isolate metabolic activities are conserved in communities remains unclear. This is because tracking population dynamics and substrate partitioning in microbial communities remains technically challenging. Here, we describe the application of a mass spectrometry-based ribosomal marker protein profiling with stable isotope probing approach that allows for concurrent monitoring of community structure dynamics and resource assimilation within a five-member synthetic soil bacterial community. Using this approach, we find that isolate substrate preferences for glutamine and phenylalanine are largely conserved in the community and can be predicted using a weighted-sum model. However, time-series monitoring revealed a significant delay in phenylalanine incorporation by two of the strains, as well as enhanced growth for Variovorax paradoxus presumably due to interspecies interactions. The unique utility of this approach to temporally probe resource incorporation and community structure enables deciphering the dynamic interactions occurring within the community. Extension of this approach to other communities under various environmental perturbations is needed to reveal the generality of microbial conservation of substrate preferences.


title: Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

abstract: DWARF14 (D14) is an [a]/{beta}-hydrolase and receptor for the plant hormone strigolactone (SL) in angiosperms. Upon SL perception, D14 works with MORE AXILLARY GROWTH2 (MAX2) to trigger polyubiquitination and degradation of DWARF53(D53)-type proteins in the SUPPRESSOR OF MAX2 1-LIKE (SMXL) family. We used CRISPR-Cas9 to generate knockout alleles of the two homoeologous D14 genes in the Nicotiana benthamiana genome. The Nbd14a,b double mutant had several phenotypes that are consistent with the loss of SL perception in other plants, including increased axillary bud outgrowth, reduced height, shortened petioles, and smaller leaves. A ratiometric fluorescent reporter system was used to monitor degradation of SMXL7 from Arabidopsis thaliana (AtSMXL7) after transient expression in N. benthamiana and treatment with the strigolactone analog GR24. AtSMXL7 was degraded after treatment with GR245DS, which has the stereochemical configuration of SLs, as well as its enantiomer GR24ent-5DS . In Nbd14a,b leaves, AtSMXL7 abundance was unaffected by GR24. Transient coexpression of AtD14 with the AtSMXL7 reporter in Nbd14a,b restored the degradation response to GR24, but required an active catalytic triad. With this platform, we evaluated the ability of several AtD14 mutants that had not been characterized in plants to target AtSMXL7 for degradation.


title: Dissecting and Tuning Primer Editing by Proofreading Polymerases

abstract: Proofreading polymerases have 3 to 5 exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples.


title: Uncoupling of Chromatin Assembly from DNA Replication in Sciara Reveals a Domain of Postreplicative Immature Chromatin

abstract: DNA replication in dividing eukaryotic cells imposes a requirement for the faithful recreation on the newly synthesized chromatids of the nucleoprotein architecture of parent chromosomes. Practically nothing is known about the structure of postreplicative immature chromatin--a very short-lived entity (< 30 min.). We report here the unexpected discovery that during DNA amplification of locus II/9A in salivary gland polytene chromosomes of the fungus fly Sciara coprophila, DNA replication fork passage is uncoupled from postreplicative chromatin assembly; this enables visualization and analysis of chromatin fibers disassembled by DNA replication. We used electron microscopy to visualize a wealth of low nucleosome density immature chromatin fibers in preparations of Sciara chromatin from amplification-stage tissue. Remarkably, as gauged by high sensitivity to micrococcal nuclease and an unusually short length of DNA associated with each histone octamer, we found that locus II/9A which undergoes amplification and is replicated once every 4-6 hrs.--but not the bulk genome or a replicatively quiescent DNA stretch--was maintained in such an ummature fiber for ca. 24 hrs. Following amplification, locus II/9A assumed conventional chromatin organization, indicating that the epigenetic mark targeting nascent DNA to the chromatin assembly machinery is stable for several hours. We propose that this very unusual prolonged maintenance of a segment of the genome in immature chromatin facilitates access by the basal transcriptional machinery to the amplified DNA, and thus is an evolutionary adaptation to the demand for high transcription from genes that reside in the amplified loci.


title: Exploiting noise, nonlinearity, and feedback to differentially control multiple synthetic cells with a single optogenetic input

abstract: Synthetic biology seeks to develop modular bio-circuits that combine to produce complex, controllable behaviors. These designs are often subject to noisy fluctuations and uncertainties, and most modern synthetic biology design processes have focused to create robust components to mitigate the noise of gene expression and reduce the heterogeneity of single-cell responses. However, deeper understanding of noise can achieve control goals that would otherwise be impossible. We explore how an "Optogenetic Maxwell Demon" could selectively amplify noise to control multiple cells using single-input-multiple-output (SIMO) feedback. Using data-constrained stochastic model simulations and theory, we show how an appropriately selected stochastic SIMO controller can drive multiple different cells to different user-specified configurations irrespective of initial condition. We explore how controllability depends on cells regulatory structures, the amount of information available to the controller, and the accuracy of the model used. Our results suggest that gene regulation noise, when combined with optogenetic feedback and non-linear biochemical auto-regulation, can achieve synergy to enable precise control of complex stochastic processes.

category: synthetic biology


title: Evaluating brain parcellations using the distance controlled boundary coefficient

abstract: One important approach to human brain mapping is to define a set of distinct regions that can be linked to unique functions. Numerous brain parcellations have been proposed, using cytoarchitectonic, structural or functional Magnetic Resonance Imaging (fMRI) data. The intrinsic smoothness of brain data, however, poses a problem for current methods seeking to compare different parcellations. For example, criteria that simply compare within-parcel to between-parcel similarity provide even random parcellations with a high value. Furthermore, the evaluation is biased by the spatial scale of the parcellation. To address this problem, we propose the Distance Controlled Boundary Coefficient (DCBC), an unbiased criterion to evaluate discrete parcellations. We employ this new criterion to evaluate existing parcellations of the human neocortex in their power to predict functional boundaries for an fMRI data set with many different tasks, as well as for resting-state data. We find that common anatomical parcellations do not perform better than chance, suggesting that task-based functional boundaries do not align well with sulcal landmarks. Parcellations based on resting-state fMRI data perform well; in some cases, as well as a parcellation defined on the evaluation data itself. Finally, multi-modal parcellations that combine functional and anatomical criteria perform substantially worse than those based on functional data alone, indicating that functionally homogeneous regions often span major anatomical landmarks. Overall, the DCBC advances the field of functional brain mapping by providing an unbiased metric that compares the predictive ability of different brain parcellations to define brain regions that are functionally maximally distinct.

category: neuroscience


title: The macrophage reprogramming ability of antifolates reveals soluble CD14 as a potential biomarker for methotrexate response in rheumatoid arthritis

abstract: The physio-pathological relevance of the one-carbon metabolism (OCM) is illustrated by the chemotherapeutic and anti-inflammatory effects of the antifolates Pemetrexed (PMX) and Methotrexate (MTX) in cancer and rheumatoid arthritis (RA). We report that OCM determines the functional and gene expression profile of human macrophages. PMX induces the acquisition of a p53-dependent proinflammatory gene signature in human monocyte-derived macrophages (GM-MO). Indeed, OCM blockade reprograms GM-MO towards a state of LPS-tolerance at the signaling and functional levels, an effect abolished by folinic acid. Importantly, OCM blockade led to reduced expression of membrane-bound and soluble CD14 (sCD14), whose exogenous addition restores LPS sensitivity. The therapeutic relevance of these results was confirmed in early RA patients, as MTX-responder RA patients exhibit lower sCD14 serum levels, with baseline sCD14 levels predicting MTX response. Our results indicate that OCM is a metabolic circuit that critically mediates the acquisition of innate immune tolerance, and positions sCD14 as a valuable tool to predict MTX-response in RA patients.


title: Spatiotemporal single cell transcriptomic analyzis of human gut macrophages revealsmultiple functional and niche-specific subsets

abstract: Macrophages are a heterogeneous population of cells involved in tissue homeostasis, inflammation and cancer. Although macrophages are densely distributed throughout the human intestine, our understanding of how gut macrophages maintain tissue homeostasis is limited. Here we show that colonic lamina propria (LpM) and muscularis macrophages (MM) consist of monocyte-like cells that differentiate into multiple transcriptionally distinct subsets. LpM comprise subsets with proinflammatory properties and subsets high antigen presenting and phagocytic capacity. The latter are strategically positioned close to the surface epithelium. Most MM differentiate along two trajectories; one that upregulates genes associated with immune activation and angiogenesis, whereas the other upregulates genes associated with neuronal homeostasis. Importantly, MM are located adjacent to neurons and vessels. Cell-cell interaction and gene network analysis indicated that survival, migration, transcriptional reprogramming, and niche-specific localization of LpM and MM are controlled by an extensive interaction with tissue-resident cells and a few key transcription factors.


title: Natural killer cell immunosuppressive function requires CXCR3-dependent redistribution within lymphoid tissues

abstract: Natural killer (NK) cell suppression of T cells is a key determinant of viral pathogenesis and vaccine efficacy. This process involves perforin-dependent elimination of activated CD4 T cells during the first three days of infection. Although this mechanism requires cell-cell contact, NK cells and T cells typically reside in different compartments of lymphoid tissues at steady state. Here, we show that NK-cell suppression of T cells is associated with a transient accumulation of NK cells within T cell-rich sites of the spleen during lymphocytic choriomeningitis virus infection. The chemokine receptor CXCR3 is required for relocation to T-cell zones and suppression of antiviral T cells. Accordingly, this NK-cell migration is mediated by type I interferon (IFN)-dependent promotion of CXCR3 ligand expression. In contrast, adenoviral vectors that weakly induce type I IFN and do not stimulate NK-cell inhibition of T cells also do not promote measurable redistribution of NK cells to T-cell zones. Provision of supplemental IFN could rescue NK-cell migration during adenoviral vector immunization. Thus, type I IFN and CXCR3 are critical for properly positioning NK cells to constrain antiviral T-cell responses. Development of strategies to curtail migration of NK cells between lymphoid compartments may enhance vaccine-elicited immune responses.


title: Cell strain-derived induced pluripotent stem cell as a genetically controlled approach to investigating aging mechanisms and viral pathogenesis

abstract: The expansion of the geographic footprint of dengue viruses (DENVs) and their mosquito vectors have affected more than half of the global population, including older adults who appear to show elevated risk of severe dengue. Despite this epidemiological trend, how age and senescence impact virus-host interactions involved in dengue pathogenesis to increase the risk of severe dengue is poorly understood. Herein, we show that conversion of diploid cells with finite lifespan into iPSCs followed by differentiation back into cell strain can be an approach to derive genetically identical cells at different stages of senescence to study virus and aging host interactions. Our findings show that cellular senescence impact the host response to infection and the ensuing outcome. We suggest iPSC-derive cell strains as a potentially useful technical approach to genetically controlled host-virus interaction studies to understand how aging impact viral pathogenesis.


title: Kinase independent function of PI3Kγ modulates calcium re-uptake by regulating phospholamban function

abstract:
RationaleGenetic deletion of Phosphoinositide 3-kinase (PI3K{gamma}) in mice (PI3K{gamma}-/-) results in increased cAMP levels and enhanced ventricular rate/contractility. Whether PI3K{gamma} plays a role in cardiac contractility by altering intracellular calcium recycling is not known.

ObjectiveTo understand the mechanism of PI3K{gamma} mediated regulation of cardiac contractility.

Methods and ResultsCaffeine treatment of adult cardiomyocytes from PI3K{gamma}-/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3K{gamma} locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3K{gamma}-/- cardiomyocytes show significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Consistently, the altered calcium regulation in the cardiomyocytes of PI3K{gamma}-/- can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyate-specific overexpression of kinase-dead PI3K{gamma} PI3K{gamma}inact) in the global PI3K{gamma}-/- cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A.

ConclusionsThese studies bring-to-fore an unrecognized regulation of PLN by PI3K{gamma} through PP2A with implications in deleterious cardiac remodeling as PI3K{gamma} is significantly upregulated following cardiac stress.


title: Engineered type six secretion systems deliver active exogenous effectors and Cre recombinase

abstract:
Genetic editing has revolutionized biotechnology but delivery of endonuclease genes as DNA can lead to aberrant integration or overexpression, leading to off-target effects. Here we develop a mechanism to deliver Cre recombinase as a protein by engineering the bacterial type six secretion system (T6SS). Using multiple T6SS fusion proteins, Aeromonas dhakensis or attenuated Vibrio cholerae donor strains, and a gain-of-function cassette for detecting Cre recombination, we demonstrate successful delivery of active Cre directly into recipient cells. Most efficient transfer was achieved using a truncated version of PAAR2 from V. cholerae, resulting in a relatively small (118 amino acid)  delivery tag. We further demonstrate the versatility of this system by delivering an exogenous effector, TseC, enabling V. cholerae to kill Pseudomonas aeruginosa. This implicates that P. aeruginosa is naturally resistant to all native effectors of V. cholerae and that the TseC chaperone protein is not required for its activity. Moreover, it demonstrates that the engineered system can improve T6SS efficacy against specific pathogens, proposing future application in microbiome manipulation or as a next-generation antimicrobial. Inexpensive and easy to produce, this protein delivery system has many potential applications ranging from studying T6SS effectors to genetic editing.

ImportanceDelivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function. Here we harness the T6SS to successfully deliver Cre recombinase to genetically edit bacteria without requiring the introduction of exogenous DNA into the recipient cells. This demonstrates a promising advantage over current genetic editing tools that require transformation or conjugation of DNA. The engineered secretion tag can also deliver a heterologous antimicrobial toxin that kills an otherwise unsusceptible pathogen, Pseudomonas aeruginosa. These results demonstrate the potential of T6SS-mediated delivery in areas including genome editing, killing drug-resistant pathogens, and studying toxin functions.


title: Detecting the ecological footprint of selection

abstract: The structure of communities is influenced by many processes, both ecological and evolutionary, but the influence of these processes on biodiversity patterns often remains unclear. The aim of this work is to distinguish the ecological footprint of differing formulations of competition and environmental filtering from that of neutral processes that are invariant to species identity. We build our work on "massive eco-evolutionary synthesis simulations" (MESS), which uses information from three biodiversity axes - species richness and abundance; population genetic diversity; and trait variation in a phylogenetic context - to distinguish between processes with a mechanistic model. We add a new form of competition to MESS that explicitly compares the traits of each pair of individuals, allowing us to distinguish between inter- and intra-specific competition. We find that this addition is essential to properly detect and characterise competition, yielding different results than the existing simpler model that only compares species traits to the community mean. We find from model selection that neutral forces receive much less support from real systems when trait data is available and incorporated into the inference algorithm.

category: ecology


title: Machine learning algorithms can predict tail biting outbreaks in pigs using feeding behaviour records

abstract: Tail biting is a damaging behaviour that impacts the welfare and health of pigs. Early detection of precursor sig outbreaks, using feeding behaviour data recorded by an electronic feeder. Prediction capacities of seven machine learning algorithms (Generalized Linear Model with Stepwise Feature Selection, random forest, Support Vector Machines with Radial Basis Function Kernel, Bayesian Generalized Linear Model, Neural network, K-nearest neighbour, and Partial Least Squares Discriminant Analysis) were evaluated from daily feeding data collected from 65 pens originating from two herds of grower-finisher pigs (25-100kg), in which 27 tail biting events occurred. Data were divided into training and testing data in two different ways, either by randomly splitting data into 75% (training set) and 25% (testing set), or by randomly selecting pens to constitute the testing set. In the first data splitting, the model is regularly updated with previous data from the pen, whereas in the second data splitting, the model tries to predict for a pen that it has never seen before. The K-nearest neighbour algorithm was able to predict 78% of the upcoming events with an accuracy of 96%, when predicting events in pens for which it had previous data. The detection of events for unknown pens was less sensitive, and the neural network model was able to detect 14% of the upcoming events with an accuracy of 63%. Our results indicate that machine learning models can be considered for implementation into automatic feeder systems for real-time prediction of tail biting events.

category: animal behavior and cognition


title: The recent evolutionary rescue of a staple crop depended on over half a century of global germplasm exchange

abstract: Rapid environmental change can lead to extinction of populations or evolutionary rescue via genetic adaptation. In the past several years, smallholder and commercial cultivation of sorghum (Sorghum bicolor), a global cereal and forage crop, has been threatened by a global outbreak of an aggressive new biotype of sugarcane aphid (SCA; Melanaphis sacchari). Here we characterized genomic signatures of adaptation in a Haitian sorghum breeding population, which had been recently founded from admixed global germplasm, extensively intercrossed, and subjected to intense selection under SCA infestation. We conducted evolutionary population genomics analyses of 296 post-selection Haitian lines compared to 767 global accessions at 159,683 single nucleotide polymorphisms. Despite intense selection, the Haitian population retains high nucleotide diversity through much of the genome due to diverse founders and an intercrossing strategy. A genome-wide fixation (FST) scan and geographic analyses suggests that adaptation to SCA in Haiti is conferred by a globally-rare East African allele of RMES1, which has also spread to other breeding programs in Africa, Asia, and the Americas. De novo genome sequencing data for SCA resistant and susceptible lines revealed putative causative variants at RMES1. Convenient low-cost markers were developed from the RMES1 selective sweep and successfully predicted resistance in independent U.S. x African breeding lines and eight U.S. commercial and public breeding programs, demonstrating the global relevance of the findings. Together, the findings highlight the potential of evolutionary genomics to develop adaptive trait breeding technology and the value of global germplasm exchange to facilitate evolutionary rescue.

category: evolutionary biology


title: Incorporating topological and age uncertainty into event-based biogeography supports paleo-islands in Galapagos and ancient connections among Neotropical dry forests

abstract: Event-based biogeographic methods, such as dispersal-extinction-cladogenesis, have become increasingly popular for attempting to reconstruct the biogeographic history of organisms. Such methods employ distributional data of sampled species and a dated phylogenetic tree to estimate ancestral distribution ranges. Because the input tree is often a single consensus tree, uncertainty in topology and age estimates are seldom taken into account, even when they may affect the outcome of biogeographic estimates. Even when such uncertainties are taken into account for estimates of ancestral ranges, they are usually ignored when researchers compare competing biogeographic hypotheses. We explore the effect of incorporating this uncertainty in a biogeographic analysis of the 21 species of sand spiders (Sicariidae: Sicarius) from Neotropical xeric biomes, based on a total-evidence phylogeny including a complete sampling of the genus. By using a custom R script made available here, we account for uncertainty in ages and topology by estimating ancestral ranges over a sample of trees from the posterior distribution of a Bayesian analysis, and for uncertainty in biogeographic estimates by using stochastic maps. This approach allows for counting biogeographic events such as dispersal among areas, counting lineages through time per area, and testing biogeographic hypotheses, while not overestimating the confidence in a single topology. Including uncertainty in ages indicates that Sicarius dispersed to the Galapagos Islands when the archipelago was formed by paleo-islands that are now drowned; model comparison strongly favors a scenario where dispersal took place before the current islands emerged. We also investigated past connections among currently disjunct Neotropical dry forests; failing to account for topological uncertainty underestimates possible connections among the Caatinga and Andean dry forests in favor of connections among Caatinga and Caribbean+Mesoamerican dry forests. Additionally, we find that biogeographic models including a founder-event speciation parameter ("+J") are more prone to suffer from the overconfidence effects of estimating ancestral ranges using a single topology. This effect is alleviated by incorporating topological and age uncertainty while estimating stochastic maps, increasing the similarity in the inference of biogeographic events between models with or without a founder-event speciation parameter. We argue that incorporating phylogenetic uncertainty in biogeographic hypothesis-testing is valuable and should be a commonplace approach in the presence of rogue taxa or wide confidence intervals in age estimates, and especially when using models including founder-event speciation.


title: Research gaps and new insights in the intriguing evolution of Drosophila seminal proteins

abstract: While the striking effects that seminal fluid proteins (SFPs) exert on females are fairly conserved among Diptera, they exhibit remarkable evolutionary lability. Consequently, most SFPs lack detectable homologs among the repertoire of SFPs of phylogenetically distant species. How such a rapidly changing proteome "manages" to conserve functions across taxa is a fascinating question. However, this and other pivotal aspects of SFPs evolution remain elusive because discoveries on these proteins have been mainly restricted to the model D. melanogaster. Here, we provide an overview of the current knowledge on the inter-specific divergence of Drosophila SFPs and compile the increasing amount of relevant genomic information from multiple species. Capitalizing the accumulated knowledge in D. melanogaster, we present novel sets of high-confidence SFP candidates and transcription factors presumptively involved in regulating the expression of SFPs. We also address open questions by performing comparative genomic analyses that failed to support the existence of conserved SFPs shared by most dipterans and indicated that gene co-option is the most frequent mechanism accounting for the origin of Drosophila SFP-coding genes. We hope our update establishes a starting point to integrate, as more species are assayed for SFPs, further data and thus, to widen the understanding of the intricate evolution of these proteins.


title: Biofilm self-patterning: mechanical force landscaping drives a collective reorientation cascade

abstract: In growing active matter systems, a large collection of engineered or living autonomous units metabolize free energy and create order at different length scales as they proliferate and migrate collectively. One such example is bacterial biofilms, which are surface-attached aggregates of bacterial cells embedded in an extracellular matrix. However, how bacterial growth coordinates with cell-surface interactions to create distinctive, long-range order in biofilms remains elusive. Here we report a collective cell reorientation cascade in growing Vibrio cholerae biofilms, leading to a differentially ordered, spatiotemporally coupled core-rim structure reminiscent of a blooming aster. Cell verticalization in the core generates differential growth that drives radial alignment of the cells in the rim, while the radially aligned rim in turn generates compressive stresses that expand the verticalized core. Such self-patterning disappears in adhesion-less mutants but can be restored through opto-manipulation of growth. Agent-based simulations and two-phase active nematic modeling reveal the strong interdependence of the driving forces for the differential ordering. Our findings provide insight into the collective cell patterning in bacterial communities and engineering of phenotypes and functions of living active matter.


title: Biofilm self-patterning: mechanical forces drive a reorientation cascade


title: Multiple ancestral and a plethora of recent gene duplications during the evolution of the green sensitive opsin genes (RH2) in teleost fishes

abstract: Vertebrates have four visual cone opsin classes that, together with a light-sensitive chromophore, provide sensitivity from the ultraviolet to the red wavelengths of light. The rhodopsin-like 2 (RH2) opsin is sensitive to the centre blue-green part of the spectrum, which is the most prevalent light underwater. While various vertebrate groups such as mammals and sharks have lost the RH2 gene, in teleost fishes this opsin has continued to proliferate. By investigating the genomes of 115 teleost species, we find that RH2 shows an extremely dynamic evolutionary history with repeated gene duplications, gene losses and gene conversion affecting entire orders, families and species. At least four ancestral duplications provided the substrate for todays RH2 diversity with duplications occurring in the common ancestors of Clupeocephala, Neoteleostei, and Acanthopterygii. Following these events, RH2 has continued to duplicate both in tandem and during lineage specific genome duplications. However, it has also been lost many times over so that in the genomes of extant teleosts, we find between zero to eight RH2 copies. Using retinal transcriptomes in a phylogenetic representative dataset of 30 species, we show that RH2 is expressed as the dominant green-sensitive opsin in almost all fish lineages. The exceptions are the Osteoglossomorpha (bony tongues and mooneyes) and several characin species that have lost RH2, and tarpons, other characins and gobies which do not or only lowly express the gene. These fishes instead express a green-shifted long-wavelength-sensitive LWS opsin. Our study highlights the strength of using modern genomic tools within a comparative framework to elucidate the detailed evolutionary history of gene families.


title: Set4 coordinates the activity of histone deacetylases and regulates stress-responsive gene expression within subtelomeric regions in yeast

abstract: The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts, although they appear to lack methyltransferase activity. The molecular functions of Set4 are relatively unexplored, likely due to its low abundance in standard growth conditions. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status has revealed a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, and the loss of Set4 decreases cell fitness and cell wall integrity in hypoxia. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for yeast Set4 in regulating repressive, heterochromatin-like environments.


title: Key Promoter Region of Wnt4 response to FSH and Genetic Effect on Several Production Traits of Its Mutations in Chicken

abstract: The signaling pathway of the wingless-type mouse mammary tumor virus integration site (Wnt) plays an important role in ovarian and follicular development. Wnt4 was shown in our previous study to be involved in the selection and development of chicken follicles by up-regulating the expression of follicle stimulating hormone receptors (FSHR), stimulating the proliferation of follicular granulosa cells and increasing the secretion of steroidal hormones. To further characterize cis-elements regulating chicken Wnt4 transcription, in this study we determined critical regulatory regions affecting chicken Wnt4 transcription, then identified a single nucleotide polymorphism (SNP) in this region, and finally analyzed the association of the SNP with chicken production traits. The results showed that the 5' regulatory region from -3354 to -2689 of the chicken Wnt4 gene had the strongest activity and greatest response to FSH stimulation, and that one SNP site -3015 (G > C) in this segment was identified as affecting the binding of NFAT5 (nuclear factor of activated T cells 5). When G was replaced by C at this site, it eliminated the binding by NFAT5. Moreover, this locus was significantly associated with the keel length and comb length of hens. Individuals with the genotype CG had longer keels while those with genotype CC had longer combs. Collectively, these data suggested that the SNP-3015 (G > C) is (i) involved in the regulation of Wnt4 gene expression by affecting the binding of NFAT5, (ii) associated with chicken keel length and comb length, and (iii) is a potential DNA marker in the molecular breeding of chickens for egg laying.


title: High-dose drug heat map based on organoid array chip for drug selection with high safety and efficacy

abstract: An organoid array chip was developed by adopting a micropillar and microwell structure to test safety and efficacy of drugs using high dose drug heat map. In the chip, we encapsulated patient-derived cells in alginate and grow them to maturity for more than 7 days to form cancer organoids. When screening drug compounds in a high-density organoid array due to lack of number of patient-derived cells, changing media without damage of organoids is a very tedious and difficult process. Organoids grown in conventional well plates needed too many cells and were also easily damaged due to multiple pipetting during maintenance culture or during experimental procedures. To solve those problem, we applied a micropillar and microwell structure to the organoid array. We used patient-derived cells from patients with Glioblastoma multiforme (GBM), the most common and lethal form of central nervous system cancer, to validate the array chip performance. After forming more than 100{micro}m-diameter organoids in 12 x 36 pillar array chip (25mm x 75mm), we tested 70 drug compounds (6 replicates) with high high-dose to find out high safety and efficacy drug candidates. Comparing the drug response of organoids derived from normal cells and cancer cells, we identified four compounds (Dacomitinib, Cediranib, Ly2835219, BGJ398) as drug candidates without toxicity to GBM cells.


title: The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ

abstract: Micro-environmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological and microenvironmental landscape across 85 multiple micro-dissected regions from 39 cases. Most somatic alterations, including whole genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified 3 immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.


title: High throughput single-cell genome sequencing gives insights into the generation andevolution of mosaic aneuploidy in Leishmania donovani

abstract:
Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two Leishmania clonal populations representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations. MA usually affected a defined subset of chromosomes, of which some display an enrichment in snoRNA genes which could represent an adaptative benefit to the amplification of these chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.

Note to the BioRxiv communityThe present preprint is a revision of an older preprint posted on 06th March 2020 on BioRxiv (https://www.biorxiv.org/content/10.1101/2020.03.05.976233v1). Here we included two extra samples in our single-cell genome sequencing (SCGS) analysis - the BPK081 cl8 clone (a nearly euploid strain) and a population consisting of a mixture of four L. donovani strains which was used as control for high levels of mosaicism in aneuploidy and for estimation of doublets. We also upgraded the bioinformatics pipeline to determine single-cell karyotypes and performed new fluorescence in situ hybridization (FISH) analysis. The new findings observed especially in the BPK081 cl8 led to a reformulation of the text, a new hypothesis for the evolution of mosaicism and a general restructuring of the article. Therefore, the older preprint is obsolete.


title: Neuronal extracellular vesicles mediate BDNF-dependent dendritogenesis and synapse maturation via microRNAs

abstract: Extracellular vesicles (EVs) have emerged as novel regulators of several biological processes, in part via the transfer of EV content such as microRNA; small non-coding RNAs that regulate protein production, between cells. However, how neuronal EVs contribute to trans-neuronal signaling is largely elusive. We examined the role of neuron-derived EVs in neuronal morphogenesis downstream signaling induced by brain-derived neurotrophic factor (BDNF). We found that EVs perpetuated BDNF induction of dendrite complexity and synapse maturation in naive hippocampal neurons, which was dependent on the activity of three microRNAs, miR-132-5p, miR-218 and miR-690. These microRNAs were up-regulated in BDNF-stimulated EVs. Moreover, supplementation with BDNF-EVs rescued the block of BDNF-induced phenotypes upon inhibition of miRNA activity. Our data therefore suggest a major role for EVs in BDNF-dependent morphogenesis, and provide new evidence for the functional transfer of microRNAs between neurons. This is not only an important step towards understanding the function of EVs in inter-neuronal signaling, but is also relevant for many disorders characterized by decreased BDNF signaling, such as major depression or cognitive impairment.


title: The pitfalls of using Gaussian Process Regression for normative modeling

abstract: Normative modeling, a group of methods used to quantify an individuals deviation from some expected trajectory relative to observed variability around that trajectory, has been used to characterize subject heterogeneity. Gaussian Processes Regression includes an estimate of variable uncertainty across the input domain, which at face value makes it an attractive method to normalize the cohort heterogeneity where the deviation between predicted value and true observation is divided by the derived uncertainty directly from Gaussian Processes Regression. However, we show that the uncertainty directly from Gaussian Processes Regression is irrelevant to the cohort heterogeneity in general.


title: Benchmarking of tools for axon length measurement in individually-labeled projection neurons

abstract:
Projection neurons are the commonest neuronal type in the mammalian forebrain and their individual characterization is a crucial step to understand how neural circuitry operates. These cells have an axon whose arborizations extend over long distances, branching in complex patterns and/or in multiple brain regions. Axon length is a principal estimate of the functional impact of the neuron, as it directly correlates with the number of synapses formed by the axon in its target regions; however, its measurement by direct 3D axonal tracing is a slow and labor-intensive method. On the contrary, axon length estimations have been recently proposed as an effective and accessible alternative, allowing a fast approach to the functional significance of the single neuron. Here, we analyze the accuracy and efficiency of the most used length estimation tools - design-based stereology by virtual planes or spheres, and mathematical correction of the 2D projected-axon length - in contrast with direct measurement, to quantify individual axon length. To this end, we computationally simulated each tool, applied them over a dataset of 951 3D-reconstructed axons (from NeuroMorpho.org), and compared the generated length values with their 3D reconstruction counterparts. Additionally, the computational results were compared with estimated and direct measurements of individual axon lengths performed on actual brain tissue sections, to analyze the practical difficulties and biases arising in real cases. The evaluated reliability of each axon length estimation method is then balanced with the required human effort, experience and know-how, and economic affordability. This work, therefore, aims to provide a constructive benchmark to help guide the selection of the most efficient method for measuring specific axonal morphologies according to the particular circumstances of the conducted research.

AUTHOR SUMMARYCharacterization of single neurons is a crucial step to understand how neural circuitry operates. Visualization of individual neurons is feasible thanks to labelling techniques that allows precise measurements at cellular resolution. This milestone gave access to powerful estimators of the functional impact of a neuron, such as axon length. Although techniques relying on direct 3D reconstruction of individual axons are the gold standard, handiness and accessibility are still an issue. Indirect estimations of axon length have been proposed as agile and effective alternatives, each offering different solutions to the accuracy-cost tradeoff. In this work we report a computational benchmarking between three experimental tools used for axon length estimation on brain tissue sections. Performance of each tool was simulated and tested for 951 3D-reconstructed axons, by comparing estimated axon lengths against direct measurements. Assessment of suitability to different research and funding circumstances is also provided, taking into consideration factors such as training expertise, economic cost and required equipment, alongside methodological results. These findings could be an important reference for research on neuronal wiring, as well as for broader studies involving neuroanatomical and neural circuit modelling.


title: Gut microbiota features associated with Clostridioides difficile colonization in dairy calves

abstract: Diarrheal disease, a major cause of morbidity and mortality in dairy calves, is strongly associated with the health and composition of the gut microbiome. Clostridioides difficile is an opportunistic pathogen that proliferates and can produce enterotoxins when the host experiences gut dysbiosis. However, even asymptomatic colonization with C. difficile can be associated with differing degrees of microbiome disruption in a range of species, including people, swine, and dogs. Little is known about the interaction between C. difficile and the gut microbiome in dairy calves. In this study, we sought to define microbial features associated with C. difficile colonization in pre-weaned dairy calves less than 2 weeks of age. We characterized the fecal microbiota of 80 calves from 23 different farms using 16S rRNA sequencing and compared the microbiota of C. difficile-positive (n=24) and C. difficile-negative calves (n=56). Farm appeared to be the greatest source of variability in the gut microbiota. When controlling for calf age, diet, and farm location, there was no significant difference in Shannon alpha diversity (P= 0.50) or in weighted UniFrac beta diversity (P=0.19) between C. difficile-positive and -negative calves. However, there was a significant difference in beta diversity as assessed using Bray-Curtiss diversity (P=0.0077), and C. difficile-positive calves had significantly increased levels of Ruminococcus (gnavus group) (Adj. P=0.052), Lachnoclostridium (Adj. P=0.060), Butyricicoccus (Adj. P=0.060), and Clostridium sensu stricto 2 compared to C. difficile-negative calves. Additionally, C. difficile-positive calves had fewer microbial co-occurrences than C. difficile-negative calves, indicating reduced bacterial synergies. Thus, while C. difficile colonization alone is not associated with dysbiosis and is therefore unlikely to result in an increased likelihood of diarrhea in dairy calves, it may be associated with a more disrupted microbiota.


title: Microbiome dysbiosis regulates the level of energy production under anaerobic condition

abstract: The microbiome of the anaerobic digester (AD) regulates the level of energy production. To assess the microbiome dysbiosis in different stages of anaerobic digestion, we analyzed 16 samples dividing into four groups (Group-I = 2; Group-II = 5; Group-III = 5 and Group-IV = 4) through whole metagenome sequencing (WMS). The physicochemical analysis revealed that highest CH4 production (74.1%, on Day 35 of digestion) was associated with decreased amount of non-metal (phosphorus and sulfur) and heavy metals (chromium, lead and nickel). The WMS generated 380.04 million reads mapped to ~ 2800 distinct bacterial, archaeal and viral genomes through PathoScope (PS) and MG-RAST (MR) analyses. The PS analysis detected 768, 1421, 1819 and 1774 bacterial strains in Group-I, Group-II, Group-III and Group-IV, respectively which were represented by Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes and Fibrobacteres (> 93.0% of the total abundances). The archaeal fraction of the AD microbiomes was represented by 343 strains, of which 95.90% strains shared across these metagenomes. The indicator species analysis showed that Methanosarcina vacuolate, Dehalococcoides mccartyi, Methanosarcina sp. Kolksee and Methanosarcina barkeri were the highly specific for energy production in Group-III and Group-IV. However, most of the indicator phylotypes displayed reduced abundance in the initial stage of biogas production (Group-I and Group-II) compared to their increased relative abundances in Group-IV (Day 35). The correlation network analysis showed that different strains of Euryarcheota and Firmicutes phyla were associated with highest level (74.1%) of energy production (Group-IV). In addition to taxonomic dysbiosis, top CH4 producing microbiomes showed increased genomic functional activities related to one carbon and biotin metabolism, oxidative stress, proteolytic pathways, MT1-MMP pericellular network, acetyl-CoA production, motility and chemotaxis. This study reveals distinct changes in composition and diversity of the AD microbiomes including different indicator species, and their genomic features that are highly specific for energy production.


title: Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative yeast two hybrid-high throughput sequencing approach

abstract:
AO_SCPLOWBSTRACTC_SCPLOWBacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35-Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35-B. thuringiensis interaction at the molecular level.

AO_SCPLOWUTHORC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSUMMARYC_SCPLOWMembers of the family Tectiviridae, composed of non-tailed icosahedral, membrane-containing bacteriophages, have been increasingly scrutinized in recent years for their possible role in the origin of dsDNA viruses. In particular, the genus Betatectivirus receives increased attention as these phages can infect clinical strains as well as industrially relevant members of the B. cereus group. However, little is known about the interactions between these temperate viruses and their hosts. Here, we present the first high-throughput study of tectivirus-host protein-protein interactions focusing on Bam35, model virus of betatectiviruses, and its host B. thuringiensis, an important entomopathogenic bacterium. We adapted the well-known technique yeast-two-hybrid and integrated high-throughput sequencing and bioinformatics for the downstream analysis of the results which enables large-scale analysis of protein-protein interactions. In total, 182 detected interactions show an enrichment in host metabolic proteins and peptidases, in contrast with the current knowledge on host-phage PPIs. Specific host-viral protein-protein interactions were also detected enabling us to propose functions for uncharacterized proteins.