title: Absence of severe COVID-19 in patients with clonal mast cells activation disorders: effective anti-SARS-CoV-2 immune response.

abstract:
Mast cells are key actors of innate immunity and Th2 adaptive immune response which counterbalance Th1 response, critical for anti-viral immunity. Clonal Mast Cells Activation Disorders (cMCADs) such as mastocytosis and clonal mast cells activation syndrome are characterized by an abnormal mast cells accumulation and/or activation. No data have been published on the anti-viral immune response of patients with cMCADs. The aims of the study were to collected, in a comprehensive way, outcomes of cMCADs patients who experienced a biologically-proven COVID-19 and to characterize both anti-endemic coronaviruses and specific anti-SARS-CoV-2 immune responses in these patients. Clinical follow-up and outcome data were collected prospectively for one year within the French rare disease network CEREMAST encompassing patients from all over the country. Anti-SARS-CoV-2 and anti-endemic coronaviruses specific T-cells were assessed with an enzyme-linked immunospot assay (EliSpot) and anti-SARS-CoV-2 humoral response with dosage of circulating levels of specific IgG, IgA and neutralizing antibodies. Overall, 32 cMCADs patients were identified. None of them required non-invasive or mechanical ventilation; two patients were hospitalized to receive oxygen and steroid therapy. In 21 patients, a characterization of the SARS-CoV-2-specific immune response has been performed. A majority of patients showed a high proportion of circulating SARS-CoV-2-specific interferon (IFN)-{gamma} producing T-cells and high levels of anti-Spike IgG antibodies with neutralizing activity. In addition, no defects in anti-endemic coronaviruses responses were found in patients with cMCADs compared to non-cMCADs controls. Patients with cMCADs frequently showed a spontaneous IFN-{gamma} T-cell production in absence of any stimulation that correlated with circulating basal tryptase levels, a marker of mast cells burden. These findings underscore that patients with cMCADs might be not at risk of severe COVID-19 and the spontaneous IFN-{gamma} production might explain this observation.

Author SummaryMast cells are immune cells involved in many biological processes including the anti-microbial response. However, previous studies suggest that mast cells may have a detrimental role in the response against viruses such as SARS-CoV-2, responsible for COVID-19. When a mutation occurs in mast cells, it can lead to a group of diseases called clonal mast cells activation disorders (cMCADs), characterized by deregulated activation of these cells. Hence, patients with cMCADs might be more susceptible to severe COVID-19 than general population.

We therefore conducted a 1-year study in France to collect data from all cMCADs patients included in the CEREMAST rare disease French network and who experienced COVID-19. Interestingly, we did not find any severe COVID-19 (i.e. requiring non-invasive or mechanical ventilation) in spite of well-known risk factors for severe COVID-19 in a part of cMCADs patients.

We then have studied the immune response against SARS-CoV-2 and other endemic coronaviruses in these patients. We did not observe any abnormalities in the immune response either at the level of T and B lymphocytes. These findings underscore that these patients might not be at risk of severe COVID-19 as one might have feared.


title: Strain-dependent selective antimicrobial action of cationic polyelectrolytes on Gram-negative bacteria

abstract: Although electrostatic modification of bacterial surfaces using polyelectrolytes (PEs) is a convenient and versatile tool for biotechnological processes, the ambiguities in toxicity of PEs between various bacteria and the insufficient understanding of the mechanism of action of cationic PEs and their nano-thick shells formed around the bacteria create a bottleneck of the approach. Here, we show how the viability of two bacterial strains, Escherichia coli and Pseudomonas stutzeri, both from the Gram-negative group differs, when the cells are exposed to cationic PEs under different conditions. Although the cell wall architecture of the strains should be structurally similar, we found that the viability of E. coli was not affected by the electrostatic deposition of polyethyleneimine (PEI) or poly(allylamine) hydrochloride (PAH), whereas for P. stutzeri the deposition resulted in high death rates. The cells of E. coli proved to be suitable templates for Layer-by-Layer (LbL) modification, while in P. stutzeri a modified protocol with mild conditions had to be used to ensure the viability of the cells. Super resolution stimulated emission depletion (STED) microscopy allowed us to clearly visualize that after PE deposition onto the surface of the cells, the PEs could penetrate inside the cells of P. stutzeri, while forming a capsule around E. coli as expected. Therefore, this knowledge will help us select the most appropriate combinations of strains and PEs, for biotechnological processes or biomedical application, preventing unwanted toxicity.

category: microbiology


title: Structural basis of Plasmodium vivax inhibition by antibodies binding to the circumsporozoite protein repeats

abstract: Malaria is a global health burden, with Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) responsible for the majority of infections worldwide. Circumsporozoite protein (CSP) is the most abundant protein on the surface of Plasmodium sporozoites, and antibodies targeting the central repeat region of CSP can prevent parasite infection. Although much has been uncovered about the molecular basis of antibody recognition of the PfCSP repeats, data remains scarce for PvCSP. Here, we performed molecular dynamics simulations for peptides comprising the PvCSP repeats from strains VK210 and VK247 to reveal how the PvCSP central repeats are highly disordered, with minor propensities to adopt turn conformations. Next, we solved eight crystal structures to unveil the interactions of two inhibitory monoclonal antibodies (mAbs), 2F2 and 2E10.E9, with PvCSP repeats. Both antibodies can accommodate subtle sequence variances in the repeat motifs and recognize largely coiled peptide conformations that also contain isolated turns. Our structural studies uncover various degrees of Fab-Fab homotypic interactions upon recognition of the PvCSP central repeats by these two inhibitory mAbs, similar to potent mAbs against PfCSP. These findings augment our understanding of host-Plasmodium interactions, and contribute molecular details of Pv inhibition by mAbs to unlock structure-based engineering of PvCSP-based vaccines.

category: molecular biology


title: Drosophila centrocortin  is dispensable for centriole duplication but contributes to centrosome separation

abstract: Centrosomes are microtubule-organizing centers that duplicate exactly once to organize the bipolar mitotic spindle required for error-free mitosis. Prior work indicated that Drosophila centrocortin (cen) is required for normal centrosome separation, although a role in centriole duplication was not closely examined. Through time-lapse recordings of rapid syncytial divisions, we monitored centriole duplication and the kinetics of centrosome separation in control versus cen null embryos. Our data suggest that although cen is dispensable for centriole duplication, it contributes to centrosome separation.

category: cell biology


title: Computational methods for the discovery and annotation of viral integrations

abstract: The transfer of genetic material between viruses and eukaryotic cells is pervasive. Somatic integrations of DNA viruses and retroviruses have been linked to persistent viral infection and genotoxic effects. Integrations into germline cells, referred to as Endogenous Viral Elements (EVEs), can be co-opted for host functions. Besides DNA viruses and retroviruses, EVEs can also derive from nonretroviral RNA viruses, which have often been observed in piRNA clusters. Here, we describe a bioinformatic framework to annotate EVEs in a genome assembly, study their widespread occurrence and polymorphism and identify sample-specific viral integrations using whole-genome sequencing data.

category: bioinformatics


title: Overcoming Tumor Heterogeneity by Ex Vivo Arming of T Cells Using Multiple Bispecific Antibodies

abstract:
PurposeTumoral heterogeneity is a hallmark of tumor evolution and cancer progression, being a longstanding challenge to targeted immunotherapy. Ex vivo armed T cells (EATs) using IgG-[L]- scFv bispecific antibodies (BsAbs) are potent tumor-specific cytotoxic effectors. To improve the anti-tumor efficacy of EATs against heterogeneous solid tumors, we explored multi-antigen targeting approaches.

MethodsEx vivo expanded T cells were armed with BsAbs built on the IgG-[L]-scFv platform, where an anti-CD3 (huOKT3) scFv was attached to the carboxyl end of both light chains of a tumor specific IgG. Multispecificity was created by combining monospecific EATs, combining BsAbs on the same T cell, or combining specificities on the same antibody. Three multi-antigens targeting EAT strategies were tested: (1) pooled EATs (simultaneous combination of monospecific EATs or alternate EATs (alternating combination of monospecific EATs), (2) dual-EATs or multi- EATs (T cells simultaneously armed with [≥] 2 BsAbs), and (3) TriAb-EATs [T cells armed with BsAb specific for two tumor targets besides CD3 (TriAb)]. The properties and efficiencies of these 3 strategies were evaluated by flow cytometry, in vitro cytotoxicity, cytokine release assays, and in vivo studies performed in BALB-Rag2-/-IL-2R-{gamma}c-KO (BRG) mice xenografted with cancer cell line (CDX) or patient-derived tumor (PDX).

ResultsMulti-EATs retained target antigen specificity and anti-tumor potency. Cytokine release with multi-EATs in the presence of tumor cells was substantially less than when multiple BsAbs were mixed with unarmed T cells. When tested against CDXs or PDXs, dual- or multi-EATs effectively suppressed tumor growth without clinical toxicities. Most importantly, dual- or multi- EATs were highly efficient in preventing clonal escape while mono- or TriAb- EATs were not as efficient.

ConclusionArming T cells with multiple BsAbs enabled multi-specific T cell immunotherapy which overcomes tumor heterogeneity without excessive cytokine release.

category: cancer biology


title: Hopanoids confer robustness to physicochemical variability in the niche of the plant symbiont Bradyrhizobium diazoefficiens

abstract:
Climate change poses a threat to soil health and agriculture, but the potential effects of climate change on soil bacteria that can help maintain soil health are understudied. Rhizobia are a group of bacteria that increase soil nitrogen content through a symbiosis with legume plants. The soil and symbiosis are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the bradyrhizobia clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens. Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in similarly successful symbioses compared to the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the in vitro growth defects of extended hopanoid deficient mutants can be at least partially compensated for by the physicochemical environment, specifically by optimal osmotic and divalent cation concentrations. Through biophysical measurements, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in vitro and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environment within root nodules. To improve the legume-rhizobia symbiosis through either bioengineering or strain selection, it will be important to consider the full lifecycle of rhizobia, from the soil to the symbiosis.

ImportanceRhizobia, such as B. diazoefficiens, play an important role in the nitrogen cycle by making nitrogen gas bioavailable through symbiosis with legume plants. As climate change threatens soil health, this symbiosis has reentered the spotlight as a more sustainable source of soil nitrogen than the energy-intensive Haber-Bosch process. Efforts to use rhizobia as biofertilizers have been effective; however, long term integration of rhizobia into the soil community has been less successful. This work represents a small step towards improving the legume-rhizobia symbiosis by identifying a cellular component--hopanoid lipids--that confers robustness to environmental stresses rhizobia are likely to encounter in soil microenvironments as sporadic desiccation and flooding events become more common.


title: Interfered Chromosome Pairing Promotes Meiosis Instability of Autotetraploid Arabidopsis by High Temperatures

abstract: Alterations of environmental temperature affect multiple meiosis processes in flowering plants. Polyploid plants derived from whole genome duplication (WGD) have enhanced genetic plasticity and tolerance to environmental stress, but meanwhile face a challenge for organization and segregation of doubled chromosome sets. In this study, we investigated the impact of increased environmental temperature on male meiosis in autotetraploid Arabidopsis thaliana. Under low to mildly-increased temperatures (5-28{degrees}C), irregular chromosome segregation universally takes place in synthesized autotetraploid Columbia-0 (Col-0). Similar meiosis lesions occur in autotetraploid rice (Oryza sativa L.) and allotetraploid canola (Brassica napus cv. Westar), but not in evolutionary-derived hexaploid wheat (Triticum aestivum). As temperature increases to extremely high, chromosome separation and tetrad formation are severely disordered due to univalent formation caused by suppressed crossing-over. We found a strong correlation between tetravalent formation and successful chromosome pairing, both of which are negatively correlated with temperature elevation, suggesting that increased temperature interferes with crossing-over prominently by impacting homolog pairing. Besides, we showed that loading irregularities of axis proteins ASY1 and ASY4 co-localize on the chromosomes of syn1 mutant, and the heat-stressed diploid and autotetraploid Col-0, revealing that heat stress affects lateral region of synaptonemal complex (SC) by impacting stability of axis. Moreover, we showed that chromosome axis and SC in autotetraploid Col-0 are more sensitive to increased temperature than that of diploid Arabidopsis. Taken together, our study provide evidence suggesting that WGD without evolutionary and/or natural adaption negatively affects stability and thermal tolerance of meiotic recombination in Arabidopsis thaliana.

category: plant biology


title: Automated Model-Predictive Design of Synthetic Promoters to Control Transcriptional Profiles in Bacteria

abstract:
Transcription rates are regulated by the interactions between RNA polymerase, sigma factor, and promoter DNA sequences in bacteria. However, it remains unclear how non-canonical sequence motifs collectively control transcription rates. Here, we combined massively parallel assays, biophysics, and machine learning to develop a 346-parameter model that predicts site-specific transcription initiation rates for any {sigma}70 promoter sequence, validated across 17396 bacterial promoters with diverse sequences. We applied the model to predict genetic context effects, design {sigma}70 promoters with desired transcription rates, and identify undesired promoters inside engineered genetic systems. The model provides a biophysical basis for understanding gene regulation in natural genetic systems and precise transcriptional control for engineering synthetic genetic systems.

One-Sentence SummaryA 346-parameter model predicted DNAs interactions with RNA polymerase initiation complex, enabling accurate transcription rate predictions and automated promoter design in bacterial genetic systems.

category: synthetic biology


title: The SARS-CoV-2 spike (S) and the orthoreovirus p15 cause neuronal and glial fusion

abstract: Numerous enveloped viruses use specialized surface molecules called fusogens to enter host cells1. During virus replication, these fusogens decorate the host cells membrane enabling them the ability to fuse with neighboring cells, forming syncytia that the viruses use to propagate while evading the immune system. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect the brain and may cause serious neurological symptoms through mechanisms which remain poorly understood2-4. Here we show that expression of either the SARS-CoV-2 spike (S) protein or p15 protein from the baboon orthoreovirus is sufficient to induce fusion between interconnected neurons, as well as between neurons and glial cells. This phenomenon is observed across species, from nematodes to mammals, including human embryonic stem cells-derived neurons and brain organoids. We show that fusion events are progressive, can occur between distant neurites, and lead to the formation of multicellular syncytia. Finally, we reveal that in addition to intracellular molecules, fusion events allow diffusion and movement of large organelles such as mitochondria between fused neurons. Our results provide important mechanistic insights into how SARS-CoV-2 and other viruses could affect the nervous system circuitries causing neurological symptoms.

category: neuroscience


title: Mixed Origins: HIV gp120-specific memory develops from pre-existing memory and naive B cells following vaccination in humans

abstract: The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naive B cell receptor, fail to bind Env suggesting that the initial responding B cell population is not exclusively comprised of a naive population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants in HVTN 105, an HIV vaccine trial. Using deep sequencing VH-lineage tracking we identified several of these mAb lineages in pre-immune peripheral blood. Several of these pre-immune lineages also persisted in the bone marrow, including CD138+ long-lived plasma cell compartment, [~]7 months after the final vaccination. The majority of the pre-immune lineage members included IgM, however IgG and IgA members were also prevalent and exhibited somatic hypermutation. These results suggest that vaccine-induced gp120-specific antibody lineages originate from both naive and cross-reactive memory B cells.

category: immunology


title: The dengue virus non-structural protein 1 (NS1) interacts with the putative epigenetic regulator DIDO1 to promote flavivirus replication.

abstract:
Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. Approximately 14% of the 817 identified proteins coincide with interactomes obtained in vertebrate cells, including ontology groups of the oligosaccharide transferase complex, the chaperonin containing TCP-1, and nuclear import and export, vesicle localization and ribosomal proteins. Notably, other protein pathways such as epigenetic regulation and RNA silencing, not previously reported in vertebrate cells, were also found in the NS1 interactome in mosquito cells. Due to the novel interaction observed for NS1 and DIDO1 (Death Inducer-Obliterator 1), we further explored the role of DIDO1 in viral replication. Interactions between NS1 and DIDO1were corroborated in infected C6/36 and Aag2 cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression in C6/36 and Aag2 cells results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected C6/36 silenced for DIDO1, revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response and necessary for flavivirus replication. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication and add to the understanding of NS1 as a multifunctional protein.

ABSTRACT IMPORTANCEDengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as epigenetic regulation and RNA silencing, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in vertebrate and mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1, revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.


title: E-cadherin redistribution mediated by Clostridioides difficile toxins increases spore-association to adherens junctions

abstract: Nearly [~]20% of patients with C. difficile infection (CDI) manifest recurrence of CDI (R-CDI). During CDI, C. difficile forms spores essential for R-CDI. Interactions of C. difficile spores with intestinal epithelial cells (IECs) contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore-adherence and internalization into IECs. C. difficile toxins TcdA/TcdB lead to adherens junctions opening and increase spore-adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA/TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By ELISA, immunofluorescence, and immunogold labelling, we observed that E-cadherin binds to C. difficile spores, specifically to the hair-like projections of the spore. Overall, these results expand our knowledge of how C. difficile spores bind to IECs and how toxin-mediated damage affects spore interactions with IECs.


title: A closed Candidatus Odinarchaeum genome exposes Asgard archaeal viruses

abstract: Asgard archaea have recently been identified as the closest archaeal relatives of eukaryotes. Their ecology remains enigmatic, and their virome, completely unknown. Here, we describe the closed genome of Ca. Odinarchaeum yellowstonii LCB_4, and, from this, obtain novel CRISPR arrays with spacer targets to several viral contigs. We find related viruses in sequence data from thermophilic environments and in the genomes of diverse prokaryotes, including other Asgard archaea. These novel viruses open research avenues into the ecology and evolution of Asgard archaea.


title: Analysing pneumococcal invasiveness using Bayesian models of pathogen progression rates

abstract:
The disease burden attributable to opportunistic pathogens depends on their prevalence in asymptomatic colonisation and the rate at which they progress to cause symptomatic disease. Increases in infections caused by commensals can result from the emergence of "hyperinvasive" strains. Such pathogens can be identified through quantifying progression rates using matched samples of typed microbes from disease cases and healthy carriers. This study describes Bayesian models for analysing such datasets, implemented in an RStan package (https://github.com/nickjcroucher/progressionEstimation). The models converged on stable fits that accurately reproduced observations from meta-analyses of Streptococcus pneumoniae datasets. The estimates of invasiveness, the progression rate from carriage to invasive disease, in cases per carrier per year correlated strongly with the dimensionless values from meta-analysis of odds ratios when sample sizes were large. At smaller sample sizes, the Bayesian models produced more informative estimates. This identified historically rare but high-risk S. pneumoniae serotypes that could be problematic following vaccine-associated disruption of the bacterial population. The package allows for hypothesis testing through model comparisons with Bayes factors. Application to datasets in which strain and serotype information were available for S. pneumoniae found significant evidence for within-strain and within-serotype variation in invasiveness. The heterogeneous geographical distribution of these genotypes is therefore likely to contribute to differences in the impact of vaccination in between locations. Hence genomic surveillance of opportunistic pathogens is crucial for quantifying the effectiveness of public health interventions, and enabling ongoing meta-analyses that can identify new, highly invasive variants.

Author summaryOpportunistic pathogens are microbes that are commonly carried by healthy hosts, but can occasionally cause severe disease. The progression rate quantifies the risk of such a pathogen transitioning from a harmless commensal to causing a symptomatic infection. The incidence of infections caused by opportunistic pathogens can rise with the emergence of "hyperinvasive" strains, which have high progression rates. Therefore methods for calculating progression rates of different pathogen strains using surveillance data are crucial for rapidly identifying emerging infectious disease threats. Existing methods typically measure progression rates relative to the overall mix of microbes in the population, but these populations can vary substantially between locations and times, making the outputs challenging to combine across studies. This work presents a new method for estimating progression rates from surveillance data that generates values useful for modelling pathogen populations, even from relatively small sample sizes.


title: Skin specimen as an alternative to brain tissue for post mortem rabies diagnosis in animals to scale up animal rabies surveillance

abstract:
BackgroundDetection of the virus or some of its specific components using WHO and OIE recommended standard laboratory tests is the only way to get a reliable diagnosis of rabies. Brain tissue is the preferred specimen for post-mortem diagnosis of rabies in both humans and animals. Higher biosecurity requirements, skill and transportation facilities required for collection and transport of brain or whole carcass to the laboratory is one of the reasons for the poor rabies surveillance in animals. Point of care testing with simple, reliable and easy to operate devices would be an ideal approach for providing rapid results.

MethodsThe study evaluated diagnostic performance of two reference tests, DFAT and RTPCR on skin specimen, to assess its suitability as an alternative of brain tissue for post mortem rabies diagnosis in animals. Brain tissue and skin sample belonging to different species of animals (n=90) collected at necropsy were compared using Fluorescent Antibody Test and RT PCR, internationally approved methods for rabies diagnosis.

ResultsValidation of RT-PCR on skin and DFAT on skin in comparison with DFAT on brain as gold standard gave a sensitivity of 98% (95% CI:94.1-100) and 80% (95% CI:71.8-88.2) respectively. Specificity was 100% in both tests.

ConclusionThe findings highlight the potential of skin specimen for improving rabies surveillance in animals especially in resource poor countries.

Author SummaryThe present study was undertaken to define a reliable protocol for diagnosing animal rabies using skin specimen, a superficial tissue sample which is collected in a non-invasive manner for post-mortem diagnosis. Our aim was to design a protocol to replace the classical post-mortem diagnostic method that uses brain biopsy with an ultimate target of stepping up rabies surveillance in animals. Brain tissue and skin sample belonging to different species of animals collected at necropsy were compared using Fluorescent Antibody Test and RT PCR, internationally approved methods for rabies diagnosis. The study established that RT PCR on skin specimen is rapid, sensitive and specific, opening its potential as an ideal rabies surveillance tool overcoming the logistical challenges of carcass transportation to reference laboratories and alleviating biosafety concerns associated with brain collection. The study highlights the potential of skin specimen for improving rabies surveillance in animals especially in resource poor countries.


title: Long-term incubation of lake water enables genomic sampling of consortia involving Planctomycetes and Candidate Phyla Radiation bacteria

abstract: Microbial communities in lakes can profoundly impact biogeochemical processes through their individual activities and collective interactions. However, the complexity of these communities poses challenges, particularly for studying rare members. Laboratory enrichments can select for subsystems of interacting organisms and enable genome recovery for enriched populations. Here, a reactor inoculated with water from Lake Fargette, France, and maintained under dark conditions at 4{degrees}C for 31 months enriched for diverse Planctomycetes and Candidate Phyla Radiation (CPR) bacteria. We reconstructed draft genomes and predicted metabolic traits for 12 diverse Planctomycetes and 9 CPR bacteria, some of which are likely representatives of undescribed families or genera. One CPR genome representing the little-studied lineage Peribacter (1.239 Mbp) was curated to completion, and unexpectedly, encodes the full gluconeogenesis pathway. Metatranscriptomic data indicate that some Planctomycetes and CPR bacteria were active under the culture conditions. We also reconstructed genomes and obtained transmission electron microscope images for numerous phages, including one with a >300 kbp genome and several predicted to infect Planctomycetes. Together, our analyses suggest that freshwater Planctomycetes may act as hubs for interaction networks that include symbiotic CPR bacteria and phages.


title: Gene signatures and host-parasite interactions revealed by dual single-cell profiling of Plasmodium vivax liver infection

abstract: Malaria-causing P. vivax parasites can linger in the human liver for weeks to years, and then reactivate to cause recurrent blood-stage infection. While an important target for malaria eradication, little is known about the molecular features of the replicative and non-replicative states of intracellular P. vivax parasites, or the human host-cell responses to them. Here, we leverage a bioengineered human microliver platform to culture Thai clinical isolates of P. vivax in primary human hepatocytes and conduct transcriptional profiling of infected cultures. By coupling enrichment strategies with bulk and single-cell analyses, we captured both parasite and host transcripts in individual hepatocytes throughout the infection course. We defined host- and state-dependent transcriptional signatures and identified previously unappreciated populations of replicative and non-replicative parasites, sharing features with sexual transmissive forms. We found that infection suppresses transcription of key hepatocyte function genes, and that P. vivax elicits an innate immune response that can be manipulated to control infection. Our work provides an extendible framework and resource for understanding host-parasite interactions and reveals new insights into the biology of malaria dormancy and transmission.


title: Artificial introns for effective expression of transgenes in mammalian cells

abstract: Introns are widely used in the assembly of genetic constructions expressing transgenic proteins in eukaryotic cells for the enhancement of this expression. However, the choice of introns that can be applied for such purposes is limited by the excessively large size of the majority of natural introns (several thousand nucleotides) and therefore they cannot be cloned in a genetic construction. With the help of site-directed mutagenesis we have generated a library of short (99 nucleotides long) introns. The efficiency of these introns in the enhancement of gene expression was analyzed. As a result, a set of 12 introns was selected. The generated intros can be used for genetic constructions with high expression level of recombinant proteins.


title: The genomic signatures of natural selection in admixed human populations

abstract:
Admixture has been a pervasive phenomenon in human history, shaping extensively the patterns of population genetic diversity. There is increasing evidence to suggest that admixture can also facilitate genetic adaptation to local environments, i.e., admixed populations acquire beneficial mutations from source populations, a process that we refer to as adaptive admixture. However, the role of adaptive admixture in human evolution and the power to detect it are poorly characterized. Here, we use extensive computer simulations to evaluate the power of several neutrality statistics to detect natural selection in the admixed population, accounting for background selection and assuming different admixture scenarios. We show that two statistics based on admixture proportions, Fadm and LAD, show high power to detect mutations that are beneficial in the admixed population, whereas iHS and FST falsely detect neutral mutations that have been selected in the source populations only. By combining Fadm and LAD into a single statistic, we scanned the genomes of 15 worldwide, admixed populations for signatures of adaptive admixture. We confirm that lactase persistence and resistance to malaria have been under adaptive admixture in West Africa and in Madagascar, North Africa and South Asia, respectively. Our approach also uncovers new cases of adaptive admixture, including the APOL1/MYH9 locus in the Fulani nomads and PKN2 in East Indonesians, involved in resistance to infection and metabolism, respectively. Collectively, our study provides new evidence that adaptive admixture has occurred in multiple human populations, whose genetic history is characterized by periods of isolation and spatial expansions resulting in increased gene flow.

Author summaryAdaptive introgression, i.e., the acquisition of adaptive traits through hybridization with another species, is a well-documented phenomenon in evolution. Conversely, adaptive admixture, i.e., the acquisition of adaptive traits through admixture between populations of the same species, is poorly described. In this study, we evaluate the importance of adaptive admixture in human recent evolutionary history. We first determine the expected signatures of adaptive admixture on patterns of genomic diversity, using realistic simulations. We then identify the methods that are the most powerful to detect such molecular signatures. Finally, by using the methods identified as the most powerful, we search for cases of adaptive admixture in the genomes of 15 admixed populations from around the globe. We find evidence that adaptive admixture has occurred in several populations from Northeast Africa, Southeast Asia and Oceania. This study suggests that admixture has indeed facilitated human genetic adaptation, particularly at genes involved in metabolism and resistance against pathogens.

category: genetics


title: Mapping interindividual dynamics of innate immune response at single-cell resolution

abstract: Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.


title: Pathogenicity effector candidates and accessory genome revealed by pan-genomic analysis of Parastagonospora nodorum

abstract: The wheat pathogen Parastagonospora nodorum has emerged as a model necrotrophic fungal species with growing genomic resources. Recent population-level pan-genome studies were leveraged to provide novel insights into pathogen evolution and effector-like gene contents relevant to local crop disease outbreaks. In this study, we examined 156 isolates representing a regional population from the Western Australian (WA) wheat-belt region, and 17 internationally sourced isolates. We observed a highly diverse local population, within which were numerous small and highly similar clusters of isolates from hotter and drier regions. Pan-genome assembly and orthologous gene datasets resulted in 3579 predicted effector candidates, 2291 of which exhibited presence-absence variation (PAV) across the population, and 1362 were specific to WA isolates. There was an abundance of mutations (including repeat-induced point mutation (RIP)), distributed in  hot-spots within the pan-genomic landscape that were rich in effector candidates. Three characterised effector loci (ToxA, Tox1 and Tox3) were located within sub- telomeric regions of lower diversity, but were nestled within larger high-diversity regions. RIP was widespread across the genome, but non-synonymous RIP-like mutations were strongly selected against. These improved bioinformatic resources for P. nodorum, represent progressive advancements in fungal pan-genomics, with a view towards supporting region- specific surveillance of host-pathogen interactions.

category: genomics


title: Hydrogen-deuterium exchange mass spectrometry of Mtr4 with diverse RNAs reveals substrate-dependent dynamics and interfaces in the arch

abstract: Mtr4 is a eukaryotic RNA helicase required for RNA decay by the nuclear exosome. Previous studies have shown how RNA enroute to the exosome threads through the highly conserved helicase core of Mtr4. Mtr4 also contains an arch domain, although details of potential interactions between the arch and RNA have been elusive. To understand the interaction of Saccharomyces cerevisiae Mtr4 with various RNAs, we have characterized RNA binding in solution using hydrogen-deuterium exchange mass spectrometry, and affinity and unwinding assays. We have identified RNA interactions within the helicase core that are consistent with existing structures and do not vary between tRNA, single-stranded RNA, and double-stranded RNA constructs. We have also identified novel RNA interactions with a region of the arch known as the fist or KOW. These interactions are important for RNA unwinding and vary in strength depending on RNA structure and length. They account for Mtr4 discrimination between different RNAs. These interactions further drive Mtr4 to adopt a closed conformation characterized by reduced dynamics of the arch arm and intra-domain contacts between the fist and helicase core.

category: biochemistry


title: Toward More General Embeddings for Protein Design: Harnessing Joint Representations of Sequence and Structure

abstract: AO_SCPLOWBSTRACTC_SCPLOWProtein embeddings learned from aligned sequences have been leveraged in a wide array of tasks in protein understanding and engineering. The sequence embeddings are generated through semi-supervised training on millions of sequences with deep neural models defined with hundreds of millions of parameters, and they continue to increase in performance on target tasks with increasing complexity. We report a more data-efficient approach to encode protein information through joint training on protein sequence and structure in a semi-supervised manner. We show that the method is able to encode both types of information to form a rich embedding space which can be used for downstream prediction tasks. We show that the incorporation of rich structural information into the context under consideration boosts the performance of the model by predicting the effects of single-mutations. We attribute increases in accuracy to the value of leveraging proximity within the enriched representation to identify sequentially and spatially close residues that would be affected by the mutation, using experimentally validated or predicted structures.


title: Spatial proteomics reveals profound subcellular reorganization in human keratinocytes exposed to UVA light

abstract: The effects of UV light on the skin have been extensively investigated. However, systematic information about how exposure to UVA light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin.


title: Spatial proteomics reveals subcellular reorganization in human keratinocytes exposed to UVA light


title: Engineering genetically-encoded synthetic biomarkers for breath-based cancer detection

abstract:
Breath analysis holds great promise for rapid, noninvasive early cancer detection; however, clinical implementation is impeded by limited signal from nascent tumors and high background expression by non-malignant tissues. To address this issue, we developed a novel breath-based reporter system for early cancer detection using D-limonene, a volatile organic compound (VOC) from citrus fruit that is not produced in humans, in order to minimize background signal and maximize sensitivity and specificity for cancer detection. We metabolically engineered HeLa human cervical cancer cells to express limonene at levels detectable by mass spectrometry by introducing a single plant gene encoding limonene synthase. To improve limonene production and detection sensitivity twofold, we genetically co-expressed a modified form of a key enzyme in the cholesterol biosynthesis pathway. In a HeLa xenograft tumor mouse model, limonene is a sensitive and specific volatile reporter of tumor presence and growth, permitting detection of tumors as small as 5 mm. Moreover, tumor detection in mice improves proportionally with breath sampling time. By continuously collecting VOCs for 10 hours, we improve sensitivity for cancer detection 100-fold over static headspace sampling methods. Whole-body physiologically-based pharmacokinetic (PBPK) modeling and simulation of tumor-derived limonene predicts detection of tumors as small as 7 mm in humans, equivalent to the detection limit of clinical imaging modalities, such as PET, yet far more economical.

Significance StatementWe developed a breath-based reporter system using the plant terpene, D-limonene - a volatile secondary metabolite that gives citrus fruit its characteristic scent but is not produced in human tissues - as a biomarker for early cancer detection. Results from this study could pave the way for in vivo gene delivery and tumor-specific expression of exogenous volatile cancer reporters with broad applicability to the early diagnosis of a wide variety of cancers.

category: bioengineering


title: Fast and robust non-negative matrix factorization for single-cell experiments

abstract: Non-negative matrix factorization (NMF) is an intuitively appealing method to extract additive combinations of measurements from noisy or complex data. NMF is applied broadly to text and image processing, time-series analysis, and genomics, where recent technological advances permit sequencing experiments to measure the representation of tens of thousands of features in millions of single cells. In these experiments, a count of zero for a given feature in a given cell may indicate either the absence of that feature or an insufficient read coverage to detect that feature ("dropout"). In contrast to spectral decompositions such as the Singular Value Decomposition (SVD), the strictly positive imputation of signal by NMF is an ideal fit for single-cell data with ambiguous zeroes. Nevertheless, most single-cell analysis pipelines apply SVD or Principal Component Analysis (PCA) on transformed counts because these implementations are fast while current NMF implementations are slow. To address this need, we present an accessible NMF implementation that is much faster than PCA and rivals the runtimes of state-of-the-art SVD. NMF models learned with our implementation from raw count matrices yield intuitive summaries of complex biological processes, capturing coordinated gene activity and enrichment of sample metadata. Our NMF implementation, available in the RcppML (Rcpp Machine Learning library) R package, improves upon current NMF implementations by introducing a scaling diagonal to enable convex L1 regularization for feature engineering, reproducible factor scalings, and symmetric factorizations. RcppML NMF easily handles sparse datasets with millions of samples, making NMF an attractive replacement for PCA in the analysis of single-cell experiments.


title: Small proline-rich proteins 1 and 2 function as antimicrobial proteins in the skin

abstract: Human skin functions as a physical barrier, preventing the entry of foreign pathogens while also accommodating a myriad of commensal microorganisms. A key contributor to the skin landscape is the sebaceous gland. Mice devoid of sebocytes are prone to skin infection, yet our understanding of how sebocytes function in host defense is incomplete. Here we show that the small proline-rich proteins, SPRR1 and SPRR2 are bactericidal in skin. SPRR1B and SPPR2A were induced in human sebocytes by exposure to the bacterial cell wall component lipopolysaccharide (LPS). Further, LPS injected into mouse skin triggered the expression of the mouse SPRR orthologous genes, Sprr1a and Sprr2a, through stimulation of MYD88. Both mouse and human SPRR proteins displayed potent bactericidal activity against MRSA (methicillin-resistant Staphylococcus aureus), Pseudomonas aeruginosa and skin commensals. Thus, Sprr1a-/-;Sprr2a-/- mice are more susceptible to MRSA and Pseudomonas aeruginosa skin infection. Lastly, mechanistic studies demonstrate that SPRR proteins exert their bactericidal activity through binding and disruption of the bacterial membrane. Taken together, these findings provide insight into the regulation and antimicrobial function of SPRR proteins in skin and how the skin defends the host against systemic infection.


title: Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption


title: MAPK cascades mediating Trichoderma brevicrassum strain TC967 against phytopathogen Rhizoctonia solani

abstract:
Trichoderma brevicrassum strain TC967 is a novel biological control agent (BCA) against the plant pathogen Rhizoctonia solani and promotes plant growth. MAPK (mitogen-activated protein kinase) cascades were involved in biocontrol by Trichoderma, but functions of each MAPK in regulating biocontrol have not been characterized in one Trichoderma. In this study, we assembled and annotated the genome of strain TC967, and identified its three MAPK gene sequences. Functions of Fus3-, Slt2- and Hog1-MAPK in strain TC967 were dissected. The three MAPKs were all involved in hyphal growth. The Hog1-MAPK was essential for conidiation and tolerance to hyperosmotic stress. The Fus3- and Slt2-MAPK both mediated cell-wall integrity (CWI) and activities of chitinase and protease. The Fus3- and Hog1-MAPK mediated response to oxidative stress. Our biocontrol assays demonstrated that the Fus3- and Slt2-MAPK mutants were considerably more effective in disease control than the wild-type strain. RNA-seq analysis revealed that MAPK collectively played a major role in regulating biocontrol-related gene expressions, including of the genes in charge of secondary metabolism, fungal cell wall-degrading enzymes (FCWDEs) and small secreted cysteine-rich proteins (SSCPs).

Author summarySoil-born fungal pathogens pose an emerging threat to crop production. Trichoderma brevicrassum strain TC967 has the ability to control the notorious phytopathogen Rhizoctonia solani and promote plant growth. In this study, we explored the functions of three-types of MAPK in mediating biocontrol process, and uncovered that Fus3-, Slt2- and Hog1-MAPK are involved in hyphal growth, conidiation, tolerance to hyperosmotic stress, cell-wall integrity, activities of chitinase and protease, and response to oxidative stress. Biocontrol ability of strain TC967 was accelerated after deletion of the Fus3- and Slt2-MAPK genes. MAPK collectively played a major role in regulating biocontrol-related gene expressions as revealed by RNA-seq analysis. To our knowledge, this is the first report of the functions of MAPKs in regulating biocontrol in one Trichoderma. Our results provide a reference for improvement of biocontrol ability of Trichoderma strains from the view of MAPK cascades.


title: A Shigella type 3 effector protein co-opts host inositol pyrophosphates for activity

abstract:
The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and dysenteric disease. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes.

ImportanceShigella spp. are important human pathogens and one of the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS), and definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB has been shown to contribute to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad.


title: The Shigella type III effector protein OspB is a cysteine protease


title: Retrovirus-induced leukemia - hijack of T-cell activation mechanisms revealed by single-cell analysis

abstract:
Human T-cell leukemia virus type 1 (HTLV-1) mainly infects CD4+ T-cells and induces chronic, persistent infection in infected individuals with some progressing to develop adult T-cell leukemia/lymphoma (ATL). Whilst HTLV-1 alters cellular differentiation, activation and survival, it is unknown whether and how these changes contribute to malignant transformation of infected T-cells. In this study, we used single-cell RNA-Seq and TCR-Seq to investigate T-cell differentiation and HTLV-1-mediated transformation processes. We analyzed 87,742 single cells from peripheral blood of 12 infected and 3 uninfected individuals. Using multiple independent bioinformatic methods, we demonstrated that naive T-cells dynamically change into activated T-cells including infected cells, which seamlessly transitioned into ATL cells characterized by clonally expanded, highly-activated T-cells. Notably, the more activated ATL cells are, the more they acquire Treg signatures. Intriguingly, HLA class II genes were uniquely induced in infected cells, further upregulated in ATL cells and was induced by viral protein Tax. Functional assays revealed that by upregulating HLA class II, HTLV-1-infected cells can act as tolerogenic antigen presenting cells (APCs) to induce anergy of antigen specific T-cells. In conclusion, our study revealed the in vivo mechanisms of HTLV-1-mediated transformation and immune escape at single-cell level.

Graphical Abstract

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title: Dual perspective proteomics infectome profiling discovers Salmonella type III secretion system effector functions in macrophages

abstract:
Intracellular bacterial pathogens have evolved sophisticated infection strategies, including the release and secretion of virulence factors to interfere with host cell functions and to perturb immune responses. For Salmonella enterica serovar Typhimurium (S. Typhimurium), the type III secretion systems encoded on Salmonella pathogenicity islands (SPI) 1 and 2 mediates invasion of the bacterium into innate immune cells and regulates bacterial replication and survival within the hostile environment of the host, respectively. Here, we explore the temporal and strain-specific dual perspective response of both the host and pathogen during cellular infection via quantitative proteomics. We report time- and pathogenicity island-specific expression and secretion of infection-associated proteins (i.e., SL1344_1263, SL1344_3112, SL1344_1563, and YnhG) and regulated immune response proteins in macrophage, including Cd86, Cd40, Casp4, C3, IL-1, and Cd69). Through intracellular macrophage and in vivo murine models of infection, we reveal a role in virulence for three of the bacterial proteins (SL1344_1263, SL1344_1563, and YnhG), defining their importance as novel T3SS effectors. We characterize the temporal intra- and extracellular production of the effectors and identify their interaction networks in host cells, representing inhibitory and stimulatory pathways mounted by invading bacterial pathogens.

Author SummaryThe relationship between a host and pathogen is intricate, and regulation of the host immune response correlates with the progressive timing of infection and tailored responses to the pathogen. Relying on detection and quantification of protein-level changes using mass spectrometry-based proteomics, we explore the production of known and novel effectors by Salmonella during intracellular survival within macrophage. Our results portray a role for these effectors in bacterial virulence using an in vivo murine model of infection, and we define a dynamic network of interaction between the effectors and host proteins. These interactions reveal opportunity for drug design to disrupt modulation of the host by the invading bacterium as a new strategy for combatting infection. Our approach is dynamic and universal, with the power to alter therapeutic discovery against infectious diseases.


title: Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast

abstract: Nuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyl-transferases (KATs) promote transcription through acetylation of chromatin-associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits to the nuclear basket the mRNA export factor Sac3, the scaffolding subunit of the Transcription and Export 2 (TREX-2) complex. Esa1-dependent nuclear export of mRNAs promotes entry into S phase, and is inhibited by the Hos3 deacetylase in G1 daughter cells to restrain their premature commitment to a new cell division cycle. This mechanism also inhibits expression of the nutrient-regulated GAL1 gene specifically in daughter cells. These results reveal how acetylation contributes to the functional plasticity of NPCs in specific cell types, and demonstrate how the evolutionarily conserved NuA4 complex regulates gene expression dually at the level of transcription and mRNA export, by modifying the nucleoplasmic entrance to nuclear pores.


title: Attention-based deep multiple instances learning for classifying circular RNA and other long non-coding RNA

abstract: Circular RNA (circRNA) is a distinguishable circular formed long non-coding RNA (lncRNA), which has specific roles in transcriptional regulation, multiple biological processes. The identification of circRNA from other lncRNA is necessary for relevant research. In this study, we designed attention-based multi-instance learning (MIL) network architecture, which can be fed with raw sequence, to learn the sparse features in sequences and accomplish the identification task for circRNAs. The model outperformed previously reported models. Following the effectiveness validation of the attention score by the handwritten digit dataset, the key sequence loci underlying circRNAs recognition were obtained based on the corresponding attention score. Moreover, the motif enrichment analysis of the extracted key sequences identified some of the key motifs for circRNA formation. In conclusion, we designed a deep learning network architecture suitable for gene sequence learning with sparse features and implemented to the circRNA identification, and the network has a strong representation capability with its indication of some key loci.


title: Parasite-probiotic interactions in the gut: Bacillus sp. and Enterococcus faecium regulate type-2 inflammatory responses and modify the gut microbiota of pigs during helminth infection

abstract: Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and faecal microbiota, notably including an enrichment of faecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.


title: CARD8 inflammasome sensitization through DPP9 inhibition enhances NNRTI-triggered killing of HIV-1-infected cells

abstract: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) induce pyroptosis of HIV-1 infected CD4+ T cells through induction of intracellular viral protease activation, which then activates the CARD8 inflammasome. Due to high concentrations of NNRTIs being required for efficient CARD8 activation and elimination of HIV-1-infected cells, it is important to elucidate ways to sensitize the CARD8 inflammasome to NNRTI-induced activation. We show that this sensitization can be done through chemical inhibition of the CARD8 negative regulator DPP9. DPP9 inhibitor Val-boroPro (VbP) can act synergistically with NNRTIs to increase their efficacy in killing HIV-1-infected cells. We also show that VbP is able to partially overcome issues with NNRTI resistance and is capable of killing infected cells without the presence of NNRTIs. This offers a promising strategy for enhancing NNRTI efficacy in elimination of HIV-1 reservoirs in patients.


title: Generating heterokaryotic cells via bacterial cell-cell fusion

abstract: Cell-cell fusion is fundamentally important for tissue repair, virus transmission, and genetic recombination, among other functions. Fusion has been mainly studied in eukaryotic cells and lipid vesicles, while cell-cell fusion in bacteria is less well characterized, due to the cell wall acting as a fusion-limiting barrier. Here we use cell wall-deficient bacteria to investigate the dynamics of cell fusion in bacteria that replicate without their cell wall. Stable, replicating cells containing differently labeled chromosomes were successfully obtained from fusion. We find that the rate of cell-cell fusion depends on the fluidity of cell membranes. Furthermore, we show that not only the efficiency but also the specificity of cell-cell fusion can be controlled via a pair of synthetic membrane-associated lipopeptides. Our results provide a molecular handle to understand and control cell-cell fusion to generate heterokaryotic cells, which was an important step in the evolution of protocells and of increasing importance for the design of synthetic cells.


title: Meta-exoproteomics identifies active plant-microbe interactions operating in the rhizosphere

abstract: The advance of DNA sequencing technologies has drastically changed our perception of the complexity and structure of the plant microbiome and its role in augmenting plant health. By comparison, our ability to accurately identify the metabolically active fraction of soil microbiota and their specific functional role is relatively limited. Here, we combined our recently developed protein extraction method and an iterative bioinformatics pipeline to enable the capture and identification of extracellular proteins (meta-exoproteomics) expressed in the rhizosphere of Brassica spp. First, we validated our method in the laboratory by successfully identifying proteins related to the host plant (Brassica rapa) and a bacterial inoculant, Pseudomonas putida BIRD-1, revealing the latter expressed numerous rhizosphere specific proteins linked to the acquisition of plant-derived nutrients. Next, we analysed natural field-soil microbial communities associated with Brassica napus L (Oil Seed rape). By combining deep-sequencing metagenomics with meta-exoproteomics, a total of 1882 proteins were identified in bulk and rhizosphere samples. Importantly, meta-exoproteomics identified a clear shift (p<0.001) in the metabolically active fraction of the soil microbiota responding to the presence of B. napus roots that was not apparent in the composition of the total microbial community (metagenome). This metabolic shift was associated with the stimulation of rhizosphere-specialised bacteria, such as Gammaproteobacteria, Betaproteobacteria and Flavobacteriia and the upregulation of plant beneficial functions related to phosphorus and nitrogen mineralisation. By providing the first meta-proteomic level assessment of the  active plant microbiome at the field-scale, this study demonstrates the importance of moving past a genomic assessment of the plant microbiome in order to determine ecologically important plant: microbe interactions driving plant growth.


title: Update on the mosquito fauna (Diptera: Culicidae) distribution in Cabo Verde: occurrence of the species complexes Anopheles gambiae and Culex pipiens (pipiens, quinquefasciatus and their hybrids)

abstract: In this study, we aimed to update the mosquito species composition and distribution based on a national entomological survey in all municipalities of Cabo Verde. This includes the sibling species of the Culex pipiens complex, namely Cx. pipiens, Cx. quinquefasciatus and their hybrids, in locations where information is not available. The entomological survey took place from October 2017 to September 2018, in all municipalities of Cabo Verde. Mosquito larvae and pupae were collected in breeding sites and samples were sent to the Laboratory of Medical Entomology of the National Institute of Public Health for the morphological identification of the species. The mosquitoes morphologically identified in Anopheles gambiae and Culex pipiens complexes of species were further molecular analysed to species confirmation. A total of 814 breeding sites were surveyed and 10 mosquito species of five genera were identified. The greatest number of mosquito species was reported in the island of Santiago. The most widespread species in the country were Aedes aegypti and Culex quinquefasciatus. Anopheles arabiensis was the only species identified in the Gambiae complex of species. The results of this study will assist decision makers in important health policies to control mosquitoes and vector-borne diseases towards a strategic approach by timely detection of changes in species diversity.

category: ecology


title: Genome-wide association analysis of cotton salt stress response-related sites

abstract: Soil salinization is the main abiotic stress factor affecting agricultural production worldwide, and salt stress has a significant impact on plant growth and development. Cotton is one of the most salt-tolerant crops. Its salt tolerance varies greatly depending on the variety, growth stage, organs, and soil salt types. Therefore, the selection and utilization of excellent salt-tolerant germplasm resources and the excavation of excellent salt-tolerant salt and salt resistance genes play important roles in improving cotton production in saline-alkali soils. In this study, we analysed the population structure and genetic diversity of 144 elite Gossypium hirsutum cultivar accessions collected from around the world, and especially from China. Illumina Cotton SNP 70K was used to obtain genome-wide single-nucleotide polymorphism (SNP) data for 149 experimental materials, and 18,432 highly consistent SNP loci were obtained by filtering. PCA (principal component analysis) indicated that 149 upland cotton materials could be divided into 2 subgroups, including subgroup 1 with 78 materials and subgroup 2 with 71 materials. Using the obtained SNP and other marker genotype test results, under salt stress, the salt tolerance traits 3d_Germination_potential, 3d_Bud_length_drop_rate, 7d_Germination_rate, 7d_Bud_length_drop_rate, 7d_Germination_weight, 3d_Bud_length, 7d_Bud_length, relative_germination_potential, Relative_germination_rate, 7d_Bud_weight_drop_rate, Salt tolerance index 3d_Germination_potential_index, 3d_Bud_length_index, 7d_Bud_length_index, 7d_Bud_weight_index, and 7d_Germination_rate_index were evaluated by genome association analysis. A total of 27 SNP markers closely related to salt tolerance traits and 15 SNP markers closely related to salt tolerance index were detected. At the SNP locus associated with the traits of the bud length decline rate at 7 days, alleles Gh_A01G0034 and Gh_D01G0028 related to plant salt tolerance were detected, and they are related to intracellular transport, membrane microtubule formation and actin network. This study provides a theoretical basis for the selection and breeding of salt-tolerant upland cotton varieties.


title: Biochemical characterisation of human transglutaminase 4

abstract: Transglutaminases are protein modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is frequently associated with pathological symptoms and particularly with cancer invasiveness. Although rodent TG4 is well characterised, bio-chemical characteristics of human TG4 that could help the understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtiter plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTP{gamma}S binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.


title: Thalamic bursts modulate cortical synchrony locally to switch between states of global functional connectivity in a cognitive task

abstract:
Performing a cognitive task requires going through a sequence of functionally diverse stages. Although it is typically assumed that these stages are characterized by distinct states of cortical synchrony that are triggered by sub-cortical events, little reported evidence supports this hypothesis. To test this hypothesis, we first identified cognitive stages in single-trial MEG data of an associative recognition task, showing with a novel method that each stage begins with local modulations of synchrony followed by a state of directed functional connectivity. Second, we developed the first whole-brain model that can simulate cortical synchrony throughout a task. The model suggests that the observed synchrony is caused by thalamocortical bursts at the onset of each stage, targeted at cortical synapses and interacting with the structural anatomical connectivity. These findings confirm that cognitive stages are defined by distinct states of cortical synchrony and explains the network-level mechanisms necessary for reaching stage-dependent synchrony states.

Author summaryA novel machine-learning method was applied to unveil the dynamics of local and cortex-wide neural coordination underlying the fundamental cognitive processes involved in a memory task. To explain how neural activity - and ultimately behavior - was coordinated throughout the task, we developed a whole-brain model that incorporates cognitive mechanisms, anatomy, and neural biophysics. Similar models are used with resting state data, however, simulating a cognitive task remained elusive. The model showed that sub-cortical pulses at the onset of cognitive processes - as hypothesized by cognitive and neurophysiological theories - were sufficient to switch between the states of neural coordination observed. These findings have implications to understand goal-directed cognitive processing and the mechanisms to reach states of neural coordination.


title: Functional connectivity gradients as a common neural architecture for predictive processing in the human brain

abstract: Predictive processing is emerging as a common computational hypothesis to account for diverse psychological functions subserved by a brain, providing a systems-level framework for characterizing structure-function relationships of its distinct substructures. Here, we contribute to this framework by examining gradients of functional connectivity as a low dimensional spatial representation of functional variation in the brain and demonstrating their computational implications for predictive processing. Specifically, we investigated functional connectivity gradients in the cerebral cortex, the cerebellum, and the hippocampus using resting-state functional MRI data collected from large samples of healthy young adults. We then evaluated the degree to which these structures share common principles of functional organization by assessing the correspondence of their gradients. We show that the organizing principles of these structures primarily follow two functional gradients consistent with the existing hierarchical accounts of predictive processing: A model-error gradient that describes the flow of prediction and prediction error signals, and a model-precision gradient that differentiates regions involved in the representation and attentional modulation of such signals in the cerebral cortex. Using these gradients, we also demonstrated triangulation of functional connectivity involving distinct subregions of the three structures, which allows characterization of distinct ways in which these structures functionally interact with each other, possibly subserving unique and complementary aspects of predictive processing. These findings support the viability of computational hypotheses about the functional relationships between the cerebral cortex, the cerebellum, and the hippocampus that may be instrumental for understanding the brains dynamics within its large-scale predictive architecture.


title: Tolerance of Pseudomonas Strain to the 2,4-D Herbicide through a Peroxidase System

abstract: Herbicides are widely used in agricultural practices for preventing the proliferation of weeds that compete with crops for survival. Upon reaching soil and water, herbicides can damage nontarget organisms, such as bacteria, which need an efficient defense mechanism to tolerate the stress induced by herbicides. 2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide that exerts increased oxidative stress among bacterial communities that consequently witness an increased toxicity in their microenvironments. Bacterial isolates were obtained from the biofilm of water that was contaminated with 2,4-D. This biofilm originated from the tanks containing washing water from the packaging of different pesticides, including 2,4-D. Moreover, several isolates were sensitive to biofilm toxicity; however, they remained alive in the presence of 2,4-D. The Pseudomonas sp. CMA-7.3 was selected because of its tolerance against biofilm agrochemicals. Therefore, the objective of this study is to evaluate the antioxidative response system of the Pseudomonas sp. CMA-7.3. This study also analyzed poorly evaluated enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase GPX, in the bacterial systems. The toxic effects of 2,4-D on bacteria were evaluated using mechanisms indicating oxidative stress, such as growth curve, cell viability, peroxide, and malondialdehyde. The Pseudomonas sp. CMA-7.3 was an efficient response system against the activity of antioxidant enzymes such as SOD, CAT, APX, and GPX in balancing the production of H2O2, even at high doses as 25x the field dose of the herbicide, thereby proving the toxicity of 2,4-D for this strain and showing the ability of the strain to tolerate 2,4-D. The adaptation of this microorganism to herbicide exposure is truly relevant for improving future metabolic studies on bacterial communities. The strain showed a great potential in the application and developmental prospects of a new product in the bioremediation process of environments contaminated by these herbicides.


title: Visualizing Antineoplastic Activity with a Whole Organism Drosophila melanogaster Screen

abstract: Cancer is a disease characterized by high mitosis rates with a loss of regulation. Many antineoplastics, those drugs used to treat cancer, act by slowing or halting mitosis. We are developing a whole-organism screening protocol to identify novel antineoplastics. After exposing Drosophila melanogaster eggs and larva to a compound, their growth rate and population decrease if mitosis inhibition or arrest occur. We screened several compounds from the National Cancer Institutes (NCI) Developmental Therapeutics Program (DTP). Our screen successfully identified two compounds, toyocamycin and stictic acid, previously identified as possible antineoplastics. Toyocamycin killed a fraction of the population proportional to the dose concentration resulting in full mortality at 100 and 200 M. At low doses, toyocamycin also slowed larval development by a mean of one day. RNAseq showed that no genes were differentially expressed in mature flies after toyocamycin exposure was halted. Stictic acid delayed larval growth by an equal or greater margin compared to toyocamycin. These results demonstrate that decreases in Drosophila growth or population can predict a compounds antineoplastic activity and toxicity.


title: Sites of active gene regulation in the prenatal frontal cortex and their role in neuropsychiatric disorders

abstract: Common genetic variation appears to largely influence risk for neuropsychiatric disorders through effects on gene regulation. It is therefore possible to shed light on the biology of these conditions by testing for enrichment of associated genetic variation within regulatory genomic regions operating in specific tissues or cell types. Here, we have used ATAC-Seq to map open chromatin (an index of active regulatory genomic regions) in bulk tissue, NeuN+ and NeuN- nuclei from the prenatal human frontal cortex, and tested enrichment of SNP heritability for 5 neuropsychiatric disorders (autism spectrum disorder, ADHD, bipolar disorder, major depressive disorder and schizophrenia) within these regions. We observed significant enrichment of SNP heritability for ADHD, major depressive disorder and schizophrenia within open chromatin regions mapped in bulk fetal frontal cortex, and for all 5 tested neuropsychiatric conditions when we restricted these sites to those overlapping histone modifications indicative of enhancers (H3K4me1) or promoters (H3K4me3) in fetal brain. SNP heritability for neuropsychiatric disorders was significantly enriched in open chromatin regions identified in fetal frontal cortex NeuN- as well as NeuN+ nuclei overlapping fetal brain H3K4me1 or H3K4me3 sites. We additionally demonstrate the utility of our mapped open chromatin regions for prioritizing potentially functional SNPs at genome-wide significant risk loci for neuropsychiatric disorders. Our data provide evidence for an early neurodevelopmental component to a range of neuropsychiatric conditions and highlight an important role for regulatory genomic regions active within both NeuN+ and NeuN- cells of the prenatal brain.


title: High-altitude adaptation and incipient speciation in geladas

abstract: Survival at high altitude requires adapting to extreme conditions such as environmental hypoxia. To understand high-altitude adaptations in a primate, we assembled the genome of the gelada (Theropithecus gelada), an endemic Ethiopian monkey, and complemented it with population resequencing, hematological, and morphometric data. Unexpectedly, we identified a novel karyotype that may contribute to reproductive isolation between gelada populations. We also identified genomic elements including protein-coding sequences and gene families that exhibit accelerated changes in geladas and may contribute to high-altitude adaptation. Our findings lend insight into mechanisms of speciation and adaptation while providing promising avenues for functional hypoxia research.


title: Right fronto-parietal networks mediate the neurocognitive benefits of enriched environments.

abstract: Exposure to enriched environments (EE) throughout a lifetime, providing so called reserve, protects against cognitive decline in later years. It has been hypothesised that high levels of alertness necessitated by EE might strengthen the right fronto-parietal networks (FPN) to facilitate this neurocognitive resilience. We have previously shown that EE offset age-related deficits in selective attention by preserving grey matter within right fronto-parietal regions. Here, using neurite orientation dispersion and density imaging (NODDI), we examined the relationship between EE, microstructural properties of fronto-parietal white matter association pathways (three branches of the superior longitudinal fasciculus, SLF), structural brain health (atrophy), and attention (alertness, orienting and executive control) in a group of older adults. We show that EE is associated with a lower orientation dispersion index (ODI) within the right SLF1 which in turn mediates the relationship between EE and alertness, as well as grey- and white-matter atrophy. This suggests that EE may induce white matter plasticity (and prevent age-related dispersion of axons) within the right FPN to facilitate the preservation of neurocognitive health in later years.


title: Amphiregulin mediates non-cell-autonomous effect of senescence on reprogramming

abstract: Cellular senescence is an irreversible growth arrest with a highly dynamic secretome, termed the senescence-associated secretory phenotype (SASP). Senescence has been implicated in somatic reprogramming to pluripotency. The cell-intrinsic proliferation arrest is a barrier for reprogramming, whereas the SASP facilitates the cell fate conversion in nonsenescent cells. However, the mechanisms by which reprogramming-induced senescence regulates cell plasticity are not well understood. Here, we have further investigated how the heterogeneity of paracrine senescence impacts reprogramming. We show that senescence promotes in vitro reprogramming in a stress-dependent manner. We identified a catalog of SASP factors and pathways potentially involved in the cell fate conversion using an unbiased proteomic analysis. Amphiregulin (AREG), a growth factor frequently secreted by the senescent cells, promotes in vitro reprogramming by accelerating proliferation and MET via the EGFR signaling pathway. Of note, AREG treatment diminished the negative effect of donor age on reprogramming. Finally, AREG enhances in vivo reprogramming in the skeletal muscle. Hence, senescence could facilitate cellular plasticity via various SASP factors to promote reprogramming and tissue repair.


title: Spatial organization of nuclear pores in Xaenopus laevis oocytes

abstract: Nuclear pores are protein assemblies inserted in the nuclear envelope of eukaryotic cells, acting as main gates for communication between nucleus and cytoplasm. So far, nuclear pores have been extensively studied to determine their structure and composition, yet their spatial organization and geometric arrangement on the nuclear surface are still poorly understood. Here, we analyze super-resolution images of the surface of Xenopus laevis oocyte nuclei during development, and characterize the arrangement of nuclear pores using tools commonly employed to study the atomic structural and topological features of soft matter. To interpret the experimental results, we hypothesize an effective interaction among nuclear pores and implemented it in extensive numerical simulations of octagonal clusters mimicking typical pore shapes. Thanks to our simple model, we find simulated spatial distributions of nuclear pores that are in excellent agreement with experiments, suggesting that an effective interaction among nuclear pores exists and could explain their geometrical arrangement. Furthermore, our results show that the statistical features of the geometric arrangement of nuclear pores do not depend on the type of pore-pore interaction, attractive or repulsive, but are mainly determined by the octagonal symmetry of each single pore. These results pave the way to further studies needed to determine the biological nature of pore-pore interactions.

category: biophysics


title: Spatial organization of nuclear pores in Xenopus laevis oocytes


title: Functional fluorescently-tagged human profilin

abstract: Profilin is an essential regulator of actin and microtubule dynamics and therefore a critical control point for the normal division, motility, and morphology of cells. Most studies of profilin have focused on biochemical investigations using purified protein because high cellular concentrations present challenges for conventional imaging modalities. In addition, past studies that employed direct labeling or conventional fusion protein strategies compromised different facets of profilin function. We engineered two versions of tagged profilin that retain native activities with respect to phosphoinositide lipids, actin monomers, formin-mediated actin assembly, and microtubule polymerization. mApple-profilin-1 directly binds to dimers of tubulin (kD = 1.7 {micro}M) and the microtubule lattice (kD = 10 {micro}M) to stimulate microtubule assembly. In cells, Halo-tagged profilin-1 fully rescues profilin-1(-/-) cells from knockout-induced perturbations to cell shape, actin filament architecture, and microtubule arrays. In cells expressing Halo-profilin, we visualized a subset of individual molecules of profilin-1 by titrating fluorescent Halo-ligands. Further, we combined this imaging approach with specific function-disrupting point-mutants in profilin to visualize dynamic profilin associated with microtubules in live-cells. Thus, these tagged profilins are reliable tools for studying the dynamic interactions of profilin with actin or microtubules in live-cell or in vitro applications.


title: Visualizing functional human profilin in cells and in vitro applications


title: Seasonal variation in the relative importance of assembly processes in marine fish communities as determined by environmental DNA analyses

abstract: Compositional variation among local communities reflects the differences in abiotic environments, though the extent of the influence varies in natural systems. Generally, the environmental filtering is expected to be strong if the environmental gradient encompasses severe habitats where species sorting is highly likely to work. However, this hypothesis has rarely been tested in dynamic systems, where the higher dispersal ability of species allows them to easily respond to stressful environments. Here, with the dynamics of fish communities in a Japanese bay revealed by environmental DNA analyses as a model case, we examined how harmful seasonal hypoxia (low concentration of oxygen in bottom waters in summer) affected the strength of environmental filtering. We found that, in summer, dissolved oxygen (DO) concentration was significantly low and fish species richness decreased in the bottom water compared to the surface, suggesting that the organisms were adversely affected by the hypoxia. At the same time, the between-depths heterogeneity in DO concentration was larger, but species composition was less divergent and the influence of DO on species composition appeared weaker during summer. These results imply that environmental filtering is weakened when the bottom water was characterized by extremely severe environments. Furthermore, there was a shift in the species occurrence from bottom to surface waters in summer that was consistent across species, suggesting that the extremely severe hypoxia adversely affected fish species irrespective of their identity. These results collectively suggest that environmental filtering is weaker during summer despite more severity and heterogeneity in environments, most likely because individual movements to avoid unpreferable environments in the bottom waters occurred quasi-neutrally. By providing evidence against the prevailing understanding that environmental filtering strongly works in severe environments, these findings invoke further investigation on how the filtering acts in various conditions.


title: Environmental filtering in marine fish communities is weakened during severe hypoxia season


title: Domestication Reshaped the Genetic Basis of Inbreeding Depression in a Maize Landrace Compared to its Wild Relative, Teosinte

abstract: Inbreeding depression is the reduction in fitness and vigor resulting from mating of close relatives observed in many plant and animal species. The extent to which the genetic load of mutations contributing to inbreeding depression is due to rare large-effect variation versus potentially more common variants with very small individual effects is unknown and may be affected by population history. We compared the effects of outcrossing and self-fertilization on 18 traits in a landrace population of maize, which underwent a population bottleneck during domestication, and a neighboring population of its wild relative teosinte. Inbreeding depression was greater in maize than teosinte for 15 of 18 traits, congruent with the greater segregating genetic load predicted from sequence data in the maize population. For many traits - and more commonly in maize - genetic variation among selffertilized families was less than expected based on additive and dominance variance estimated in outcrossed families, suggesting that a negative covariance between additive and homozygous dominance effects limits the variation available to selection under partial inbreeding. We identified quantitative trait loci (QTL) representing large-effect rare variants carried by only a single parent, which were more important in teosinte than maize. Teosinte also carried more putative juvenile-acting lethal variants identified by segregation distortion. These results suggest a mixture of mostly polygenic, smalleffect recessive variation underlying inbreeding depression, with an additional contribution from rare larger-effect variants that was more important in teosinte but depleted in maize following to the domestication bottleneck. Purging associated with the maize domestication bottleneck may have selected against large effect variants, but polygenic load is harder to purge and segregating mutational burden increased in maize compared to teosinte.

category: evolutionary biology


title: Multi-omic profiling of histone variant H3.3 lysine 27 methylation reveals a distinct role from canonical H3 in stem cell differentiation

abstract: Histone variants, such as histone H3.3, replace canonical histones within the nucleosome to alter chromatin accessibility and gene expression. Although the biological roles of selected histone post-translational modifications (PTMs) have been extensively characterized, the potential differences in the function of a given PTM on different histone variants is almost always elusive. By applying proteomics and genomics techniques, we investigate the role of lysine 27 tri-methylation specifically on the histone variant H3.3 (H3.3K27me3) in the context of mouse embryonic stem cell pluripotency and differentiation as a model system for development. We demonstrate that while the steady state overall levels of methylation on both H3K27 and H3.3K27 decrease during differentiation, methylation dynamics studies indicate that methylation on H3.3K27 is maintained more than on H3K27. Using a custom-made antibody, we identify a unique enrichment of H3.3K27me3 at lineage-specific genes, such as olfactory receptor genes, and at binding motifs for the transcription factors FOXJ2/3. REST, a predicted FOXJ2/3 target that acts as a transcriptional repressor of terminal neuronal genes, was identified with H3.3K27me3 at its promoter region. H3.3K27A mutant cells confirmed an upregulation of FOXJ2/3 targets upon the loss of methylation at H3.3K27. Thus, while canonical H3K27me3 has been characterized to regulate the expression of transcription factors that play a general role in differentiation, our work suggests H3.3K27me3 is essential for regulating distinct terminal differentiation genes. This work highlights the importance of understanding the effects of PTMs not only on canonical histones but also on specific histone variants, as they may exhibit distinct roles.


title: Genomically hardwired regulation of gene activity orchestrates cellular iron homeostasis in Arabidopsis

abstract: Iron (Fe) is an essential micronutrient that plays pivotal roles as electron donor and catalyst across organisms. In plants, variable, often insufficient Fe supply necessitates mechanisms that constantly attune Fe uptake rates and recalibrate cellular Fe homeostasis. Here, we show that short-term (0.5, 6, and 12 h) exposure of Arabidopsis thaliana plants to Fe deficiency triggered massive changes in gene activity governed by transcription and alternative splicing (AS), regulatory layers that were to a large extent mutually exclusive. Such preclusion was not observed for genes that are directly involved in the acquisition of Fe, which appears to be concordantly regulated by both expression and AS. Generally, genes with lower splice site strengths and higher intron numbers were more likely to be regulated by AS, no dependence was on gene architecture was observed for transcriptionally controlled genes. Conspicuously, specific processes were associated with particular genomic features and biased towards either regulatory mode, suggesting that genomic hardwiring is functionally biased. Early changes in splicing patterns were, in many cases, congruent with later changes in transcript or protein abundance, thus contributing to the pronounced transcriptome-proteome discordance observed in plants.