title: A dynamic kissing model for enhancer-promoter communication on the surface of transcriptional condensate

abstract: Enhancers are key DNA elements that regulate gene transcription by activating promoters in eukaryotes. Many potential mechanistic models have been proposed to account for enhancer-promoter communication, including the formation of the condensed enhancer-promoter clusterings that may result from liquid-liquid phase separation. However, the detailed kinetic mechanisms by which condensates may mediate enhancer-promoter contacts remain unclear. Here, we characterized enhancer-promoter communication in condensates using a self-avoiding polymer chain model under several possible spatial constraints. When the chain was confined to the spherical surface or interior, we discovered a distinct relationship between average end-to-end distance and contour length, as well as a distinct distribution of end-to-end distance. Experimental molecular tracking data showed strong preference in support of the spherical surface confinement hypothesis. Based on such observation, we proposed a novel enhancer-promoter communication model in which enhancers dynamically "kiss" promoters along the surface of transcriptional condensate. The theoretical results may guide future experiments, as researchers continue to elucidate the regulation dynamics of gene expression.


title: Amplification of Olfactory Signals by Anoctamin 9 is Essential for Mammalian Olfaction: a Risk Factor for the Covid-19-associated Anosmia

abstract: Sensing smells of foods, prey, or predators determines animal survival. Olfactory sensory neurons in the olfactory epithelium (OE) detect odorants, where cAMP and Ca2+ play a significant role in transducing odorant inputs to electrical activity. Here we show Anoctamin 9, a cation channel activated by cAMP/PKA pathway, is expressed in the OE and amplifies olfactory signals. Ano9- deficient mice had reduced olfactory behavioral sensitivity, electro-olfactogram signals, and neural activity in the olfactory bulb. In line with the difference in olfaction between birds and other vertebrates, chick ANO9 failed to respond to odorants, whereas chick CNGA2, a major transduction channel, showed greater responses to cAMP. Importantly, single-cell transcriptome data from Covid-19 patients revealed that Ano9 transcripts were markedly suppressed among genes in the olfactory signal pathway. The signal amplification by ANO9 is essential for mammalian olfactory transduction, whose downregulation may be a risk factor for the olfactory dysfunction in Covid-19 patients.

category: cell biology


title: Lack of antiviral activity of probenecid in Vero E6 cells and Syrian golden hamsters: a need for better understanding of inter-lab differences in preclinical assays.

abstract: Antiviral interventions are urgently required to support vaccination programmes and reduce the global burden of COVID-19. Prior to initiation of large-scale clinical trials, robust preclinical data in support of candidate plausibility are required. The speed at which preclinical models have been developed during the pandemic are unprecedented but there is a vital need for standardisation and assessment of the Critical Quality Attributes. This work provides cross-validation for the recent report demonstrating potent antiviral activity of probenecid against SARS-CoV-2 in preclinical models (1). Vero E6 cells were pre-incubated with probenecid, across a 7-point concentration range, or control media for 2 hours before infection with SARS-CoV-2 (SARS-CoV-2/Human/Liverpool/REMRQ0001/2020, Pango B; MOI 0.05). Probenecid or control media was then reapplied and plates incubated for 48 hours. Cells were fixed with 4% v/v paraformaldehyde, stained with crystal violet and cytopathic activity quantified by spectrophotometry at 590 nm. Syrian golden hamsters (n=5 per group) were intranasally inoculated with virus (SARS-CoV-2 Delta variant B.1.617.2; 103 PFU/hamster) for 24 hours prior to treatment. Hamsters were treated with probenecid or vehicle for 4 doses. Hamsters were ethically euthanised before quantification of total and sub-genomic pulmonary viral RNAs. No inhibition of cytopathic activity was observed for probenecid at any concentration in Vero E6 cells. Furthermore, no reduction in either total or sub-genomic RNA was observed in terminal lung samples from hamsters on day 3 (P > 0.05). Body weight of uninfected hamsters remained stable throughout the course of the experiment whereas both probenecid- (6 - 9% over 3 days) and vehicle-treated (5 - 10% over 3 days) infected hamsters lost body weight which was comparable in magnitude (P > 0.5). The presented data do not support probenecid as a SARS-CoV-2 antiviral. These data do not support use of probenecid in COVID-19 and further analysis is required prior to initiation of clinical trials to investigate the potential utility of this drug.

category: pharmacology and toxicology


title: Soluble signal inhibitory receptor on leukocytes-1 is released from activated neutrophils by proteinase 3 cleavage

abstract: Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes which dampens antimicrobial functions. We previously showed that sputum neutrophils from infants with severe respiratory syncytial virus (RSV) bronchiolitis have decreased SIRL-1 surface expression compared to blood neutrophils, and that SIRL-1 surface expression is rapidly lost from in vitro activated neutrophils. This led us to hypothesize that activated neutrophils lose SIRL-1 by ectodomain shedding. Here, we developed an ELISA and measured the concentration of soluble SIRL-1 (sSIRL-1) in RSV bronchiolitis and hospitalized COVID-19 patients, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared to plasma of RSV bronchiolitis patients, and in serum of hospitalized COVID-19 patients compared to control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage, which can be prevented by proteinase 3 inhibition. Finally, we found that SIRL-1 shedding is prevented by extracellular adherence protein (Eap) from S. aureus. Notably, we recently showed that SIRL-1 is activated by PSM3 from S. aureus, suggesting that S. aureus may counteract SIRL-1 shedding to benefit from preserved inhibitory function of SIRL-1. In conclusion, we are the first to report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.

category: immunology


title: Microbial invasion of a toxic medium is facilitated by a resident community but inhibited as the community co-evolves

abstract: Predicting whether microbial invaders will colonize an environment is critical for managing natural and engineered ecosystems, and controlling infectious disease. Invaders often face competition by resident microbes. But how invasions play out in communities dominated by facilitative interactions is less clear. We previously showed that growth medium toxicity can promote facilitation between four bacterial species, as species that cannot grow alone rely on others to survive. Following the same logic, here we allowed other bacterial species to invade the four-species community, and found that invaders could more easily colonize a toxic medium when the community was present. In a more benign environment instead, invasive species that could survive alone colonized more successfully when the residents were absent. Next, we asked whether early colonists could exclude future ones through a priority effect, by inoculating the invaders into the resident community only after its members had co-evolved for 44 weeks. Compared to the ancestral community, the co-evolved resident community was more competitive toward invaders, and less affected by them. Our experiments show how communities may assemble by facilitating one another in harsh, sterile environments, but that arriving after community members have co-evolved can limit invasion success.

category: microbiology


title: A novel biosynthetic gene cluster across the Pantoea species complex is important for pathogenicity in onion

abstract:
Onion center rot is caused by at least four species of Pantoea (P. ananatis, P. agglomerans, P. allii, and P. stewartii subsp. indologenes). Critical onion pathogenicity determinants for P. ananatis were recently described but whether those determinants are common among other onion-pathogenic Pantoea species remains unknown. In this work, we report onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic gene clusters in different strains of onion pathogenic P. stewartii subsp. indologenes. One cluster is similar to the previously described HiVir phosphonate biosynthetic cluster of P. ananatis and another is a novel putative phosphonate biosynthetic gene cluster, which we name "Halophos". The Halophos gene cluster was also identified in P. allii strains. Both clusters are predicted to be phosphonate biosynthetic clusters based on the presence of a characteristic phosphenolpyruvate phosphomutase (pepM) gene. The deletion of pepM gene from either the P. stewartii subsp. indologenes HiVir or Halophos clusters caused loss of necrosis on onion leaves and red onion scales, and resulted in significantly lower bacterial populations compared to the corresponding wildtype and complemented strains. Seven (halB-halH) out of eleven genes (halA-halK) in the Halophos gene cluster are required for onion necrosis phenotypes. The onion non-pathogenic strain PNA15-2 gained the capacity to cause foliar necrosis on onion via exogenous expression of a minimal seven gene Halophos cluster (halB -halH). Furthermore, cell-free culture filtrates of PNA14-12 expressing the intact Halophos-gene cluster caused necrosis on onion leaves consistent with the presence of a secreted toxin. Together, these observations indicated that pepM genes in both phosphonate biosynthetic gene clusters (HiVir and Halophos) are important for Pantoea spp. onion pathogenicity and the biosynthetic product of the Halophos cluster causes necrosis on onion leaf tissue. Overall, this is the first report of onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii.

Author summaryOnion center rot is caused by multiple Pantoea species including P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic clusters associated with onion pathogenic strains, the validated HiVir phosphonate cluster and a putative phosphonate biosynthetic cluster that we named as Halophos based on the associated "halo" phenotype on the red onion scales. We found that pepM genes from each cluster (HiVir and Halophos) are required for onion infection by P. stewartii subsp. indologenes and P. allii but not for millet infection by P. stewartii subsp. indologenes. Conversely, the T3SS was important for millet infection by P. stewartii subsp. indologenes but not onion infection. Induction of the intact Halophos cluster was associated with the accumulation of a necrosis-inducing factor in culture, which suggests it might be a secreted phytotoxin. Seven of the eleven Halophos cluster genes are required for onion necrosis phenotypes and expression of this minimal cluster conferred a limited onion necrosis phenotype to an onion-non-pathogenic Pantoea strain. We provide evidence of a Halophos biosynthetic gene cluster to be associated with onion pathogenicity in strains of P. stewartii subsp. indologenes and P. allii.


title: Synchronic resisto-genesis effects identify most prevalent mechanism in Acinetobacter baumannii

abstract: Acinetobacter baumannii as deadliest infection agent to ICU patients challenge experimental treatment in lack of new antibiotic and non-optimal recommendations. Despite confirm importance of infectious ICU sources, IC2 strains, ISAba/blaOXA genes and colistin in adequate concentrations or combination therapy due to potential heteroresistance; this study reveals unusual hypersensitive colistin- and sensitive aminoglycosides-resistant strains using designed methods, antibio-dendrogram classification and a computational table. Accordingly, lipopolysaccharide loss-mediated penetration and upregulated AdeAB pump-mediated compounds exclusion, are found to be causes of these abnormalities. Consequently, these simultaneous resistances termed synchronic resisto-genesis effects, drive population and create abnormal multidrug-resistant strains; while, delay extensively drug-resistant phenotype. This phenomenon predicts the predominant determinant mechanisms and makes abnormal behavior comprehensible among antibiotic-phenotypic levels or in vitro and vivo observations. Therefore, this method provides preclinical information to address therapeutic resistance in future microbial or cancer cell studies, and improves drug choosing in combination therapies, like use of inhibitory drugs.


title: Z-RNA and the flipside of the SARS Nsp13 helicase

abstract: We present evidence that the severe acute respiratory syndrome coronavirus (SARS) non-structural protein 13 (Nsp13) modulates the Z-RNA dependent regulated cell death pathways [1]. We show that Z-prone sequences (called flipons [2]) exist in coronavirus and provide a signature (Z-sig) that enables identification of the animal viruses from which the human pathogens arose. We also identify a potential RIP Homology Interaction Motif (RHIM) in the helicase Nsp13 that resembles those present in proteins that initiate Z-RNA-dependent cell death through interactions with the Z-RNA sensor protein ZBP1. These two observations allow us to suggest a model in which Nsp13 down regulates Z-RNA activated innate immunity by two distinct mechanisms. The first involves a novel ATP-independent Z-flipon helicase (flipase) activity in Nsp13 that differs from that of canonical A-RNA helicases. This flipase prevents formation of Z-RNAs that would otherwise activate cell death pathways. The second mechanism likely inhibits the interactions between ZBP1 and the Receptor Interacting Proteins Kinases RIPK1 and RIPK3 by targeting their RHIM domains. Together the described Nsp13 RHIM and flipase activities have the potential to alter the host response to coronaviruses and impact the design of drugs targeting the Nsp13 protein. The Z-sig and RHIM domains may provide a way of identifying previously uncharacterized viruses that are potentially pathogenic for humans.


title: Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize and Purify a Detergent-Sensitive Membrane Protein Assembly

abstract: The peptidisc membrane mimetic enables global reconstitution of the bacterial membrane proteome into water-soluble detergent-free particles, termed peptidisc libraries. We present here a method that combines peptidisc libraries and chromosomal-level gene tagging technology with affinity purification and mass spectrometry (AP/MS) to stabilize and identify fragile membrane protein complexes that exist at native expression levels. This method circumvents common artifacts caused by bait protein overproduction and protein complex dissociation due to lengthy exposure to detergents during protein isolation. Using the E. coli Sec system as a case study, we identify an expanded version of the translocon, termed the HMD complex, consisting of 9 different integral membrane subunits. This complex is stable in peptidiscs but dissociates in detergent. Guided by this native-level proteomic information, we design and validate a procedure that enables purification of the HMD complex with minimal protein dissociation. These results highlight the utility of peptidiscs and AP/MS to discover and stabilize fragile membrane protein assemblies.

category: biochemistry


title: Roles of the second-shell amino acid R266 in other members of the MLE subgroup of the enolase superfamily

abstract: Catalytic promiscuity is the coincidental ability to catalyze non-biological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies such a pre-adaptive residue in an N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzymes from the NSAR/OSBS subfamily. Previously, we identified a point mutation, R266Q, in the catalytically promiscuous Amycolatopsis sp. T-1-60 NSAR/OSBS that has a deleterious effect on NSAR activity with a lesser effect on OSBS activity (Truong et al., in preparation). We demonstrated that R266 was a pre-adaptive feature that enabled the emergence and evolution of NSAR activity in AmyNSAR/OSBS. We examined the role of the residue R266 in the evolution of NSAR activity by examining the effects of the single substitution R266Q in other members of the NSAR/OSBS subfamily including Enterococcus faecalis NSAR/OSBS, Roseiflexus castenholzii NSAR/OSBS, Lysinibacillus varians NSAR/OSBS, and Listeria innocua NSAR/OSBS, which have been previously characterized to carry out both OSBS and NSAR activities efficiently. RcNSAR/OSBS, LvNSAR/OSBS, EfNSAR/OSBS, and LiNSAR/OSBS are 49, 48, 32, and 28% identical, respectively, to AmyNSAR/OSBS. We found that while the R266Q mutation decreases NSAR activity more than OSBS activity, as expected, in most NSAR/OSBS members, the differential effects of the R266Q substitution on NSAR and OSBS activities are not as striking as observed in AmyNSAR/OSBS. In some homologs, the R266Q mutation has very deleterious effects on both OSBS and NSAR activities. Furthermore, the mutation unexpectedly decreases OSBS activity more than NSAR activity in LiNSAR/OSBS. Thus, the effects of R266Q on NSAR and OSBS activities depend on differences in sequence context between members of the NSAR/OSBS subfamily, demonstrating the complex role of epistasis in the evolution of NSAR activity in the NSAR/OSBS subfamily.


title: Structural basis of flagellar filament asymmetry and supercoil templating by Leptospira spirochete sheath proteins

abstract: Several Leptospira species are bacterial agents of leptospirosis, a neglected tropical disease responsible for ~1 million cases and 50,000 deaths each year worldwide. Leptospira, like other members of the Spirochaeta phylum, possess specially adapted flagella that remain confined within the periplasm. These appendages drive a unique, corkscrew-like swimming style that enables efficient motility and pathogenesis. However, the composition, function, and molecular architecture of spirochetal flagellar filaments remain poorly understood. We solved single-particle cryo-EM structures of isolated Leptospira flagellar filaments, comparing the wild-type form to two mutant forms with different missing components and abrogated motilities. The structures reveal a complex proteinaceous sheath surrounding a conserved core composed of the FlaB flagellin homolog. Sheath proteins were found to fall into two distinct categories, both of which are required for motility. Filament  coiling proteins, FcpA and FcpB, exert force on the filament when they bind its surface, causing the filament to stretch. In contrast, we identify sheath components FlaAP (newly discovered in this study) and FlaA2 as  template factors, which have little effect on filament shape by themselves, but partition the coiling proteins to one side of the filament. In this way, the two types of Leptospira sheath factors operate collectively on the flagellar filament to bend it from a  relaxed form associated with cell immobility, to a motility-competent shape that is tightly supercoiled. Our structures also indicate that core-sheath interactions are largely mediated by carbohydrate moieties from flagellin core side chain O-glycosylations. The supercoiling mechanism presented here provides a benchmark for studies with other bacteria, for which near-atomic resolution structures of flagellar filament in native supercoiled forms, are still lacking.

category: biophysics


title: Partial gene suppression improves identification of cancer vulnerabilities when CRISPR-Cas9 knockout is pan-lethal

abstract: Hundreds of genome-wide loss-of-function screens have been performed, as part of efforts such as The Cancer Dependency Map, to create a catalog of genetic dependencies in a diverse set of cancer contexts. In recent years, large-scale screening efforts have shifted perturbation technology from RNAi to CRISPR-Cas9, due to the superior efficacy and specificity of CRISPR-Cas9-mediated approaches. However, questions remain about the extent to which partial suppression of gene targets could result in selective dependency across cell lines, potentially revealing a larger set of targetable cancer vulnerabilities than can be identified using CRISPR knockout alone. Here, we use CRISPR-Cas9 and RNAi screening data for more than 400 shared cell lines to represent knockout and partial suppression genetic perturbation modalities and evaluate the utility of each for therapeutic target discovery and the inference of gene function. We find that CRISPR screens identify more dependencies, and yield more accurate predictive models and co-dependency relationships overall. However, RNAi outperforms CRISPR in identifying associations (omics, drug, co-dependencies) with genes that are common dependencies for most cell lines (pan-dependencies). As pan-dependencies occur frequently in the CRISPR dataset (~2,000 genes), using results from both RNAi and CRISPR analyses facilitates the discovery of predictive models and associated co-dependencies for a wider range of gene targets than could be detected using either dataset alone. These findings can aid in the interpretation of contrasting results obtained from CRISPR and RNAi screens and reinforce the importance of partial gene suppression methods in building a cancer dependency map.

category: cancer biology


title: One-pot synthesis of prenylated proteins utilizing E. coli cell-free expression

abstract: Bottom-up synthetic biology is a powerful tool for uncovering the mechanisms underlying vital biological processes, such as signaling and cell polarization. The core principle of reconstituting cellular functions in their minimal forms can be achieved through modular protein design. However, assembling multiple purified proteins into a functional and synchronized system remains a technical challenge. The fact that many regulatory proteins show direct or indirect membrane interactions further exacerbates the complications. Here, we introduce the Cell-Free prenylated Protein Synthesis (CFpPS) system which enables the production of prenylated proteins in a single reaction mix, through reconstituted prenylation machinery. Not only does the CFpPS system offer a fast and reliable method for producing solubilized prenylated proteins, but it can also produce the protein of interest directly in the vicinity of biomimetic membranes, thus enabling microscopy-based functional assessment. As proof of principle, we demonstrate synthesis and solubilization of various important signaling proteins from the Ras superfamily, as well as membrane binding and extraction of the key polarity regulator Cdc42. Furthermore, our method can be used to confer membrane affinity to any protein, simply by adding a 4-peptide motif to the C-terminus of the protein. In sum, the CFpPS system offers a versatile and effective platform for designing peripheral membrane proteins for synthetic biology applications.

category: synthetic biology


title: Taxonomic and functional shifts in the perinatal gut microbiome of rhesus macaques

abstract: Pregnancy and the postpartum period result in some of the most dramatic metabolic, hormonal, and physiological changes that can be experienced by an otherwise healthy adult. The timing and magnitude of these changes is key for both maternal and fetal health. One of the factors believed to critically modulate these physiological changes is the maternal gut microbiome. However, the dynamic changes in this community during the perinatal period remain understudied. Clinical studies can be complicated by confounding variables such as diet and other drivers of heterogeneity in the human microbiome. Therefore, in this study, we conducted a longitudinal analysis of the fecal microbiome obtained during the perinatal and post-partum periods in 25 rhesus macaques using 16S rRNA gene amplicon sequencing and shotgun metagenomics. Shifts at both the taxonomic and functional potential level were detected when comparing pregnancy to postpartum samples. Taxonomically, Alloprevotella, Actinobacillus, and Anaerovibrio were enriched during pregnancy while Treponema, Lachnospiraceae, and Methanosphaera were more abundant post-partum. Functionally, pregnancy was associated with increased abundance in the pathway to produce the beneficial short chain fatty acid (SCFA), butyrate, while pathways associated with starch degradation and folate transformation were more abundant postpartum. These data demonstrate dramatic changes in the maternal gut even in the absence of dietary changes and suggest that rhesus macaques could provide a valuable model to determine how changes in the microbiome correlate to other physiological changes in pregnancy.


title: Impact of fungal hyphae on growth and dispersal of obligate anaerobic bacteria in aerated habitats

abstract: Anoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes even in well-areated environments. Here, we analyzed whether and to which degree fractal hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches allowing for spore germination, growth and fermentative activity of the obligate anaerobe in an otherwise oxic environment. Hypoxic liquid films containing 80 {+/-} 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Our results suggest that fungal biomass typical in soil (<550 g g-1soil) may create anoxic microniches and enable activity as well as dispersal of obligate anaerobes near hyphae in an otherwise inhabitable environment.


title: Profiling the plasmid conjugation potential of urinary E. coli

abstract: Escherichia coli is often associated with urinary tract infection (UTI). Antibiotic resistance in E. coli is an ongoing challenge in managing UTI. Extrachromosomal elements - plasmids - are vectors for clinically relevant traits, such as antibiotic resistance, with conjugation being one of the main methods for horizontal propagation of plasmids in bacterial populations. Targeting of conjugation components has been proposed as a strategy to curb spread of plasmid-borne antibiotic resistance. Understanding the types of conjugative systems present in urinary E. coli isolates is fundamental to assessing the viability of these strategies. In this study, we profile the two well studied conjugation systems (F-type and P-type) in the draft genomes of 65 urinary isolates of E. coli obtained from bladder urine of adult women with and without UTI-like symptoms. Most of these isolates contained plasmids and we found that conjugation genes are abundant/ubiquitous, diverse, and often associated with IncF plasmids. To validate conjugation of these urinary plasmids, we conjugated the plasmids from two urinary isolates, UMB1223 (predicted to have F-type genes) and UMB1284 (predicted to have P-type genes), into the K-12 E. coli strain MG1655. Overall, the findings of this study support the notion that care should be taken in targeting any individual component of a urinary E. coli isolates conjugation system, given the inherent mechanistic redundancy, gene diversity, and different types of conjugation systems in this population.


title: Transcriptional response of Candida auris to the Mrr1 inducers methylglyoxal and benomyl

abstract:
Candida auris is an urgent threat to human health due to its rapid spread in healthcare settings and its repeated development of multidrug resistance. Diseases that put individuals at a higher risk for C. auris infection, such as diabetes, kidney failure, or immunocompromising conditions, are associated with elevated levels of methylglyoxal (MG), a reactive dicarbonyl compound derived from several metabolic processes. In other Candida species, expression of MG reductase enzymes that catabolize and detoxify MG are controlled by Mrr1, a multidrug resistance-associated transcription factor, and MG induces Mrr1 activity. Here, we used transcriptomics and genetic assays to determine that C. auris MRR1a contributes to MG resistance, and that the main Mrr1a targets are an MG reductase and MDR1, which encodes an drug efflux protein. The C. auris Mrr1a regulon is smaller than Mrr1 regulons described in other species. In addition to MG, benomyl (BEN), a known Mrr1 stimulus, induces C. auris Mrr1 activity, and characterization of the MRR1a-dependent and independent transcriptional responses revealed substantial overlap in genes that were differentially expressed in response to each compound. Additionally, we found that an MRR1 allele specific to one C. auris phylogenetic clade, clade III, encodes a hyperactive Mrr1 variant, and this activity correlated with higher MG resistance. C. auris MRR1a alleles were functional in Candida lusitaniae and were inducible by BEN, but not by MG, suggesting that the two Mrr1 inducers act via different mechanisms. Together, the data presented in this work contribute to the understanding Mrr1 activity and MG resistance in C. auris.

ImportanceCandida auris is a fungal pathogen that has spread since its identification in 2009 and is of concern due to its high incidence of resistance against multiple classes of antifungal drugs. In other Candida species, the transcription factor Mrr1 plays a major role in resistance against azole antifungals and other toxins. More recently, Mrr1 has been recognized to contribute to resistance to methylglyoxal (MG), a toxic metabolic byproduct. Here, we show that C. auris MRR1a, the closest ortholog to MRR1 in other species, contributes to resistance to MG, and that Mrr1a strongly co-regulates expression of MGD1, encoding a methylglyoxal reductase enzyme and MDR1, encoding an efflux protein involved in resistance to azole drugs, antimicrobial peptides and bacterial products. We found that one major clade of C. auris has a constitutively active Mrr1 despite high azole resistance due to other mutations, and that this high Mrr1a activity correlates with higher MG resistance. Finally, we gain insights into the activities of MG and another Mrr1 inducer, benomyl, to better understand C. auris regulation of phenotypes relevant in vivo.


title: De-heterogeneity of the eukaryotic viral reference database (EVRD) improves the accuracy and efficiency of viromic analysis

abstract: Widespread in public databases, the notorious contamination in virus reference databases often leads to confusing even wrong conclusions in applications like viral disease diagnosis and viromic analysis, highlighting the need of a high-quality database. Here, we report the comprehensive scrutiny and the purification of the largest viral sequence collections of GenBank and UniProt by detection and characterization of heterogeneous sequences (HGSs). A total of 766 nucleotide- and 276 amino acid-HGSs were determined with length up to 6,605 bp, which were widely distributed in 39 families, with many involving highly public health-related viruses, such as hepatitis C virus, Crimea-Congo hemorrhagic fever virus and filovirus. Majority of these HGSs are sequences of a wide range of hosts including humans, with the rest resulting from vectors, misclassification and laboratory components. However, these HGSs cannot be simply considered as exotic contaminants, since part of which are resultants of natural occurrence or artificial engineering of the viruses. Nevertheless, they significantly disturb the genomic analysis, and hence were deleted from the database. A further augmentation was implemented with addition of the risk and vaccine sequences, which finally results in a high-quality eukaryotic virus reference database (EVRD). EVRD showed higher accuracy and less time-consuming without coverage compromise by reducing false positives than other integrated databases in viromic analysis. EVRD is freely accessible with favorable application in viral disease diagnosis, taxonomic clustering, viromic analysis and novel virus detection.


title: Live-cell imaging and physical modeling reveal control of chromosome folding dynamics by cohesin and CTCF

abstract: Physical proximity between genomic sequences in mammalian chromosomes controls key biological processes such as transcriptional regulation and DNA repair. Yet it is currently unknown if chromosomal contacts are rare and stable or instead frequent and dynamic, and how they depend on the loop extrusion activity of cohesin or barriers such as CTCF. By imaging chromosomal locations at high spatial and temporal resolution over several hours in living cells, we show that sequences within topological associating domains (TADs) frequently come into physical proximity during the course of a cell cycle and remain close to each other only for a few minutes. Such contacts become nonetheless substantially longer and more frequent in the presence of convergent CTCF sites, resulting in a suppression of variability in chromosome folding in single cells across time. Supported by physical models of chromosome dynamics, our data additionally suggests that individual CTCF-anchored loops last around 10 minutes. The estimates of chromosomal contact dynamics in our study provide a novel quantitative framework to link chromosome structure to function and show that cohesin and CTCF stabilize otherwise highly dynamic chromosome structures to facilitate selected subsets of chromosomal interactions.

category: molecular biology


title: TENT2, TUT4, and TUT7 selectively regulate miRNA sequence and abundance

abstract: TENTs generate miRNA isoforms by 3 tailing. However, little is known about how tailing regulates miRNA function. Here, we generated isogenic HEK293T cell lines in which TENT2, TUT4 and TUT7 were knocked out individually or in combination. Together with rescue experiments, we characterized TENT-specific effects by deep sequencing, Northern blot and in vitro assays. We found that 3 tailing is not random but highly specific. In addition to its known adenylation, TENT2 contributes to guanylation and uridylation on mature miRNAs. TUT4 uridylates most miRNAs whereas TUT7 is dispensable. Removing adenylation has a marginal impact on miRNA levels. By contrast, abolishing uridylation leads to dysregulation of a set of miRNAs. Besides let-7, miR-181b and miR-222 are negatively regulated by TUT4/7 via distinct mechanisms while the miR-888 cluster is upregulated specifically by TUT7. Our results uncover the selective actions of TENTs in generating 3 isomiRs and pave the way to investigate their functions.


title: Stress promotes RNA G-quadruplex folding in human cells

abstract:
Guanine-rich nucleic acids can fold into G-quadruplex (G4) structures. Although endogenous RNAs contain sequences that can fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. We show that the folding of putative rG4s in human cells into bona fide rG4 structures is dynamically regulated by stress. By using a high-throughput approach based on differential reactivity of dimethyl sulfate (DMS) towards Gs within folded vs unfolded rG4s, we identified hundreds of endogenous rG4s whose folding is promoted by cellular stress and validated them using a newly developed rG4-specific ligand. Stress-dependent rG4s are enriched in mRNA 3'-untranslated regions, suggesting their role in regulating mRNA stability under stress. Lastly, rG4 folding is reversible upon stress removal or adaptation. Our study show that rG4s function as regulatory elements in regulating mRNA stability and cellular stress response.

One-Sentence SummaryRNA G-quadruplexes assemble under stress in human cells


title: Endothelial FOXC1 and FOXC2 promote intestinal regeneration after ischemia-reperfusion injury

abstract: Intestinal ischemia induces mucosal damage while simultaneously activating intestinal stem cells (ISCs), which subsequently regenerate the damaged intestinal epithelium. However, whether paracrine factors secreted from vascular endothelial cells (ECs) - blood and lymphatic ECs (BECs and LECs, respectively) - regulate ISC-mediated regeneration have yet to be elucidated. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in regeneration of the small intestine after ischemia-reperfusion (I/R) injury. EC- and LEC-specific deletions of Foxc1, Foxc2, or both in mice augment I/R-induced intestinal damage by causing defects in vascular regrowth, expression of the chemokine CXCL12 and the Wnt activator R- spondin 3 in BECs and LECs, respectively, and activation of Wnt signaling in ISCs. Treatment with CXCL12 and R-spondin 3 rescues the I/R-induced intestinal damage in EC- and LEC-Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling.


title: Rapid single-molecule characterisation of nucleic-acid enzymes

abstract: The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lacking statistical power. We present a highly generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different nucleic-acid enzyme activities: digestion by the phage{lambda} exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe a previously unknown activity of the UvrD helicase to remove proteins tightly bound to the ends of DNA.


title: A domesticated fungal cultivar recycles its cytoplasmic contents as nutritional rewards for its leafcutter ant farmers

abstract: Leafcutter ants farm a fungal cultivar (Leucoagaricus gongylophorus) that converts inedible vegetation into food that sustains colonies with millions of workers. Like fruits of crops domesticated by humans, L. gongylophorus has evolved specialized nutritional rewards--tiny swollen hyphal cells called gongylidia that package metabolites eaten by ant farmers. Yet, little is known about how gongylidia form, and whether ants regulate this formation through plant-fragment provisioning. We used microscopy and in vitro manipulations to explain the cellular mechanisms governing gongylidium formation. First, L. gongylophorus is polykaryotic (up to 17 haploid nuclei/cell) and our results suggest intracellular nucleus distributions govern gongylidium morphology with their absence in expanding edges arresting apical growth and their presence mediating complex branching patterns. Second, nanoscale TEM imaging shows that the cultivar recycles its own cellular material (e.g. cytosol, mitochondria) through a process called  autophagy and stores the resulting metabolites in gongylidia. This autophagic pathway is further supported by gongylidium inhibition when isolated fungal cultures are grown on media with autophagy inhibitors (chloroquine, 3-methyladenine). We hypothesize that autophagic nutritional reward production is the ultimate cultivar service and reflects a higher-level organismality adaptation enabled by strict symmetric lifetime commitment between ant farmers and their fungal crop.


title: A chromosome-scale hybrid genome assembly of the extinct Tasmanian tiger (Thylacinus cynocephalus)

abstract:
The extinct Tasmanian tiger or thylacine (Thylacinus cynocephalus) was a large marsupial carnivore native to Australia. Once ranging across parts of the mainland, the species remained only on the island of Tasmania by the time of European colonization. It was driven to extinction in the early 20th century and is an emblem of native species loss in Australia. The thylacine was a striking example of convergent evolution with placental canids, with which it shared a similar skull morphology. Consequently, it has been the subject of extensive study. While the original thylacine assemblies published in 2018 enabled the first exploration of the species genome biology, further progress is hindered by the lack of high-quality genomic resources. Here, we present a new chromosome-scale hybrid genome assembly for the thylacine, which compares favorably with many recent de novo marsupial genomes. Additionally, we provide homology-based gene annotations, characterize the repeat content of the thylacine genome and show that, consistent with demographic decline, the species possessed a low rate of heterozygosity even compared to extant, threatened marsupials.

SignificanceThe lack of high-quality genomes for extinct species inhibits research into their biology. Moreover, marsupials are underrepresented among sequenced genomes. Here, we present a new, chromosome-scale thylacine genome. This high-quality assembly is a valuable new resource for studies on marsupial carnivores.

category: genomics


title: Modulation of the Biophysical and Biochemical Properties of Collagen by Glycation for Tissue Engineering Applications

abstract: The structural and functional properties of collagen are modulated by the presence of intramolecular and intermolecular crosslinks. Advanced Glycation End-products (AGEs) can produce intermolecular crosslinks by bonding the free amino groups of neighboring proteins. In this research, the following hypothesis is explored: The accumulation of AGEs in collagen decreases its proteolytic degradation rates while increasing its stiffness. Fluorescence Lifetime Imaging (FLIM) and Fourier-transform infrared spectroscopy (FTIR) detect biochemical changes in collagen scaffolds during the glycation process. The accumulation of AGEs increases exponentially in the collagen scaffolds as a function of Methylglyoxal (MGO) concentration by performing autofluorescence measurement and competitive ELISA. Glycated scaffolds absorb water at a much higher rate confirming the direct affinity between AGEs and interstitial water within collagen fibrils. In addition, the topology of collagen fibrils as observed by Atomic Force Microscopy (AFM) is a lot more defined following glycation. The elastic modulus of collagen fibrils decreases as a function of glycation, whereas the elastic modulus of collagen scaffolds increases. Finally, the enzymatic degradation of collagen by bacterial collagenase shows a sigmoidal pattern with a much slower degradation rate in the glycated scaffolds. This study identifies unique variations in the properties of collagen following accumulation of AGEs.

category: bioengineering


title: Intrinsic Protein Disorder, Conditional Folding and AlphaFold2

abstract: Intrinsically disordered regions (IDRs) defying the traditional protein structure-function paradigm have been difficult to analyze. AlphaFolds recent breakthrough in predicting protein structures accurately offers a fresh perspective on IDR prediction as assessed on the CAID dataset. Surprisingly, AlphaFold is highly competitive for predicting both IDRs and conditionally folded regions, demonstrating the plasticity of the disorder to structure continuum.

category: bioinformatics


title: Collagen-VI expression is negatively mechanosensitive in pancreatic cancer cells and supports the metastatic niche

abstract:
Pancreatic cancer is a deadly disease with high rates of metastasis, though how tumor cells establish metastatic lesions is not fully understood. A key feature of primary pancreatic tumors is extensive fibrosis due to deposition of extracellular matrix. While pancreatic cancer cells are programmed by stimuli derived from a stiff ECM, metastasis requires loss of attachment as well as adaptation to a softer microenvironment upon reaching distant sites. Growing evidence suggests that stiff ECM influences pancreatic cancer cell behaviour. Here we argue that this influence is reversible and that pancreatic cancer cells can be reprogrammed upon sensing of soft substrates. Through use of engineered polyacrylamide hydrogels with tuneable mechanical properties, we show that Collagen-VI is specifically upregulated on soft substrates, due to a lack of integrin engagement and low YAP1 activity. Collagen-VI supports migration in vitro and metastasis formation in vivo. Metastatic nodules formed by pancreatic cancer cells lacking Col6a1 expression, were characterised by stromal cell-derived collagen-VI deposition, suggesting that collagen-VI, either cancer or stroma derived, is an essential component of the metastatic niche.

Summary StatementCollagen-VI is expressed by pancreatic tumors and metastases in a mechanosensitive way to promote niche colonisation.


title: PCYT2 controls muscle health and muscle aging

abstract: Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing, affecting hundreds of millions of people. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases represents an important goal in improving human health. Here, we show that phosphatidylethanolamine cytidyltransferase (PCYT2/ECT), the critical enzyme of the Kennedy branch of phosphatidylethanolamine (PE) synthesis pathway, has an essential role in muscle health and lifespan. Human genetic deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. Pcyt2 mutant zebrafish recapitulate patient phenotypes, indicating that the role of PCYT2/PE in muscle is evolutionary conserved. Muscle specific Pcyt2 knockout mice exhibited failure to thrive, impaired muscle development, progressive muscle weakness, muscle loss and accelerated ageing. Interestingly, from several organs tested, this pathology is muscle specific. Mechanistically, in muscle deficiency of PCYT2 triggers dramatic alterations of physicochemical properties of the myofiber membrane lipid bilayer, compromising membrane stability and durability under strain. We also show that PCYT2 activity declines in the aging muscles of humans and mice, and Pcyt2 gene-therapy in aged mice improved muscle strength. AAV-based delivery of PCYT2 also rescued muscle weakness in Pcyt2 knock-out mice, offering a feasible novel therapeutic avenue for rare disease patients and to alleviate muscle aging. Thus, PCYT2 plays a fundamental, specific, and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2 synthesized PE lipids to severe muscle dystrophy, exercise intolerance and aging.

category: genetics


title: Systematic hyper-variation and evolution at a lipopolysaccharide locus in the population of Xanthomonas species that infect rice and sugarcane

abstract: Advent of high throughput sequencing and population genomics is enabling researchers to investigate selection pressure at hyper-variable genomic loci encoding pathogen-associated molecular patter (PAMP) molecules like lipopolysaccharide (LPS) in an unprecedented manner. Xanthomonas is a model group of phytopathogenic bacteria that infects host in tissue-specific manner. Our in-depth investigation revealed that the successful emergence of lineages infecting major cereals and grasses like rice, sugarcane, and wheat was mediated by acquisition and later replacement of an ancestral type (BXO8) of LPS cassette by distinct one. In the population of the rice xylem pathogen, X. oryzae pv. oryzae (Xoo), the BXO8 is replaced by a distinct BXO1 type of cassette. Alternatively, in diverse Xanthomonas species that infect sugarcane, the BXO8 ancestral cassette has been replaced by yet another kind of Xvv type of LPS cassette, suggesting convergent evolution at an LPS locus mediated by horizontal gene transfer (HGT) events. Aside from xylem, two closely related lineages of X. oryzae that infect parenchyma tissue of rice and Leersia hexandra grass have acquired an LPS cassette from Xanthomonas pathogens that infect citrus, walnut, and strawberry parenchyma, indicating yet another instance of parallel evolution facilitated by HGT. Our targeted and mega-population-based genome dynamic studies revealed potential role of acquisition of specific types of LPS cassettes in the emergence and evolution of tissue specificity in Xanthomonas. Additional cellular, molecular, genetic, and plant studies will help us figure out how a distinct type of LPS help Xanthomonas pathovars and lineages adapt to parenchyma and xylem tissues.


title: The mitochondrial genome of Oryza sativa L. cv. Taichung 65 (Poaceae)

abstract: A complete mitochondrial genome sequence was determined by a hybrid approach with long- and short-read sequences in a japonica rice cultivar Taichung 65. The assembled mitochondrial genome was 401,060 bases in length and an overall GC content of 43.88% and predicted to harbor 57 protein-encoding, 18 transfer RNA, and 3 ribosomal RNA genes. Phylogenetic and sequence polymorphism analysis indicated that Taichung 65 is classified as typical japonica. The mitochondrial genome resource would serve as a fundamental tool to investigate cytoplasmic male sterility in rice.


title: Evaluation of N6-adenine DNA-immunoprecipitation-based genomic profiling in eukaryotes

abstract: The detection of low-abundance DNA N6-methyladenine (DNA-m6A) remains challenging, limiting our understanding of this novel base in eukaryotes. To address this, we introduce an approach for systematically validating the selectivity and sensitivity of antibody-based DNA-m6A methods, revealing most commercial antibodies as poorly selective towards DNA-m6A. Finally, using a validated highly selective anti-DNA-m6A antibody we expose distinct patterns of DNA-m6A in C. reinhardtii, A. thaliana, and D. melanogaster.


title: Organoid-based human stomach micro-physiological system to recapitulate the dynamic mucosal defense mechanism

abstract: Several stomach diseases are attributed to the dysregulation of physiological function of gastric mucosal barrier by pathogens. Gastric organoids are a promising tool to develop treatment strategies for gastric infections. However, their functional features of in vivo gastric mucosal barrier and host-microbe interactions are limited due to the lack of physiological stimuli. Herein, we describe the first human stomach micro-physiological system (hsMPS) with physiologically relevant gastric mucosal defense system based on the combination of organoid and MPS technology. A fluid flow enhanced epithelial-mesenchymal interaction in the hsMPS enables functional maturation of gastric epithelial cells, which allows for the recreation of mesh-like mucus layer containing high level of mucus protective peptides and well-developed epithelial junctional complexes. Furthermore, gastroprotection mechanisms against Helicobacter pylori are successfully demonstrated in our system. Therefore, hsMPS represents a new in vitro tool for research where gastric mucosal defense mechanism is pivotal for developing therapeutic strategies.


title: GTEM cell design for controlled exposure to electromagnetic fields in biological system

abstract: This study describes a structure design of the Gigaherz Transverse Electromagnetic (GTEM) cell for electromagnetic tests in biological systems. Using electromagnetic simulation software we show that the optimization of final structure assures the criteria imposed on a low level of reflection towards the radio frequency generator, and a fairly uniform field in the test zone. A sensitivity study was also conducted when the cell was loaded with a few samples under test. We report that the level of reflection remains adequate as long as the test volume remains limited in relation to the size of the cell. The first estimation results on the SAR values are consistent with the distribution of the fields in the cell. A cell was fabricated with the design data, and tested for the reflection level. The measurements confirm the results of simulations.


title: Using machine learning to understand microgeographic determinants of the Zika vector, Aedes aegypti

abstract: There are limited data on why the 2016 Zika outbreak in Miami-Dade County, Florida was confined to certain neighborhoods. In this research, Aedes aegypti, the primary vector of Zika virus, are studied to examine neighborhood-level differences in their population dynamics and underlying processes. Weekly mosquito data were acquired from the Miami-Dade County Mosquito Control Division from 2016 to 2020 from 172 traps deployed around Miami-Dade County. Using Random Forest, a machine learning method, predictive models of spatiotemporal dynamics of Ae. aegypti in response to meteorological conditions and neighborhood-specific socio-demographic and physical characteristics, such as land-use and land-cover type and income level, were created. The study area was divided into two groups: areas affected by local transmission of Zika during the 2016 outbreak and unaffected areas. Aedes aegypti populations in areas affected by Zika were more strongly influenced by 14- and 21-day lagged weather conditions. In the unaffected areas, mosquito populations were more strongly influenced by land-use and day-of-collection weather conditions. There are neighborhood-scale differences in Ae. aegypti population dynamics. These differences in turn influence vector-borne disease diffusion in a region. These results have implications for vector control experts to lead neighborhood-specific vector control strategies and for epidemiologists to guide vector-borne disease risk preparations, especially for containing the spread of vector-borne disease in response to ongoing climate change.

category: ecology


title: Development of a mouse salivary gland-derived mesenchymal cell line for immunological studies of murine cytomegalovirus

abstract: The salivary glands are a crucial site of replication for human cytomegalovirus (HCMV) and its murine counterpart, murine cytomegalovirus (MCMV). Studies of MCMV often involve the use of BALB/c strain mice, but most in vitro assays are carried out in the NIH 3T3 cell line, which is derived from Swiss Albino mice. This report describes a BALB/c-derived mouse salivary gland cell line immortalized using the SV40 large T antigen. Cells stained positive for PDGFR1 and negative for E-cadherin and PECAM-1, indicating mesenchymal origin. This cell line, which has been named murine salivary gland mesenchymal (mSGM), shows promise as a tool for ex vivo immunological assays due to its MHC haplotype match with the BALB/c mouse strain. In addition, plaque assays using mSGM rather than NIH 3T3 cells are significantly more sensitive for detecting low concentrations of MCMV particles. Finally, it is demonstrated that mSGM cells express all 3 BALB/c MHC class I isotypes and are susceptible to T cell-mediated ex vivo cytotoxicity assays, leading to many possible uses in immunological studies of MCMV.


title: A promoter-proximal silencer modifies the activity of a shared enhancer to mediate divergent expression of nub and pdm2 paralogs in wing development

abstract: Duplication of genes and their associated cis-regulatory elements, or enhancers, is a key contributor to genome evolution and biological complexity. Moreover, many paralogs, particularly tandem duplicates, are fixed for long periods of time under the control of shared enhancers. However, in most cases the mechanism by which gene expression and function diverge following duplication is not known. Here we dissect the regulation and function of the paralogous nubbin/pdm2 genes during wing development in Drosophila melanogaster. We show that these paralogs play a redundant role in the wing and that their expression relies on a single shared wing enhancer. However, the two genes differ in their ability to respond to this enhancer, with nub responding in all wing progenitor cells and pdm2 only in a small subset. This divergence is a result of a pdm2-specific silencer element at the pdm2 promoter that receives repressive input from the transcription factor Rotund. Repression through this silencer also depends on nub, allowing pdm2 to fully respond to the wing enhancer when nub expression is perturbed and functional compensation to occur. Thus, expression divergence downstream of a shared enhancer arises as a consequence of silencing the promoter of one paralog.

category: developmental biology


title: Single-cell Spatial Proteomic Imaging for Human Neuropathology

abstract: Neurodegenerative disorders are characterized by phenotypic changes and hallmark proteopathies. Quantifying these in archival human brain tissues remains indispensable for validating animal models and understanding disease mechanisms. We present a framework for nanometer-scale, spatial proteomics with multiplex ion beam imaging (MIBI) for capturing neuropathological features. MIBI facilitated simultaneous, quantitative imaging of 36 proteins on archival human hippocampus from individuals spanning cognitively normal to dementia. Customized analysis strategies identified cell types and proteopathies in the hippocampus across stages of Alzheimers disease (AD) neuropathologic change. We show microglia-pathologic tau interactions in hippocampal CA1 subfield, in AD dementia. Data driven, sample independent creation of spatial proteomic regions identified persistent neurons in pathologic tau neighborhoods expressing mitochondrial protein MFN2, regardless of cognitive status, suggesting a survival advantage. Our study revealed unique insights from multiplexed imaging and data-driven approaches for neuropathologic analysis and serves as a baseline for mechanistic and interventional understanding in human neurodegeneration.

category: systems biology


title: Concurrent mapping of brain ontogeny and phylogeny within a common connectivity space

abstract: Developmental and evolutionary effects on brain organisation are complex, yet linked, as evidenced by the striking correspondence in cortical expansion changes. However, it is still not possible to study concurrently the ontogeny and phylogeny of cortical areal connections, which is arguably more relevant to brain function than allometric changes. Here, we propose a novel framework that allows the integration of connectivity maps from humans (adults and neonates) and non-human primates (macaques) onto a common space. We use white matter bundles to anchor the definition of the common space and employ the uniqueness of the areal connection patterns to these bundles to probe areal specialisation. This enables us to quantitatively study divergences and similarities in cortical connectivity over both evolutionary and developmental scales. It further allows us to map brain maturation trajectories, including the effect of premature birth, and to translate cortical atlases between diverse brains.

category: neuroscience


title: GABAergic modulation of conflict adaptation and response inhibition

abstract:
Adaptive behavior is only possible by stopping stereotypical actions to generate new plans according to internal goals. It is response inhibition --the ability to stop actions automatically triggered by exogenous cues-- that allows for the flexible interplay between bottom-up, stimulus driven behaviors, and top-down strategies. In addition to response inhibition, cognitive control draws on conflict adaptation, the facilitation of top-down actions following high conflict situations. It is currently unclear whether and how response inhibition and conflict adaptation depend on GABAergic signaling, the main inhibitory neurotransmitter in the human brain. Here, we applied a recently developed computational model (SERIA) to data from two studies (N=150 & 50) of healthy volunteers performing Simon and antisaccade tasks. One of these datasets was acquired under placebo-controlled pharmacological enhancement of GABAergic transmission (lorazepam, an allosteric modulator of the GABA-A receptor). Our model-based results suggest that enhanced GABA-A signaling boosts conflict adaptation but impairs response inhibition. More generally, our computational approach establishes a unified account of response inhibition and conflict adaptation in the Simon and antisaccade tasks and provides a novel tool for quantifying specific aspects of cognitive control and their modulation by pharmacology or disease.

Author SummaryOur capacity to prepare for situations that afford conflicting responses (conflict adaptation) and to stop our immediate impulses in these scenarios (response inhibition) are the hallmark of cognitive control. As these abilities require both the stopping or slowing of response tendencies, a natural question is whether they are mediated by inhibitory neurotransmission in the brain. Here, we combined computational modeling with two experiments to investigate how conflict adaptation and response inhibition interact with each other (experiment 1) and how these are modulated by lorazepam (experiment 2), a positive modulator of the GABA-A receptor, one of the main inhibitory receptors in the human brain. Using our computational model to disentangle conflict adaptation and response inhibition, our results indicate that while lorazepam impaired response inhibition, it improved conflict adaptation. Thus, our results suggests that conflict adaptation is mediated by GABA-A neurotransmission.


title: What are oxytocin assays measuring? Epitope mapping, metabolites, and comparisons of wildtype & knockout mouse urine

abstract: Oxytocin has become a popular analyte in behavioral endocrinology in recent years, due in part to its roles in social behavior, stress physiology, and cognition. Urine samples have the advantage of being non-invasive and minimally disruptive to collect, allowing for oxytocin measurements even in some wild populations. However, methods for urinary oxytocin immunoassay have not been sufficiently optimized and rigorously assessed for their potential limitations. Using samples from oxytocin knockout (KO) and wildtype (WT) mice, we find evidence of considerable interference in unextracted urine samples, with similar distributions of measured oxytocin in both genotypes. Importantly, although this interference can be reduced by a reversed-phase solid-phase extraction (SPE), this common approach is not sufficient for eliminating false-positive signal on three immunoassay kits. To better understand the source of the observed interference, we conducted epitope mapping of the Arbor Assays antibody and assessed its cross-reactivity with known, biologically active fragments of oxytocin. We found considerable cross-reactivity (0.5-52% by-molarity) for three fragments of oxytocin that share the core epitope, with more cross-reactivity for longer fragments. Given the presence of some cross-reactivity for even the tripeptide MIF-1, it is likely that many small protein metabolites might be sufficiently similar to the epitope that at high concentrations they interfere with immunoassays. We present a new mixed-mode cation-exchange SPE method that minimizes interference--with knockout samples measuring below the assays limit of detection--while effectively retaining oxytocin from the urine of wildtype mice. This method demonstrates good parallelism and spike recovery across multiple species (mice, dogs, sifakas, humans). Our results suggest that immunoassays of urine samples may be particularly susceptible to interference, even when using common extraction protocols, but that this interference can be successfully managed using a novel mixed-mode cation exchange extraction.


title: Feedback Inhibition Underlies New Computational Functions of Cerebellar Interneurons

abstract: The function of a feedback inhibitory circuit between cerebellar Purkinje cells and molecular layer interneurons (MLIs) was defined by combining optogenetics, neuronal activity recordings both in cerebellar slices and in vivo, and computational modeling. Purkinje cells inhibit a subset of MLIs in the inner third of the molecular layer. This inhibition is non-reciprocal, short-range (less than 200 m) and is based on convergence of 1-2 Purkinje cells onto MLIs. During learning-related eyelid movements in vivo, the activity of a subset of MLIs progressively increases at the same time that Purkinje cell activity decreases, with Purkinje cells usually leading the MLIs. Computer simulations indicate that these relationships are best explained by the feedback circuit from Purkinje cells to MLIs and that this feedback circuit plays a central role in making cerebellar learning efficient.


title: Inferring cell-specific gene regulatory networks from single cell gene expression data

abstract:
Single cell gene expression datasets have been used to uncover differences between single cells, leading to discoveries of new cell types and cell identities, which are usually defined by the transcriptome profiles of cells. Biological networks, in particular, gene regulatory networks (GRNs), can be viewed as another feature of the cells, that contributes to the uniqueness of each single cell. However, methods that reconstruct cell-specific GRNs are still missing.

We propose CeSpGRN (Cell Specific GRN), which infers cell-specific GRNs from single cell gene expression data. CeSpGRN uses a Gaussian weighted kernel which allows the GRN of a given cell to be learned from the gene expression profile of this cell and cells that are upstream and downstream of this cell in the developmental process. CeSpGRN can be applied to infer cell-specific GRNs in cell populations of any trajectory or cluster structure, and it does not require time information of cells as additional input. We compared the performance of CeSpGRN and baseline methods on simulated datasets obtained under various settings. CeSpGRN showed superior performance in reconstructing the GRN for each cell, as well as in detecting the regulatory interactions that differ between cells. We also applied CeSpGRN to real datasets including THP-1 human myeloid monocytic leukemia cells and mouse embryonic stem cells, where CeSpGRN suggested a set of interactions between genes that rewire during the differentiation process in these cells. CeSpGRN is available at https://github.com/PeterZZQ/CeSpGRN.


title: Determining the optimal SARS-CoV-2 mRNA vaccine dosing interval for maximum immunogenicity

abstract:
ObjectiveEmerging evidence indicates that longer SARS-CoV-2 vaccine dosing intervals results in an enhanced immune response. However, the optimal vaccine dosing interval for achieving maximum immunogenicity is unclear.

MethodsThis study included samples from adult paramedics in Canada who received two doses of either BNT162b2 or mRNA-1273 vaccines and provided blood samples 6 months (170 to 190 days) after the first vaccine dose. The main exposure variable was vaccine dosing interval (days), categorized as "short" (first quartile), "moderate" (second quartile), "long" (third quartile), and "longest" interval (fourth quartile). The primary outcome was total spike antibody concentrations, measured using the Elecsys SARS-CoV-2 total antibody assay. Secondary outcomes included: spike and RBD IgG antibody concentrations, and inhibition of angiotensin-converting enzyme 2 (ACE-2) binding to wild-type spike protein and several different Delta variant spike proteins. We fit a multiple log-linear regression model to investigate the association between vaccine dosing intervals and the antibody concentrations.

ResultsA total of 564 adult paramedics (mean age 40 years, SD=10) were included. Compared to "short interval" ([≤]30 days), higher dosing interval quartiles (moderate: 31-38 days; long: 39-73 days and longest: [≥]74 days) were all associated with increased Elescys spike total antibody concentration. Compared to the short interval, "long" and "longest" interval quartiles were associated with higher spike and RBD IgG antibody concentrations. Similarly, increasing dosing intervals increased inhibition of ACE-2 binding to viral spike protein, regardless of the vaccine type.

ConclusionIncreased mRNA vaccine dosing intervals longer than 30 days result in higher levels of circulating antibodies and viral neutralization when assessed at 6 months.


title: SlS5H silencing reveals specific pathogen-triggered salicylic acid metabolism in tomato

abstract:
The phytohormone salicylic acid (SA or 2-hydroxybenzoic acid) plays an important role in plant biotic and abiotic responses. Gentisic acid (GA or 2,5-Dihydroxybenzoic acid, 2,5-DHBA) is the product of the SA 5-hydroxylation which is catalysed by the S5H enzyme, also known as DMR6. GA has been described to accumulate at high levels in compatible plant-pathogen interactions such as tomato plants infected by Citrus Exocortis Viroid (CEVd), and to a much lesser extend upon Pseudomonas syringae DC3000 pv. tomato (Pst) infection. Here we describe the specific effect that tomato SlS5H impairment produces on both plant-pathogen interactions. The induction of SlS5H in tomato plants by different pathogens was corroborated by qRT-PCR and correlated with previously described 2,5-DHBA accumulations. Transient SlS5H over-expression assays in Nicotiana benthamiana confirmed that SA is a substrate for SlS5H in vivo. RNAi_SlS5H tomato transgenic plants were generated and characterized upon CEVd and Pst infections. Transgenic tomato plants displayed an activation of defences and therefore a loss of susceptibility against both pathogens, and alternative SA homeostasis seems to occur for each specific interaction. Metabolomic assays revealed that whilst the glycosylated form of SA was the most discriminant metabolite found in CEVd infected RNAi_SlS5H transgenic plants, trans-feruloyldopamine, feruloylquinic acid, feruloylgalactarate and 2-hydroxyglutarate were the most accumulated compounds in the Pst-infected transgenic tomato leaves. Transgenic lines also displayed hyper susceptibility to Botrytis cinerea, as well as a smaller size and early senescence. Collectively, our results reveal a novel mechanism by which tomato plants specifically set SA homeostasis upon different pathogen attacks.

One sentence summaryThe impairment of SA hydroxylation in tomato plants uncovers specific SA homeostasis upon CEVd or Pseudomonas syringae infections.


title: A gut-derived hormone switches dietary preference after mating in Drosophila

abstract: Animals must adapt their dietary choices to meet their nutritional needs. How these needs are detected and translated into nutrient-specific appetites that drive food-choice behaviors is poorly defined. Here, we show that the enteroendocrine cells (EECs) of the adult female Drosophila midgut sense nutrients and in response release neuropeptide F (NPF), an ortholog of mammalian NPY-family gut-brain hormones. Gut-derived NPF acts via effects on glucagon-like adipokinetic hormone (AKH) signaling to induce sugar satiety and to drive hunger for protein-rich food, and on adipose tissue to promote storage of ingested nutrients. Suppression of gut NPF leads to overconsumption of dietary sugar while decreasing intake of protein-rich yeast. Furthermore, we show a female-specific function of gut-derived NPF in the suppression of AKH signaling after mating. This induces a dietary switch that promotes preference for protein-containing food to support reproduction. Together, our findings suggest that the gut NPF-AKH axis regulates appetite that drives specific food choices to ensure homeostatic consumption of nutrients, providing insight into the hormonal mechanisms that underlie nutrient-specific hungers.

category: animal behavior and cognition


title: Bumblebees navigate using path integration while walking

abstract: Path integration is a computational strategy that allows an animal to maintain an internal estimate of its position relative to a point of origin. Many species use path integration to navigate back to specific locations, typically their homes, after lengthy and convoluted excursions. Hymenopteran insects are impressive path integrators, directly returning to their hives after hundreds of meters of outward travel. Recent neurobiological insights have established hypotheses for how path integration may be mediated by the brains of bees, but clear ways to test these hypotheses in the laboratory are currently unavailable. Here we report that the bumblebee, Bombus terrestris, uses path integration while walking over short distances in an indoor arena. They estimate accurate vector distances after displacement and orient by artificial celestial cues. Walking bumblebees also exhibited systematic search patterns when home vectors failed to lead them accurately back to the nest, closely resembling searches performed by other species in natural conditions. We thus provide a robust experimental system to test navigation behavior in the laboratory that reflects most aspects of natural path integration. Importantly, we established this assay in an animal that is both readily available and resilient to invasive manipulations. In the future, our behavioral assay therefore can be combined with current electrophysiological techniques, opening a path towards directly probing the neural basis of the sophisticated vector navigation abilities of bees.


title: Unruly octopuses are the rule: Octopus vulgaris use multiple and individually variable strategies in an episodic-like memory task

abstract:
The evolution of complex cognition can be explained by different hypotheses, mutually non-exclusive: the social intelligence hypothesis, the ecological intelligence hypothesis and the predator-prey interaction hypothesis. Episodic-like memory can be used as a proxy to study complex cognition. This ability has mainly been studied through experimental tasks where subjects have to remember what they ate, where and when or in which context. Seemingly quite common in mammals and corvids, episodic-like memory abilities have been observed in only one invertebrate species: the common cuttlefish, a cephalopod mollusc. To explore if this ability is common to all cephalopods or if it has emerged to face specific constraints, we conducted an episodic-like memory task with seven Octopus vulgaris. Only one individual learnt the replenishing rates during the training and subsequently showed episodic-like memory abilities, whereas the other individuals favoured simpler foraging strategies, such as avoidance of familiarity and alternation, use of win-stay strategy and risk-sensitivity. A high variability in the use of these strategies was observed between and within individuals throughout the training. Since octopuses seem to live under lighter environmental pressure than cuttlefish, they may not need to rely on episodic-like memory abilities to optimize foraging as cuttlefish do. These results highlight the differences in the use of complex cognitive abilities between cuttlefish and octopuses, which might be linked with different environmental, predatory and social constraints.

Summary statementWhen trained in an episodic-like memory task, common octopuses favour individually variable foraging strategies rather than keeping track of time to solve the task as cuttlefish do.


title: Temperature Stressed Caenorhabditis elegans Males Prioritize Feeding over Mating Resulting in Sterility

abstract: Heat stress negatively impacts fertility in sexually reproducing organisms at sublethal temperatures. These temperature stress effects are typically more pronounced in males. In some species, sperm production, quality, and motility are the primary cause of male infertility under temperature stress. However, this is not the case in the model nematode Caenorhabditis elegans where changes in mating behavior are the primary cause of fertility loss. We report that temperature stressed C. elegans males experience a dramatic upset in the balance of their food drive and their mating drive such that they prioritize feeding over mating. This change in priorities is due partially to increased expression of the chemoreceptor odr-10 in the AWA sensory neurons. Increased odr-10 expression in the presence of ample food demonstrates that males are unable to experience satiety, thus they never leave a food source and engage in mate searching behavior. These results demonstrate that moderate temperature stress may have profound and previously underappreciated effects on reproductive behaviors. As climate change associated temperature variability becomes more commonplace, it will be imperative to understand how temperature stress affects conserved behavioral elements critical to reproduction.


title: Sexual deprivation induces a CRF independent stress response and decreases resistance to stressors in Drosophila via a subpopulation of Neuropeptide F receptor-expressing neurons

abstract: Living in a changing environment composed of other behaving animals entails both opportunities and challenges to obtain resources and mating partners. Actions that promote survival and reproduction are reinforced by the brain reward systems, whereas coping with the challenges associated with obtaining these rewards are mediated by stress response pathways. The activation of the latter can impair health and shorten lifespan. Although similar responses to social opportunity and challenge exist across the animal kingdom, little is known about the mechanisms that process reward and stress under different social conditions. Here, we studied the interplay between deprivation of sexual reward and stress response in Drosophila melanogaster and discovered that repeated failures to obtain sexual reward induces a frustration-like state that is characterized by increased arousal, persistent sexual motivation, and impaired ability to cope with starvation and oxidative stressors. We show that this increased arousal and sensitivity to starvation is mediated by disinhibition of neurons that express receptors for the fly homologue of neuropeptide Y (neuropeptide F, NPF). We furthermore demonstrate the existence of an anatomical overlap between stress and reward systems in the fly brain in the form of neurons that co-express receptors for NPF (NPFR) and the corticotropin-releasing factor (CRF)-like homologue Diuretic hormone 44 (Dh44), and that deprivation of sexual reward leads to translocation of forkhead box-subgroup O (FoxO) to the cytoplasm in these neurons. Nevertheless, the activity of Dh44 neurons alone does not mediate sensitivity to starvation and aroused behavior following sexual deprivation, instead, these responses are mediated by disinhibition of ~12-16 NPFR-expressing neurons via a dynamin-independent synaptic signaling mechanism, suggesting the existence of a NPFR mediated stress pathway which is Dh44-independent. This paves the path for using simple model organisms to dissect mechanisms behind anticipation of reward, and more specifically, to determine what happens when expectations to obtain natural and drug rewards are not met.


title: EMC10 reduction in human neurons and adult mouse brain rescues cellular and behavioral deficits linked to 22q11.2 deletion

abstract: Up-regulation of Mirta22/Emc10 is a major transcriptional effect of the 22q11.2-associated microRNA dysregulation and underlies key cellular as well as behavioral deficits. EMC10 is a component of the ER membrane complex, which promotes membrane insertion of a subset of polytopic and tail-anchored membrane proteins. Here we show that EMC10 expression is elevated in hiPSC-derived neurons from 22q11.2 deletion carriers and that reduction of EMC10 levels restores defects in neurite outgrowth and calcium signaling. Moreover, using injection of Antisense Oligonucleotides, we demonstrate that normalization of Emc10 levels in adult mouse brain rescues social memory deficits. The observations that elevated EMC10 expression is deleterious in 22q11.2 deletion carriers and that sustained elevation of EMC10 throughout the adult life can interfere with neural processes point to manipulations of EMC10 levels and downstream targets as a specific venue to ameliorate or even prevent disease progression in 22q11.2 deletion syndrome.


title: Global ecological and biogeochemical impacts of pelagic tunicates

abstract: The pelagic tunicates, gelatinous zooplankton that include salps, doliolids, and appendicularians, are filter feeding grazers thought to produce a significant amount of particulate organic carbon (POC) detritus. However, traditional sampling methods (i.e., nets), have historically underestimated their abundance, yielding an overall underappreciation of their global biomass and contribution to ocean biogeochemical cycles relative to crustacean zooplankton. As climate change is projected to decrease the average plankton size and POC export from traditional plankton food webs, the ecological and biogeochemical role of pelagic tunicates may increase; yet, pelagic tunicates were not resolved in the previous generation of global earth system climate projections. Here we present a global ocean study using a coupled physical-biogeochemical model to assess the impact of pelagic tunicates in the pelagic food web and biogeochemical cycling. We added two tunicate groups, a large salp/doliolid and a small appendicularian to the NOAA-GFDL Carbon, Ocean Biogeochemistry, and Lower Trophics version 2 (COBALTv2) model, which was originally formulated to represent carbon flows to crustacean zooplankton. The new GZ-COBALT simulation was able to simultaneously satisfy new pelagic tunicate biomass constraints and existing ecosystem constraints, including crustacean zooplankton observations. The model simulated a global tunicate biomass of 0.10 Pg C, annual production of 0.49 Pg C y-1 in the top 100 m, and export flux of 0.7 Pg C y-1, representing 11% of the total export flux past 100 m. Overall export from the euphotic zone remained largely constant, with the GZ-COBALT pe-ratio only increasing 5.3% (from 0.112 to 0.118) compared to the COBALTv2 control. While the bulk of the tunicate-mediated export production resulted from the rerouting of phytoplankton- and mesozooplankton-mediated export, tunicates also shifted the overall balance of the upper oceans away from recycling and towards export. Our results suggest that pelagic tunicates play important trophic roles in both directly competing with microzooplankton and indirectly shunting carbon export away from the microbial loop.


title: Sequence conservation need not imply purifying selection: evidence from mammalian stop codon usage

abstract: The assumption that conservation of sequence implies the action of purifying selection is central to diverse methodologies to infer functional importance. In mammals, however, GC-biased gene conversion (gBGC), a meiotic mismatch repair bias strongly favouring GC over AT, can in principle mimic the action of selection. As mutation is GC[->]AT biased, to demonstrate that gBGC does indeed cause false signals requires confidence that an AT-rich residue is selectively optimal compared to its more GC-rich allele, while showing also that the GC-rich alternative is conserved. We propose that mammalian stop codon evolution provides a robust test case. Although in most taxa TAA is the optimal stop codon, TGA is both abundant and conserved in mammalian genomes. We show that this mammalian exceptionalism is well explained by gBGC mimicking purifying selection and that TAA is the selectively optimal codon. Supportive of gBGC, we observe (i) TGA usage trends are consistent at the focal stop and elsewhere (in UTR sequences), (ii) that higher TGA usage and higher TAA[->]TGA substitution rates are predicted by high recombination rate and (iii) across species the difference in TAA <-> TGA rates between GC rich and GC poor genes is largest in genomes that possess higher between-gene GC variation. TAA optimality is supported both by enrichment in highly expressed genes and trends associated with effective population size. High TGA usage and high TAA[->]TGA rates in mammals are thus consistent with gBGCs predicted ability to "drive" deleterious mutations and supports the hypothesis that sequence conservation need not be indicative of purifying selection. A general trend for GC-rich trinucleotides to reside at frequencies far above their mutational equilibrium in high recombining domains supports generality of these results.

category: evolutionary biology


title: Rapid and transient evolution of local adaptation to seasonal host fruits in an invasive fly

abstract:
Both adaptive phenotypic plasticity and local adaptation can influence the match between phenotypic traits and local environmental conditions. Theory predicts that coarse-grained environments, which are stable for multiple generations, promote local adaptation, while fine-grained environments, in which individuals encounter more than one environment in their lifetime, favor adaptive phenotypic plasticity. When the heterogeneity of the environment is spatially and/or temporarily intermediate, with periods of environmental stability from one to only a few generations, the relative contributions of local adaptation and adaptive phenotypic plasticity in enabling individuals phenotypes to match the environments they encounter remains unclear. Here, we used Drosophila suzukii as a model system to evaluate the relative influence of genetic and plastic effects on this match in heterogeneous environments with an intermediate grain. This pest insect can develop within different fruits, and persists throughout the year in a given location on a succession of different host fruits, each one being available for only a few generations. Using reciprocal common environment experiments of natural D. suzukii populations collected from cherry, strawberry and blackberry, we found that both oviposition preference and offspring performance were higher on medium made with the fruit from which the population originated, than on media made with alternative fruits. This pattern remained after two generations in the laboratory, suggesting that genetic effects predominate over plastic effects. Our study demonstrates that spatially and temporally variable selection does not prevent the rapid evolution of local adaptation in natural populations. The speed and strength of adaptation may be facilitated by several mechanisms including a large effective population size and strong selective pressures imposed by host plants.

Impact SummaryNatural populations often exhibit good "fit" to the environment they are in. However, environments change over both time and space, and following change, the fit between a population and its environment may be poor. A question of long-standing interest to evolutionary biologists is, how do populations track changing environments to maintain fitness? Two main mechanisms are known: (i) plastic shifts, or adaptive phenotypic plasticity, in which traits immediately change in response to environmental change, and (ii) genetic shifts in the form of local adaptation, in which traits change over time through differences in fitness of individuals harboring different genetic variants. Plasticity is common when environments change over the course of an individual lifetime, while adaptation is common when environments change over the course of multiple generations. However, many environments change at an intermediate pace, and it is unclear whether plasticity or adaptation are more vital to maintaining fitness under such conditions.

Drosophila suzukii is well-suited to evaluating the relative importance of plasticity and adaptation in response to an intermediate pace of environmental change. This species experiences an environment that shifts every 1-4 generations as host fruits change over time and space. Here, we studied natural populations of D. suzukii collected from different hosts. Using reciprocal common environment experiments, we evaluated their fitness on their source and alternative hosts.

Drosophila suzukii populations were most fit on their source host, successfully tracking an intermediate pace of environmental change. We developed a new statistical method to quantify the contributions of adaptive plasticity and local adaptation in determining fitness. We found that fitness was maintained via local adaptation to each new host in succession. This study provides a novel statistical tool that can be applied to other systems, and highlights that spatially and temporally variable selection does not prevent local adaptation and, on the contrary, illustrates how rapid the adaptive process can be.


title: Comprehensive analysis of cis- and trans-acting factors affecting Break-Induced Replication

abstract: Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating BIR efficiency, we found that BIR efficiency is the highest close to chromosome ends. The variations of BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of BIR depends on the length of DNA already synthesized. BIR is more susceptible to disruption during the synthesis of the first [~]35-40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway.


title: Gap selection and steering during obstacle avoidance in pigeons

abstract: The ability of birds to fly through cluttered environments has inspired biologists interested in understanding its underlying mechanisms, and engineers interested in applying its underpinning principles. To analyse this problem empirically, we break it down into two distinct, but related, questions: How do birds select which gaps to aim for? And how do they steer through them? We answered these questions by using a combined experimental and modelling approach, in which we released pigeons (Columbia livia domestica) inside a large hall whose open exit was separated from the release point by a curtain creating two vertical gaps--one of which was obstructed by an obstacle. We tracked the birds using a high-speed motion capture system, and found that their gap choice seemed to be biased by their intrinsic handedness, rather than determined by extrinsic cues such as the size of the gap or its alignment with the destination. We modelled the pigeons steering behaviour algorithmically by simulating their flight trajectories under a set of six candidate guidance laws, including those used previously to model targeted flight behaviours in birds. We found that their flights were best modelled by delayed proportional navigation commanding turning in proportion to the angular rate of the line-of-sight from the pigeon to the midpoint of the gap. Our results are consistent with this being a two-phase behaviour, in which the pigeon first heads forward from the release point before steering towards the midpoint of whichever gap it chooses to aim for under closed-loop guidance. Our findings have implications for the sensorimotor mechanisms that underlie clutter negotiation in birds, uniting this with other kinds of target-oriented behaviours including aerial pursuit.


title: Deconstructing a visual signal: the role of motion and colour in predator deterrence

abstract: Visual animal communication, whether to the same species or to other species, is largely conducted through dynamic and colourful signals. For a signal to be effective, the signaller must capture and retain the attention of the receiver. Signal efficacy is also dependent on the sensory limitations of the receiver. However, most signalling studies consider movement and colour separately, resulting in a partial understanding of the signal in question. We explored the structure and function of predator-prey signalling in the jumping spider-tephritid fly system, where the prey performs a wing waving display that deters an attack from the predator. Using a custom-built spider retinal tracker combined with visual modelling, and behavioural assays, we studied the effect of fly wing movement and colour on the jumping spiders visual system. We show that jumping spiders track their prey less effectively during wing display and this can be attributed to a series of fluctuations in chromatic and achromatic contrasts arising from the wing movements. These results suggest that displaying flies deter spider attacks by manipulating the movement biases of the spiders visual system. Our results emphasise the importance of receiver attention on the evolution of interspecific communication.


title: Macro-Chemical Biology: Engineering Biomimetic Trogocytosis with Farnesylated Chemically Self-Assembled Nanorings

abstract: With the recent success of cell-based therapies, there has been a rapidly emerging interest in the engineering of cell-cell interactions and communications. Inspired by the natural intercellular material transfer process of trans-endocytosis or trogocytosis, we proposed that targeted farnesylated chemically self-assembled nanorings (farnesyl-CSANs) could serve as a biomimetic trogocytosis vehicle for engineering directional cargo transfer between cells; thus, allowing cell-cell interactions to be monitored, as well as facilitating communication between the cells by delivery of bioactive species. The membranes of sender cells were stably modified by hydrophobic insertion with the targeted farnesyl-CSANs and to be efficiently transferred to receiver cells expressing the appropriate receptor by endocytosis. CSAN-assisted cell-cell cargo transfer (C4T) was demonstrated to be receptor-specific and dependent on direct cell-cell interactions, the rate of receptor internalization and the amount of receptor expression. In addition, C4T was shown to facilitate cell-to-cell delivery of an apoptosis inducing drug, as wells as antisense oligonucleotides (ASO). Taken together, the C4T approach is a potentially versatile biomimetic trogocytosis platform that can be used to monitor cell-cell interactions, as well as the engineering of cell-cell communications, such as cell-based drug delivery.


title: Spatial Transcriptomics-based Identification of Molecular Markers for Nanomedicine Distribution in Tumor Tissue

abstract: The intratumoral accumulation of nanomedicine has been considered a passive process, referred to as the enhanced permeability and retention (EPR) effect. Recent studies have suggested that the tumor uptake of nanomedicines follows an energy-dependent pathway rather than being a passive process. Herein, to explore the factor candidates that are associated with nanomedicine tumor uptake, we developed a molecular marker identification platform by integrating microscopic fluorescence images of a nanomedicine distribution with spatial transcriptomics (ST) information. When this approach is applied to PEGylated liposomes, molecular markers related to hypoxia, glucose metabolism and apoptosis can be identified as being related to the intratumoral distribution of the nanomedicine. We expect that our method can be applied to explain the distribution of a wide range of nanomedicines and that the data obtained from this analysis can enhance the precise utilization of nanomedicines.


title: The network makeup artist (NORMA-2.0): Distinguishing annotated groups in a network using innovative layout strategies

abstract:
MotivationNetwork biology is a dominant player in todays multi-omics era. Therefore, the need for visualization tools which can efficiently cope with intra-network heterogeneity emerges.

ResultsNORMA-2.0 is a web application which uses efficient layouts to group together areas of interest in a network. In this version, NORMA-2.0 utilizes three different strategies to make such groupings as distinct as possible while it preserves all of the properties from its first version where one can handle multiple networks and annotation files simultaneously.

AvailabilityThe web resource is available at http://norma.pavlopouloslab.info/

The source code is freely available at https://github.com/PavlopoulosLab/NORMA

Contactpavlopoulos@fleming.gr


title: Automatic determination of the handedness of Single-Particle maps of macromolecules solved by CryoEM

abstract:
Single-Particle Analysis by Cryo-Electron Microscopy is a well-established technique to elucidate the three-dimensional (3D) structure of biological macromolecules. The orientation of the acquired projection images must be initially estimated without any reference to the final structure. In this step, algorithms may find a mirrored version of all the orientations resulting in a mirrored 3D map. It is as compatible with the acquired images as its unmirrored version from the image processing point of view, only that it is not biologically plausible.

In this article, we introduce HaPi (Handedness Pipeline), the first method to automatically determine the hand of electron density maps of macromolecules solved by CryoEM. HaPi is built by training two 3D convolutional neural networks. The first determines -helices in a map, and the second determines whether the -helix is left-handed or right-handed. A consensus strategy defines the overall map hand. The pipeline is trained on simulated and experimental data. The handedness can be detected only for maps whose resolution is better than 5[A]. HaPi can identify the hand in 89% of new simulated maps correctly. Moreover, we evaluated all the maps deposited at the Electron Microscopy Data Bank and 11 structures uploaded with the incorrect hand were identified.