A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type virus and Delta variant Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.
Immunogenicity of SARS-CoV-2 trimetric spike protein associated to Poly(I:C) plus Alum The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (spike protein with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.
Development of an in-situ printing system with human platelet lysate-based bio-adhesive to treat corneal perforation PurposeCorneal perforation is a clinical emergency. Tissue glue to seal the perforation, and supplementary topical medication represents existing standard treatment. Previously, our group developed a transparent human platelet lysate (hPL)-based biomaterial that showed good cell compatibility and accelerated corneal epithelial cells healing in-vitro. This study aims to develop a novel treatment method for corneal perforation using this biomaterial.

MethodsRheometry was used to measure the hPL-based biomaterial behaviour at room and corneal surface temperatures. Its adhesiveness to porcine skin and burst pressure limit were also measured. Based on rheological behaviour, a hand-held biopen was developed to extrude it onto the cornea. An animal trial (5 New Zealand white rabbits) to compare impact of the biomaterial and cyanoacrylate glue (control group) on a 2mm perforation was conducted to evaluate safety and efficacy.

ResultsThe hPL-based biomaterial showed higher adhesiveness compared to commercial fibrin glue and withstood burst pressure approximately 6.4x higher than routine intraocular pressure. Treatment rabbits had lower pain scores and faster recovery, despite generating similar scar-forming structure compared to controls. No secondary corneal ulcer was generated in rabbits treated with the bio-adhesive.

ConclusionsThis study reports a novel in-situ printing system capable of delivering a hPL-based, transparent bio-adhesive and successfully treating small corneal perforations. Bio-adhesive-treated rabbits recovered faster and required no additional analgesia. Both groups showed scarred corneal tissue after healing, however no infection and inflammation was observed by 3 weeks. The delivery system was easy to use and may represent an alternative treatment for corneal perforation.

HighlightsO_LIThis study presents a novel in situ printing system to treat corneal perforation
C_LIO_LIThe system is comprised of a human platelet lysate-based bio-adhesive and a pen-like hand held delivery system
C_LIO_LIMechanical tests showed our transparent bio-adhesive has a higher adhesiveness compared to existing treatments and burst pressure threshold approximately 6.4 times higher than normal intraocular pressure.
C_LIO_LIIn vivo rabbit trial showed that compared to cyanoacrylate glue, the bio-adhesive was safer, faster healing and led to less pain in rabbits.
C_LI Patterned apoptosis has an instructive role for local growth and tissue shape in a fast-growing epithelium What regulates organ size and shape remains one of the fundamental mysteries of modern biology. So far, research in this area has primarily focused on deciphering the regulation in time and space of growth and cell division, while the contribution of cell death has been much more neglected. This includes studies of the Drosophila wing imaginal disc, the prospective fly wing which undergoes massive growth during larval stage, and represents one of the best characterised systems for the study of growth and patterning. So far, it has been assumed that cell death was relatively neglectable in this tissue and as a result the pattern of growth was usually attributed to the distribution of cell division. Here, using systematic mapping and registration combined with quantitative assessment of clone size and disappearance, we show for the first time that cell death is not neglectable, and outline a persistent pattern of cell death and clone elimination in the disc. Local variation of cell death is associated with local variation of clone size, pointing to an impact of cell death on local growth which is not fully compensated by proliferation. Using morphometric analyses of adult wing shape and genetic perturbations, we provide evidence that patterned death affects locally and globally adult wing shape and size. This study describes a roadmap for accurate assessment of the contribution of cell death to tissue shape, and outlines for the first time an important instructive role of cell death in modulating quantitatively local growth and the morphogenesis of a fast-growing tissue. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. The mutation effect reaction norm (Mu-RN) highlights environmentally dependent mutation effects and genetic interactions Since the modern synthesis, the fitness effects of mutations and epistasis have been central yet provocative concepts in evolutionary and population genetics. Studies of how the interactions between parcels of genetic information can change as a function of environmental context have added a layer of complexity to these discussions. Here I introduce the "mutation effect reaction norm" (Mu-RN), a new instrument through which one can analyze the phenotypic consequences of mutations and interactions across environmental contexts. It embodies the fusion of measurements of genetic interactions with the reaction norm, a classic depiction of the performance of genotypes across environments. I demonstrate the utility of the Mu-RN through the signature of a "compensatory ratchet" mutation that undermines reverse evolution of antimicrobial resistance. More broadly, I argue that the mutation effect reaction norm may help us resolve the dynamism and unpredictability of evolution, with implications for theoretical biology, genetic modification technology, and public health. Linked machine learning classifiers improve species classification of fungi when using error-prone long-reads on extended metabarcodes BackgroundThe increased usage of error-prone long-read sequencing for metabarcoding of fungi has not been matched with adequate public databases and concomitant analysis approaches. We address this gap and present a proof-of-concept study for classifying fungal taxa using linked machine learning classifiers. We demonstrate the capability of linked machine learning classifiers to accurately classify species and strains using real-world and simulated fungal ribosomal DNA datasets, including plant and human pathogens. We benchmark our new approach in comparison to current alignment and k-mer based methods based on synthetic mock communities. We also assess real world applications of species identification in complex unlabelled datasets.

ResultsOur machine learning approach assigned individual nanopore long-read amplicon sequences to fungal species with high recall rates and low false positive rates. Importantly, our approach successfully distinguished between closely-related species and strains when individual read errors were higher than the genetic distance between individual taxa, which the alignment and k-mer methods could not do. The machine learning approach showed an ability to identify key species with high recall rates, even in complex samples of unknown species composition.

ConclusionsA proof of concept machine learning approach using a tree-descent approach on a decision tree of classifiers can identify known taxa with high accuracy, and precisely detect known target species from complex samples with high recall rates. We propose this approach is suitable for detecting the known knowns of pathogens or invasive species in any environment of mostly unknown composition, including agriculture and wild ecosystems. Liver Endothelium Promotes HER3-mediated Cell Survival in KRAS Wild-type and Mutant Colorectal Cancer Cells We previously showed that liver endothelial cells (ECs) secreted soluble factors in a paracrine fashion and activated human epidermal growth factor receptor 3 (HER3, also known as ERBB3) for promoting colorectal cancer (CRC) growth and chemoresistance. However, RAS proteins play a critical role in receptor tyrosine kinase signaling pathways, and KRAS mutations mediate CRC resistance to therapies targeting EGFR, another HER protein. Therefore, the role of KRAS mutation status in EC-induced HER3 activation and CRC survival was investigated as it has therapeutic implications. We used CRC cell lines and patient-derived xenografts harboring KRAS wild-type or mutant genes and demonstrated that liver EC-secreted factors promoted HER3-mediated CRC cell growth independent of KRAS mutation status. Also, blocking HER3 in CRC cells by siRNAs or a HER3 antibody seribantumab blocked EC-induced CRC survival. Our findings highlight the potential of utilizing HER3 targeted therapies for treating patients with mCRC independent of RAS mutational status. Three membrane fusion pore families determine the pathway to pore dilation During exocytosis secretory vesicles fuse with a target membrane and release neurotransmitters, hormones or other bioactive molecules through a membrane fusion pore. The initially small pore may subsequently dilate for full contents release, as commonly observed in amperometric traces. The size, shape and evolution of the pore is critical to the course of contents release, but exact fusion pore solutions accounting for membrane tension and bending energy constraints have not been available. Here we obtained exact solutions for fusion pores between two membranes. We find three families: a narrow pore, a wide pore and an intermediate tether-like pore. For high tensions these are close to the catenoidal and tether solutions recently reported for freely hinged membrane boundaries. We suggest membrane fusion initially generates a stable narrow pore, and the dilation pathway is a transition to the stable wide pore family. The unstable intermediate pore is the transition state that sets the energy barrier for this dilation pathway. Pore dilation is mechanosensitive, as the energy barrier is lowered by increased membrane tension. Finally, we study fusion pores in nanodiscs, powerful systems for the study of individual pores. We show that nanodiscs stabilize fusion pores by locking them into the narrow pore family.

SignificanceDuring neurotransmission, hormone release and other fundamental processes, secretory vesicles fuse their membranes with target membranes to release contents through an initially small membrane fusion pore that subsequently dilates. Dilation is assisted by proteins such as SNAREs and synaptotagmin. While macroscopic soap film shapes are well characterized, finding exact solutions for microscopic cellular membrane surfaces is made more complex by bending energy constraints. Here, computational analysis revealed three families of fusion pores between two membranes. Our work suggests membrane fusion generates a member of the narrow pore family, and pore dilation is a transition to the wide pore family. The energy barrier that SNAREs or synaptotagmin must surmount to achieve dilation is set by a third unstable intermediate pore family. Naming of human diseases on the wrong side of history BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Probing Patterning in Microbial Consortia with picCASO: a Cellular Automaton for Spatial Organisation Microbial consortia exhibit spatial patterning across diverse environments. Since probing the self-organization of natural microbial communities is limited by their inherent complexity, synthetic models have emerged as attractive alternatives. In this study, we develop novel frameworks of bacterial communication and explore the emergent spatiotemporal organization of microbes. Specifically, we build quorum sensing-mediated models of microbial growth that are utilized to characterize the dynamics of communities from arbitrary initial configurations and establish the effectiveness of our communication strategies in coupling the growth rates of microbes. Our simulations indicate that the behavior of quorum sensing-coupled consortia can be most effectively modulated by the rates of secretion of AHLs. Such a mechanism of control enables the construction of desired relative populations of constituent species in spatially organized populations. Our models accurately recapitulate previous experiments that have investigated pattern formation in synthetic multi-cellular systems. Additionally, our software tool enables the easy implementation and analysis of our frameworks for a variety of initial configurations and simplifies the development of sophisticated gene circuits facilitating distributed computing. Overall, we demonstrate the potential of spatial organization as a tunable parameter in synthetic biology by introducing a communication paradigm based on the location and strength of coupling of microbial strains.

Author SummaryInteracting microorganisms that coexist in a given environment tend to have well-defined spatial arrangements. While the emergence of such organization is seen across different microbiomes in nature, it is hitherto not well understood. Decoding the inherent spatial patterning of microbes is constrained by the complexity of their natural habitats. Here, we take advantage of synthetic models of microbial communities to study the dynamics of emergent spatial organization. Our framework of bacterial communication utilizes modular synthetic devices to couple growth rates. In addition to uncovering potential principles of spatial organization, this work enables the construction of complex genetic circuits distributed across communicating strains. Additionally, we have developed a software tool, picCASO, that streamlines the investigation of microbial systems communicating through such frameworks. The N terminus of adhesion G protein-coupled receptor GPR126/ADGRG6 as allosteric force integrator The adhesion G protein-coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown.

In this study we provide this information by combining classic second messenger detection with single cell atomic force microscopy. We establish a monoclonal antibody targeting the N terminus to stimulate GPR126 and compare it to the activation through its known ECM ligands collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be viewed as an allosteric module that can fine-tune receptor activation in a context-specific manner. Contrasting resistance and resilience to light variation of the coupled oxic and anoxic componentsof an experimental microbial ecosystem Understanding how microbial communities of aquatic ecosystems respond to environmental change remains a critical challenge in microbial ecology. In this study, we used phototrophic oxic-anoxic micro-ecosystems to understand how the functioning and diversity of aerobic and anaerobic lake analog communities is affected by light deprivation. Continuous measurements were performed to describe oxygen dynamics (mean/min/max/amplitude) and time-series of full-length 16S rRNA sequencing were used to quantify changes in alpha- and beta-diversity. In the top oxic layer, oxygen concentration decreased significantly under light deprivation, but showed resilience in mean, minimum and maximum after light conditions were restored. Only the amplitude of diurnal fluctuations in oxygen concentrations did not recover fully, and instead tended to remain lower in treated ecosystems. Alpha-diversity of the top oxic layer communities showed a delayed increase after light conditions were restored, and was not resilient. In contrast, alpha-diversity of the anoxic bottom layer communities increased due to the stressor, but was resilient. Community composition changed significantly during light deprivation, showed resilience in the oxic layer and lack of resilience in the anoxic layer. Alpha-diversity and the amplitude of oxygen within and among treatments were strongly correlated, suggesting that higher diversity could lead to less variable oxygen concentrations, or vice versa. Our experiment showed that light deprivation induces multifaceted responses of community function and structure, hence focusing on a single stability component could potentially be misleading. Natural variations in reproductive timing of brood size and X-chromosome nondisjunction in Caenorhabditis elegans Reproductive timing underlies self-sperm production and X-chromosome nondisjunction rate in Caenorhabditis elegans. These traits can be measured as brood size and male-production rate, and brood size decreases and male-production rate increases as worms age. These phenotypic changes occur simultaneously; however, whether these changes share common underlying genetic architectures still remains unclear. To enhance our understanding of reproductive timing in C. elegans, we measured and compared these two reproductive timing-related traits of 96 wild strains during early, late and total reproductive periods. We found that the two traits exhibited natural phenotypic variation with few outliers and a similar reproductive timing pattern as previous reports. The brood size and the male-production rate were not genetically correlated, implying that they might have different genetic architectures and that meiosis quality can be maintained despite increased progeny-production in the late reproductive period caused by more self-sperm. We also identified loci and candidate genetic variants significantly associated with male-production rate in the late and total reproductive periods. Our results provide an insight into life history traits in wild C. elegans strains. Full-length Genome of a Ogataea polymorpha strain CBS4732 ura3Δ reveals large duplicated segments in subtelomeric regions BackgroundCurrently, methylotrophic yeasts (e.g., Pichia pastoris, Ogataea polymorpha, and Candida boindii) are subjects of intense genomics studies in basic research and industrial applications. In the genus Ogataea, most research is focused on three basic O. polymorpha strains--CBS4732, NCYC495, and DL-1. However, the relationship between CBS4732, NCYC495, and DL-1 remains unclear, as the genomic differences between them have not be exactly determined without their high-quality complete genomes. As a nutritionally deficient mutant derived from CBS4732, the O. polymorpha strain CBS4732 ura3{Delta} (named HU-11) is being used for high-yield production of several important proteins or peptides. HU-11 has the same reference genome as CBS4732 (noted as HU-11/CBS4732), because the only genomic difference between them is a 5-bp insertion.

ResultsIn the present study, we have assembled the full-length genome of O. polymorpha HU-11/CBS4732 using high-depth PacBio and Illumina data. Long terminal repeat (LTR) retrotransposons, rDNA, 5 and 3 telomeric, subtelomeric, low complexity and other repeat regions were curated to improve the genome quality. Particularly, we detected large duplicated segments (LDSs) in the subtelomeric regions and exactly determined all the structural variations (SVs) between CBS4732 and NCYC495.

New findings mainly include(1) the genomic differences between HU-11/CBS4732 and NCYC495 include single nucleotide polymorphisms, small insertions and deletions, and only three SVs; (2) six genes were incorporated into CBS4732 from Cyberlindnera jadinii by horizontal gene transfer and may bring HU-11/CBS4732 new biological functions or physiological properties; (3) many recombination events may have occurred on chromosome 4 and 5 of CBS4732 and NCYC495 ancestors and two large segments were acquired by CBS4732 and NCYC495 from chromosome 6 and C. jadinii during recombination, respectively; and (4) the genome expansion in methylotrophic yeasts is mainly driven by large segment duplication in subtelomeric regions.

ConclusionsThe present study preliminarily revealed the complex relationship between CBS4732, NCYC495, and DL-1. The new findings provide new opportunities for in-depth understanding of genome evolution in methylotrophic yeasts and lay the foundations for the industrial applications of O. polymorpha CBS4732, NCYC495, DL-1, and their derivative strains. The full-length genome of the O. polymorpha strain HU-11/CBS4732 should be included into the NCBI RefSeq database for future studies of Ogataea spp.. Multi-omics investigation of tacrolimus nephrotoxicity BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics. First detection of herpesvirus and prevalence of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. Nonideal polar residues in the core of the coiled coil of FtsLB are critical for modulating its stability and activation in cell division The FtsLB complex is a key regulator of bacterial cell division, existing in either an off or on state which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a "detuned" structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction. Engineered bacteria detect tumor DNA Advances in bacterial engineering have catalysed the development of living cell diagnostics and therapeutics1-3, including microbes that respond to diseases such as gut inflammation4, intestinal bleeding5, pathogens6 and hypoxic tumors7. Bacteria can easily access the entire gastrointestinal tract via oral administration8, and they can produce outputs that can be noninvasively measured in stool4 or urine7. Cellular memory, such as bistable switches4,9,10 or genomic rearrangement11, has been used to allow bacteria to store information over time. However, living biosensors have not yet been engineered to detect specific DNA sequences or mutations from outside the cell. Here, we engineer naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells and organoids. We characterize the functionality of the biosensors in vitro with co-culture assays and then validate in vivo with sensor bacteria delivered orally or rectally into mice injected with orthotopic donor CRC organoids. We observe horizontal gene transfer from the tumor to the sensor bacteria in vivo, allowing their detection in stool. The sensor bacteria achieved 100% discrimination between mice with and without CRC using both delivery methods. Our findings establish a framework for biosensing applications that require the detection of mutations or organisms within environments that are difficult to sample. In addition, the platform can be readily expanded to include in situ production and delivery of therapeutic payloads at the detection site. Multilevel multinomial models as a tool to extract population spatial networks from capture-recapture data O_LIEstimating the impacts of anthropogenic disturbances requires an understanding of the habitat use patterns of individuals within a population. This is especially the case when disturbances are localized within a populations spatial range, as variation in habitat-use within a population can drastically alter the distribution of impacts.
C_LIO_LIHere, we illustrate the potential for multilevel multinomial models to generate spatial networks from capture-recapture data, a common data source use in wildlife studies to monitor population dynamics and habitat use. These spatial networks capture which regions of a populations spatial distribution share similar/dissimilar individual usage patterns, and can be especially useful for detecting structured habitat use within the populations spatial range.
C_LIO_LIUsing simulations and 18 years of capture-recapture data from St. Lawrence Estuary (SLE) beluga, we show that this approach can successfully estimate the magnitude of similarities/dissimilarities in individual usage patterns across sectors, and identify sectors that share similar individual usage patterns that differ from other sectors, i.e., structured habitat use. In the case of SLE beluga, this method identified multiple clusters of individuals, each preferentially using restricted areas within their summer range of the SLE.
C_LIO_LISynthesis and applications. Multilevel multinomial models can be effective at estimating spatial structure in habitat use within wildlife populations sampled by capture-recapture of individuals. Our finding of a structured habitat use within the SLE beluga summer range has direct implications for estimating individual exposures to localized stressors, such as underwater noise from shipping or other activities.
C_LI Different computations over the same inputs produce selective behavior in algorithmic brain networks A key challenge in systems neuroscience remains to understand where, when and now particularly how brain networks compute over sensory inputs to achieve behavior. We used XOR, OR and AND functions as behavioral tasks, because each requires a different computation over the same inputs to produce correct outputs. In each task, source-localized magnetoencephalographic activity progresses through four systems-level computations identified within individual participants (N = 10/task): (1) linear discrimination of each visual input, first contra-laterally in occipital cortex then (2) jointly in midline occipital cortex and right fusiform gyrus, followed by (3) nonlinear task-dependent input integration in temporal-parietal cortex and finally (4) behavioral response representation in post-central gyrus. Our results show how network algorithms differently compute over the same inputs to produce different behaviors.

One sentence summaryFour stages of task-specific computations over the same visual inputs achieve different behaviors in dynamic brain networks Placental uptake and metabolism of 25(OH)Vitamin D determines its activity within the fetoplacental unit Pregnancy 25-hydroxyvitamin D (25(OH)D) concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy. Barbary ground squirrels do not have a sentinel system but instead synchronise vigilance Coordinated behavior, such as hunting in lions and coordinated vigilance as antipredator behavior, are examples of benefits of group-living. Instead of asynchronous vigilance, some social species synchronize their vigilance bouts or take turns acting as sentinels. To increase our knowledge on the evolution of vigilance behavior, we studied whether vigilance is coordinated in Barbary ground squirrels, Atlantoxerus getulus. We show that vigilance was synchronized instead of taking turns. Multiple non-mutually exclusive hypotheses could explain synchronization: Barbary ground squirrels may perch because 1) neighbors are perched (copying effect), 2) perch synchrony may be an emergent property of the ecology as all squirrels may be satiated at the same time (collective behavior), or 3) the benefits are large in terms of evading ambush predators and scanning effectiveness (watch each others back). Particularly, in habitats where the field of view is obstructed by man-made structures and multiple individuals may be necessary to watch for terrestrial predators, synchronized vigilance may have greater fitness benefits than sentinel behavior. We conclude that it is essential to test assumptions of coordination and, thus, to analyze coordination to describe sentinel systems.

Significance StatementVigilance behavior can be vital to an animals survival. Taking turns acting as sentinels or synchronizing vigilance bouts reduces the cost of the trade-off between feeding and predation risk. A sentinel system assumes that sentinels are vigilant from raised positions, warn group members of danger, and alternate vigilance bouts. However, the assumption of alternating vigilance bouts remains poorly tested. We tested this assumption in invasive Barbary ground squirrels. We found that instead of alternating, individuals synchronized their vigilance bouts. Perch synchrony may be 1) a response to perching group members (copying effect), 2) an emergent property of the species ecology, and 3) an adaptation to anthropogenically altered habitats (watch each others back). The cortical bone metabolome of C57BL/6J mice is sexually dimorphic Cortical bone quality, which is sexually dimorphic, depends on bone turnover and therefore the activities of remodeling bone cells. However, sex differences in cortical bone metabolism are not yet defined. Adding to the uncertainty about cortical bone metabolism, the metabolomes of whole bone, isolated cortical bone without marrow, and bone marrow have not been compared. We hypothesized that the metabolome of isolated cortical bone would be distinct from that of bone marrow and would reveal sex differences. Metabolite profiles from LC-MS of whole bone, isolated cortical bone, and bone marrow were generated from humeri from 20-week-old female C57Bl/6J mice. The cortical bone metabolomes were then compared for 20-week-old female and male C57Bl/6J mice. Femurs from male and female mice were evaluated for flexural material properties and were then categorized into bone strength groups. The metabolome of isolated cortical bone was distinct from both whole bone and bone marrow. We also found sex differences in the isolated cortical bone metabolome. Based on metabolite pathway analysis, females had higher lipid metabolism, and males had higher amino acid metabolism. High-strength bones, regardless of sex, had greater tryptophan and purine metabolism. For males, high strength bones had upregulated nucleotide metabolism, whereas lower strength bones had greater pentose phosphate pathway metabolism. Since the higher strength groups (females compared with males, high strength males compared with lower strength males) had higher serum CTX1/P1NP, we estimate that the metabolomic signature of bone strength in our study at least partially reflects differences in bone turnover. These data provide novel insight into bone bioenergetics and the sexual dimorphic nature of bone material properties in C57Bl/6 mice. Positive Selection and Heat-Response Transcriptomes Reveal Adaptive Features of the Brassicaceae Desert Model, Anastatica hierochuntica O_LIPlant adaptation to a desert environment and its endemic heat stress is poorly understood at the molecular level. The naturally heat-tolerant Brassicaceae species Anastatica hierochuntica is an ideal extremophyte model to identify genetic adaptations that have evolved to allow plants to tolerate heat stress and thrive in deserts.
C_LIO_LIWe generated an A. hierochuntica reference transcriptome and pinpointed extremophyte adaptations by comparing Arabidopsis thaliana and A. hierochuntica transcriptome responses to heat and identifying positively selected genes in A. hierochuntica.
C_LIO_LIThe two species exhibit similar transcriptome adjustment in response to heat and the A. hierochuntica transcriptome does not exist in a constitutive heat "stress-ready" state. Furthermore, the A. hierochuntica global transcriptome as well as heat-responsive orthologs, display a lower basal and higher heat-induced expression than in A. thaliana. Genes positively selected in multiple extremophytes are associated with stomatal opening, nutrient acquisition, and UV-B induced DNA repair while those unique to A. hierochuntica are consistent with its photoperiod-insensitive, early-flowering phenotype.
C_LIO_LIWe suggest that evolution of a flexible transcriptome confers the ability to quickly react to extreme diurnal temperature fluctuations characteristic of a desert environment while positive selection of genes involved in stress tolerance and early flowering could facilitate an opportunistic desert lifestyle.
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