Differential molecular profiles and associated functionalities characterize connective tissue grafts obtained at different locations and depths in the human palate.

The present study aimed to assess the molecular profiles of subepithelial connective tissue grafts (CTGs) obtained at different locations and depths in the human palate. Sixty-four CTGs belonging to anterior deep (AD), anterior superficial (AS), posterior deep (PD), and posterior superficial (PS) groups were subjected to RNA-Sequencing and their transcriptomes were analyzed computationally. Functional correlations characterizing the CTG groups were validated by cell biological experiments using primary human palatal fibroblasts (HPFs) extracted from the CTGs. A clearly more pronounced location-dependent than depth-dependent difference between the grafts, with a minimal number of genes (4) showing no dependence on the location, was revealed. Epithelial, endothelial, and monocytic cell migration was strongly (P < 0.001) potentiated by AD- and PS-HPFs. Moreover, significantly increased expression of genes encoding C-C and C-X-C motif chemokine ligands as well as significantly (P < 0.01) activated p38 signaling suggested immunomodulatory phenotype for AD- and PS-HPFs. Increased growth factor gene expression and significantly activated (P < 0.001) Erk and Akt signaling in HPFs originating from A-CTGs implied their involvement in cell survival, proliferation, and motility. Prominent collagen-rich expression profile contributing to high mechanical stability, increased osteogenesis-related gene expression, and strongly activated (P < 0.001) Smad1/5/8 signaling characterized HPFs originating from P-CTGs. The present data indicate that in humans, differences between palatal CTGs harvested from different locations and depths appear to be location- rather than depth-dependent. Our findings provide the basis for future personalization of the therapeutic strategy by selecting an optimal graft type depending on the clinical indications.

Effect of preharvest conditions on cut-flower quality.

The cut flower industry has a global reach as flowers are often produced in countries around the equator and transported by plane or ship (reefer) mostly to the global north. Vase-life issues are often regarded as linked to only postharvest conditions while cultivation factors are just as important. Here, we review the main causes for quality reduction in cut flowers with the emphasis on the importance of preharvest conditions. Cut flower quality is characterised by a wide range of features, such as flower number, size, shape, colour (patterns), fragrance, uniformity of blooming, leaf and stem colour, plant shape and developmental stage, and absence of pests and diseases. Postharvest performance involves improving and preserving most of these characteristics for as long as possible. The main causes for cut flower quality loss are reduced water balance or carbohydrate availability, senescence and pest and diseases. Although there is a clear role for genotype, cultivation conditions are just as important to improve vase life. The role of growth conditions has been shown to be essential; irrigation, air humidity, and light quantity and quality can be used to increase quality. For example, xylem architecture is affected by the irrigation scheme, and the relative humidity in the greenhouse affects stomatal function. Both features determine the water balance of the flowering stem. Light quality and period drives photosynthesis, which is directly responsible for accumulation of carbohydrates. The carbohydrate status is important for respiration, and many senescence related processes. High carbohydrates can lead to sugar loss into the vase water, leading to bacterial growth and potential xylem blockage. Finally, inferior hygiene during cultivation and temperature and humidity control during postharvest can lead to pathogen contamination. At the end of the review, we will discuss the future outlook focussing on new phenotyping tools necessary to quantify the complex interactions between cultivation factors and postharvest performance of cut flowers.

Targeted removal of macrophage-secreted interleukin-1 receptor antagonist protects against lethal Candida albicans sepsis.

Invasive fungal infections are associated with high mortality rates, and the lack of efficient treatment options emphasizes an urgency to identify underlying disease mechanisms. We report that disseminated Candida albicans infection is facilitated by interleukin-1 receptor antagonist (IL-1Ra) secreted from macrophages in two temporally and spatially distinct waves. Splenic CD169+ macrophages release IL-1Ra into the bloodstream, impeding early neutrophil recruitment. IL-1Ra secreted by monocyte-derived tissue macrophages further impairs pathogen containment. Therapeutic IL-1Ra neutralization restored the functional competence of neutrophils, corrected maladapted hyper-inflammation, and eradicated the otherwise lethal infection. Conversely, augmentation of macrophage-secreted IL-1Ra by type I interferon severely aggravated disease mortality. Our study uncovers how a fundamental immunoregulatory mechanism mediates the high disease susceptibility to invasive candidiasis. Furthermore, interferon-stimulated IL-1Ra secretion may exacerbate fungal dissemination in human patients with secondary candidemia. Macrophage-secreted IL-1Ra should be considered as an additional biomarker and potential therapeutic target in severe systemic candidiasis.

A single MYB transcription factor with multiple functions during flower development.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.

Evaluation of a Viscoelastic Coagulation Monitoring System (VCM Vet®) and Its Correlation with Thromboelastometry (ROTEM®) in Diseased and Healthy Dogs.

Thromboelastometry provides a real-time assessment of global coagulation in whole blood. A novel bed-side viscoelastic coagulation monitor (VCM) has been developed for use in small animals. The aims of the study were to determine inter-device agreement of two VCM devices, to evaluate the correlation between VCM and rotational thromboelastometry as a reference method (ROTEM), and to determine the sensitivity and specificity of VCM to diagnose hypo-, normo-, and hypercoagulability. ROTEM (extrinsic and intrinsic activation) analysis was performed using citrated blood and VCM analysis using native blood. Twenty healthy and forty diseased dogs with and without coagulopathies were enrolled. The VCM inter-device agreement was moderate to strong for most of the parameters, depending on the grading scale. Correlation between VCM and ROTEM was moderate to strong for parameters of clotting time and clot strength. The VCM most likely detects true hypocoagulability and reliably rules out hypocoagulability. The VCM has a high sensitivity in diagnosing normocoagulability, but incorrectly classified dogs with abnormal coagulation as normocoagulable. The VCM was not able to detect hypercoagulability. ROTEM and VCM cannot be used interchangeably.

Molecular characterization of cell types in the squid Loligo vulgaris.

Cephalopods are set apart from other mollusks by their advanced behavioral abilities and the complexity of their nervous systems. Because of the great evolutionary distance that separates vertebrates from cephalopods, it is evident that higher cognitive features have evolved separately in these clades despite the similarities that they share. Alongside their complex behavioral abilities, cephalopods have evolved specialized cells and tissues, such as the chromatophores for camouflage or suckers to grasp prey. Despite significant progress in genome and transcriptome sequencing, the molecular identities of cell types in cephalopods remain largely unknown. We here combine single-cell transcriptomics with in situ gene expression analysis to uncover cell type diversity in the European squid Loligo vulgaris. We describe cell types that are conserved with other phyla such as neurons, muscles, or connective tissues but also cephalopod-specific cells, such as chromatophores or sucker cells. Moreover, we investigate major components of the squid nervous system including progenitor and developing cells, differentiated cells of the brain and optic lobes, as well as sensory systems of the head. Our study provides a molecular assessment for conserved and novel cell types in cephalopods and a framework for mapping the nervous system of L. vulgaris.

Microbial uptake in oral mucosa-draining lymph nodes leads to rapid release of cytotoxic CD8+ T cells lacking a gut-homing phenotype.

The gastrointestinal (GI) tract constitutes an essential barrier against ingested microbes, including potential pathogens. Although immune reactions are well studied in the lower GI tract, it remains unclear how adaptive immune responses are initiated during microbial challenge of the oral mucosa (OM), the primary site of microbial encounter in the upper GI tract. Here, we identify mandibular lymph nodes (mandLNs) as sentinel lymphoid organs that intercept ingested Listeria monocytogenes (Lm). Oral Lm uptake led to local activation and release of antigen-specific CD8+ T cells that constituted most of the early circulating effector T cell (TEFF) pool. MandLN-primed TEFF disseminated to lymphoid organs, lung, and OM and contributed substantially to rapid elimination of target cells. In contrast to CD8+ TEFF generated in mesenteric LN (MLN) during intragastric infection, mandLN-primed TEFF lacked a gut-seeking phenotype, which correlated with low expression of enzymes required for gut-homing imprinting by mandLN stromal and dendritic cells. Accordingly, mandLN-primed TEFF decreased Lm burden in spleen but not MLN after intestinal infection. Our findings extend the concept of regional specialization of immune responses along the length of the GI tract, with CD8+ TEFF generated in the upper GI tract displaying homing profiles that differ from those imprinted by lymphoid tissue of the lower GI tract.

Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance.

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

Detecting quantitative trait loci and exploring chromosomal pairing in autopolyploids using polyqtlR.

MOTIVATION: The investigation of quantitative trait loci (QTL) is an essential component in our understanding of how organisms vary phenotypically. However, many important crop species are polyploid (carrying more than two copies of each chromosome), requiring specialized tools for such analyses. Moreover, deciphering meiotic processes at higher ploidy levels is not straightforward, but is necessary to understand the reproductive dynamics of these species, or uncover potential barriers to their genetic improvement. RESULTS: Here, we present polyqtlR, a novel software tool to facilitate such analyses in (auto)polyploid crops. It performs QTL interval mapping in F1 populations of outcrossing polyploids of any ploidy level using identity-by-descent probabilities. The allelic composition of discovered QTL can be explored, enabling favourable alleles to be identified and tracked in the population. Visualization tools within the package facilitate this process, and options to include genetic co-factors and experimental factors are included. Detailed information on polyploid meiosis including prediction of multivalent pairing structures, detection of preferential chromosomal pairing and location of double reduction events can be performed. AVAILABILITYAND IMPLEMENTATION: polyqtlR is freely available from the Comprehensive R Archive Network (CRAN) at http://cran.r-project.org/package=polyqtlR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

polymapR-linkage analysis and genetic map construction from F1 populations of outcrossing polyploids.

Motivation: Polyploid species carry more than two copies of each chromosome, a condition found in many of the world's most important crops. Genetic mapping in polyploids is more complex than in diploid species, resulting in a lack of available software tools. These are needed if we are to realize all the opportunities offered by modern genotyping platforms for genetic research and breeding in polyploid crops. Results: polymapR is an R package for genetic linkage analysis and integrated genetic map construction from bi-parental populations of outcrossing autopolyploids. It can currently analyse triploid, tetraploid and hexaploid marker datasets and is applicable to various crops including potato, leek, alfalfa, blueberry, chrysanthemum, sweet potato or kiwifruit. It can detect, estimate and correct for preferential chromosome pairing, and has been tested on high-density marker datasets from potato, rose and chrysanthemum, generating high-density integrated linkage maps in all of these crops. Availability and implementation: polymapR is freely available under the general public license from the Comprehensive R Archive Network (CRAN) at http://cran.r-project.org/package=polymapR. Supplementary information: Supplementary data are available at Bioinformatics online.

An ultra-dense integrated linkage map for hexaploid chrysanthemum enables multi-allelic QTL analysis.

KEY MESSAGE: We constructed the first integrated genetic linkage map in a polysomic hexaploid. This enabled us to estimate inheritance of parental haplotypes in the offspring and detect multi-allelic QTL. Construction and use of linkage maps are challenging in hexaploids with polysomic inheritance. Full map integration requires calculations of recombination frequency between markers with complex segregation types. In addition, detection of QTL in hexaploids requires information on all six alleles at one locus for each individual. We describe a method that we used to construct a fully integrated linkage map for chrysanthemum (Chrysanthemum × morifolium, 2n = 6x = 54). A bi-parental F1 population of 406 individuals was genotyped with an 183,000 SNP genotyping array. The resulting linkage map consisted of 30,312 segregating SNP markers of all possible marker dosage types, representing nine chromosomal linkage groups and 107 out of 108 expected homologues. Synteny with lettuce (Lactuca sativa) showed local colinearity. Overall, it was high enough to number the chrysanthemum chromosomal linkage groups according to those in lettuce. We used the integrated and phased linkage map to reconstruct inheritance of parental haplotypes in the F1 population. Estimated probabilities for the parental haplotypes were used for multi-allelic QTL analyses on four traits with different underlying genetic architectures. This resulted in the identification of major QTL that were affected by multiple alleles having a differential effect on the phenotype. The presented linkage map sets a standard for future genetic mapping analyses in chrysanthemum and closely related species. Moreover, the described methods are a major step forward for linkage mapping and QTL analysis in hexaploids.

Conclusive evidence for hexasomic inheritance in chrysanthemum based on analysis of a 183 k SNP array.

BACKGROUND: Cultivated chrysanthemum is an outcrossing hexaploid (2n = 6× = 54) with a disputed mode of inheritance. In this paper, we present a single nucleotide polymorphism (SNP) selection pipeline that was used to design an Affymetrix Axiom array with 183 k SNPs from RNA sequencing data (1). With this array, we genotyped four bi-parental populations (with sizes of 405, 53, 76 and 37 offspring plants respectively), and a cultivar panel of 63 genotypes. Further, we present a method for dosage scoring in hexaploids from signal intensities of the array based on mixture models (2) and validation of selection steps in the SNP selection pipeline (3). The resulting genotypic data is used to draw conclusions on the mode of inheritance in chrysanthemum (4), and to make an inference on allelic expression bias (5). RESULTS: With use of the mixture model approach, we successfully called the dosage of 73,936 out of 183,130 SNPs (40.4%) that segregated in any of the bi-parental populations. To investigate the mode of inheritance, we analysed markers that segregated in the large bi-parental population (n = 405). Analysis of segregation of duplex x nulliplex SNPs resulted in evidence for genome-wide hexasomic inheritance. This evidence was substantiated by the absence of strong linkage between markers in repulsion, which indicated absence of full disomic inheritance. We present the success rate of SNP discovery out of RNA sequencing data as affected by different selection steps, among which SNP coverage over genotypes and use of different types of sequence read mapping software. Genomic dosage highly correlated with relative allele coverage from the RNA sequencing data, indicating that most alleles are expressed according to their genomic dosage. CONCLUSIONS: The large population, genotyped with a very large number of markers, is a unique framework for extensive genetic analyses in hexaploid chrysanthemum. As starting point, we show conclusive evidence for genome-wide hexasomic inheritance.

Partial preferential chromosome pairing is genotype dependent in tetraploid rose.

It has long been recognised that polyploid species do not always neatly fall into the categories of auto- or allopolyploid, leading to the term 'segmental allopolyploid' to describe everything in between. The meiotic behaviour of such intermediate species is not fully understood, nor is there consensus as to how to model their inheritance patterns. In this study we used a tetraploid cut rose (Rosa hybrida) population, genotyped using the 68K WagRhSNP array, to construct an ultra-high-density linkage map of all homologous chromosomes using methods previously developed for autotetraploids. Using the predicted bivalent configurations in this population we quantified differences in pairing behaviour among and along homologous chromosomes, leading us to correct our estimates of recombination frequency to account for this behaviour. This resulted in the re-mapping of 25 695 SNP markers across all homologues of the seven rose chromosomes, tailored to the pairing behaviour of each chromosome in each parent. We confirmed the inferred differences in pairing behaviour among chromosomes by examining repulsion-phase linkage estimates, which also carry information about preferential pairing and recombination. Currently, the closest sequenced relative to rose is Fragaria vesca. Aligning the integrated ultra-dense rose map with the strawberry genome sequence provided a detailed picture of the synteny, confirming overall co-linearity but also revealing new genomic rearrangements. Our results suggest that pairing affinities may vary along chromosome arms, which broadens our current understanding of segmental allopolyploidy.