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Chytrid fungi play key ecological roles in aquatic ecosystems, and some species cause a devastating skin disease in frogs and salamanders. Additionally, chytrids occupy a unique phylogenetic position– sister to the well-studied Dikarya (the group including yeasts, sac fungi, and mushrooms) and related to animals– making chytrids useful for answering important evolutionary questions. Despite their importance, little is known about the basic cell biology of chytrids. A major barrier to understanding chytrid biology has been a lack of genetic tools with which to test molecular hypotheses. Medina and colleagues recently developed a protocol for Agrobacterium -mediated transformation of Spizellomyces punctatus. In this manuscript, we describe the general procedure including planning steps and expected results. We also provide in-depth, step-by-step protocols and video guides for performing the entirety of this transformation procedure on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v1).

The Global Panzootic Lineage (GPL) of the pathogenic fungusBatrachochytrium dendrobatidis(Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly availableBdgenome sequences, we identified multiple non-GPLBdisolates that contain a circular Rep-encoding single stranded DNA-like virus which we named BdDV-1. We further sequenced and constructed genome assemblies with long read sequences to find that the virus is likely integrated into the nuclear genome. Attempts to cure virus positive isolates were unsuccessful, however, phenotypic differences between naturally virus positive and virus negativeBdisolates suggested that BdDV-1 decreases the growth of its hostin vitrobut increases the virulence of its hostin vivo. BdDV-1 is the first described CRESS DNA mycovirus to increase host virulence with a distribution inversely associated with the emergence of the panzootic lineage.

Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments like freshwater must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify basic principles governing contractile vacuole function, we here investigate the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages—the discobanNaegleria gruberi, and the amoebozoan slime moldDictyostelium discoideum. Using quantitative cell biology we find that, although these species respond differently to osmotic challenges, they both use actin for osmoregulation, as well as vacuolar-type proton pumps for filling contractile vacuoles. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolateParamecium multimicronucleatum. Because these three lineages diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.

Early-postemergence herbicide applications in the USA often include residual herbicides such as S-metolachlor to suppress late late-emerging Amaranthus spp. Although this practice benefits weed control, herbicide tankmixes can influence spray droplet size and drift potential during applications. The addition of S-metolachlor products to dicamba spray solutions generally decreases spray droplet size and increases spray drift potential. Advances in formulation technology fostered the development of products with reduced spray drift potential, especially for herbicide premixes containing multiple active ingredients. The objective of this study was to compare the drift potential of a novel dicamba plus S-metolachlor premix formulation (capsule suspension) against a tankmix containing dicamba (soluble liquid) and S-metolachlor (emulsifiable concentrate) using different venturi nozzles.The MUG nozzle had greater DStudy results evidence that advances in pesticide formulation can improve pesticide drift mitigation. © 2021 Society of Chemical Industry.

Pelvic tilt affects acetabular anteversion, and thus total hip arthroplasty (THA) dislocation risk. The pubic symphysis-sacrococcygeal distance (PSCD) is an indicator of pelvic tilt, and a PSCD0 mm (ie, excessive posterior pelvic tilt) is associated with a 3.7-fold increase in postoperative dislocation rate. However, it is not known if the direct anterior (DA) approach might reduce this dislocation rate, specifically in high-risk populations such as negative PSCD.Standing anteroposterior radiographs were reviewed for 510 consecutive DA THAs to determine PSCD. Patients were separated into 2 groups: (1) PSCD0 mm (PSCD[+]) and (2) PSCD0 mm (PSCD[-]). Incidence of dislocation was determined. We recorded if patients had spinal deformity or lumbar fusion. Continuous variables were analyzed using Student's t-test, categorical variables were analyzed using Fisher's exact test, and a sample size calculation was performed.Three hundred fifty-eight hips (70.2%) were PSCD[+], while 152 hips (29.8%) were PSCD[-]. Three dislocations (3/510 hips, 0.6%) occurred. Two dislocators were in the PSCD[-] group (2/152 hips, 1.3%) and 1 dislocator was in the PSCD[+] group (1/358 hips, 0.3%) (P = .21). Twenty-four patients had degenerative scoliosis (24/510, 4.7%), of which 1 had a dislocation (1/24, 4.2%); 2 dislocations occurred in nonscoliosis patients (2/486, 0.4%) (P = .134). Twenty-seven patients had lumbar spinal fusion (27/510, 5.3%), of which there were no dislocations (0/27, 0.0%); all dislocations were in nonfusion patients (3/483, 0.6%) (P = 1.0).We demonstrate no increased risk for THA dislocation in patients with a PSCD0 mm who have undergone a DA approach. These data would suggest a protective effect of the DA approach against dislocation, even in historically high-risk populations.

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

Leo I is considered one of the youngest dwarf spheroidals (dSph) in the Local Group. Its isolation, extended star formation history (SFH), and recent perigalacticon passage (~1 Gyr ago) make Leo~I one of the most interesting nearby stellar systems. Here, we analyse deep photometric Hubble Space Telescope data via colour-magnitude diagram fitting techniques to study its global and radially-resolved SFH. We find global star formation enhancements in Leo I ~13, 5.5, 2.0, and 1.0 Gyr ago, after which it was substantially quenched. Within the context of previous works focused on Leo I, we interpret the most ancient and the youngest ones as being linked to an early formation (surviving reionisation) and the latest perigalacticon passage (transition from dIrr to dSph), respectively. We clearly identify the presence of very metal poor stars ([Fe/H]~-2) ageing ~5-6 and ~13 Gyr old. We speculate with the possibility that this metal-poor population in Leo I is related to the merging with a low mass system (possibly an ultra-faint dwarf). This event would have triggered star formation (peak of star formation ~5.5 Gyr ago) and accumulated old, metal poor stars from the accreted system in LeoI. Some of the stars born during this event would also form from accreted gas of low-metallicity (giving rise to the 5-6 Gyr low-metallicity tail). Given the intensity and extension of the 2.0 Gyr burst, we hypothesise that this enhancement could also have an external origin. Despite the quenching of star formation around 1 Gyr ago (most probably induced by ram pressure stripping with the Milky Way halo at pericentre), we report the existence of stars as young as 300-500 Myr. We also distinguish two clear spatial regions: the inner ~190 pc presents an homogeneous stellar content (size of the gaseous star forming disc in LeoI from ~4.5 to 1 Gyr ago), whereas the outer regions display a clear positive age gradient., 20 pages, 11+5 figures, accepted for publication in MNRAS

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars., Comparison of multiple genome assemblies from wheat reveals extensive diversity that results from the complex breeding history of wheat and provides a basis for further potential improvements to this important food crop.

Key message The first cytological characterization of the 2NvS segment in hexaploid wheat; complete de novo assembly and annotation of 2NvS segment; 2NvS frequency is increasing 2NvS and is associated with higher yield. Abstract The Aegilops ventricosa 2NvS translocation segment has been utilized in breeding disease-resistant wheat crops since the early 1990s. This segment is known to possess several important resistance genes against multiple wheat diseases including root knot nematode, stripe rust, leaf rust and stem rust. More recently, this segment has been associated with resistance to wheat blast, an emerging and devastating wheat disease in South America and Asia. To date, full characterization of the segment including its size, gene content and its association with grain yield is lacking. Here, we present a complete cytological and physical characterization of this agronomically important translocation in bread wheat. We de novo assembled the 2NvS segment in two wheat varieties, ‘Jagger’ and ‘CDC Stanley,’ and delineated the segment to be approximately 33 Mb. A total of 535 high-confidence genes were annotated within the 2NvS region, with > 10% belonging to the nucleotide-binding leucine-rich repeat (NLR) gene families. Identification of groups of NLR genes that are potentially N genome-specific and expressed in specific tissues can fast-track testing of candidate genes playing roles in various disease resistances. We also show the increasing frequency of 2NvS among spring and winter wheat breeding programs over two and a half decades, and the positive impact of 2NvS on wheat grain yield based on historical datasets. The significance of the 2NvS segment in wheat breeding due to resistance to multiple diseases and a positive impact on yield highlights the importance of understanding and characterizing the wheat pan-genome for better insights into molecular breeding for wheat improvement.

Este articulo sostiene que las escenas cinegeticas de Amadis de Gaula (Garci Rodriguez de Montalvo, 1508) funcionan como aparatos altuserianos de interpelacion, al reclutar a los nobles a traves de representaciones de comportamientos recreativos propios de su estamento, reafirmando asi una ideologia estamental. Esta interpretacion facilita asimismo un entendimiento de los motivos ideologicos de los moralistas que condenaban al Amadis. Estas condenas pueden entenderse como parte de un esfuerzo general para lograr una mejor movilidad social a traves de la difusion de valores meritocraticos y de la critica a conceptualizaciones tradicionales de identidad del estamento noble.

BACKGROUND Evolution and spread of resistance to glyphosate in kochia [Bassia scoparia (L.) A.J. Scott] is a major challenge for the sustainability of glyphosate-resistant crop technology in this region. Dicamba offers a viable option to manage glyphosate-resistant kochia. However, the recent and rapid evolution of dicamba resistance in glyphosate-resistant kochia populations in Kansas (KS), and other states in the USA is a threat to the management of this weed. Our previous research suggests that two distinct mechanisms confer dicamba resistance in KS (KSUR) and NE (CSUR) kochia. CSUR kochia is dicamba-resistant due to a double mutation in an auxin and dicamba coreceptor gene (Aux/IAA16), and CSUR kochia plants show reduced dicamba translocation. However, the mechanism of dicamba resistance in KSUR is not known. The objective of this research was to determine if dicamba resistance in KSUR is due to a different mechanism and therefore evolved independently from CSUR by measuring whether the resistance traits are chromosomally linked. RESULTS The F1 and F2 progenies from KSUR × CSUR were generated. Single dicamba rate tests were conducted using the F1 and F2 progeny. The results indicate that two different genes confer dicamba resistance in KSUR and CSUR; importantly, these two genes are not linked. CONCLUSION This research provides evidence that different populations of kochia have independently evolved resistance to dicamba by different mechanisms, and we confirmed that the genes conferring resistance to the same herbicide in different populations are not chromosomally linked.

Wheat (Triticum aestivum L.) rusts are a worldwide production problem. Plant breeders have used genetic resistance to combat these fungi. However, single-gene resistance is rapidly overcome as a result of frequent occurrence of new virulent fungal strains. Thus, a supply of new resistance sources is continually needed, and new resistance sources are limited within hexaploid wheat genetic stocks. Wild relatives are able to be a resource for new resistance genes but are hindered because of chromosome incapability with domesticated wheats. Twenty-eight double-haploid hexaploid wheat/Amblyopyrum muticum (Boiss.) Eig introgression lines, with introgressions covering the majority of the T genome, were evaluated for resistance to Puccinia triticina Erikss., P. graminis Pers.:Pers. f.sp. tritici Erikss. & E. Henning, and P. striiformis Westend. f.sp. tritici Erikss.. At the seedling level, four lines were resistant to races of P. triticina, six lines were resistant to P. graminis, and 15 lines were resistant to P. striiformis. At the adult stage, 16 lines were resistant to P. triticina. Line 355 had resistance to all three rusts and line 161 had resistance to all tested races of P. triticina. Some of these lines will require further work to reduce the size of the introgressed segment; however, lines 92 and 355 have very small fragments and can be used directly as new resistance donors.

Introgression from wild relatives is a valuable source of novel allelic diversity for breeding. We investigated the genomic patterns of introgression fromAegilops tauschii, the diploid ancestor of the wheat D genome, into winter wheat (Triticum aestivum) cultivars. The population of 351 BC1F3:5lines was selected based on phenology from crosses between six hexaploid wheat lines and 21 wheat-Ae. tauschiioctoploids. SNP markers developed for this population and a diverse panel of 116Ae. tauschiiaccessions by complexity-reduced genome sequencing were used to detect introgression based on the identity-by-descent analysis. Overall, introgression frequency positively correlated with recombination rate, with a high incidence of introgression at the ends of chromosomes and low in the pericentromeric regions, and was negatively related to sequence divergence between the parental genomes. Reduced introgression in the pericentromeric low-recombining regions spans nearly 2/3 of each chromosome arm, suggestive of the polygenic nature of introgression barriers that could be associated with multilocus negative epistasis between the alleles of wild and cultivated wheat. On the contrary, negative selection against the wild allele ofTg, controlling free-threshing trait and located in the high-recombining chromosomal region, led to reduced introgression only within ∼10 Mbp region aroundTg. These results are consistent with the effect of selection on linked variation described by the Hill-Robertson effect, and offer insights into the introgression population development for crop imrpovement to ensure retention of introgressed diversity across entire genome.

Eukaryotic cells use a number of diverse mechanisms to swim through liquid or crawl across solid surfaces. The two most prevalent forms of eukaryotic cell motility are flagellar-dependent swimming and actin-dependent cell migration, both of which are used by animal cells and unicellular eukaryotes alike. Evolutionary cell biologists have used morphological and molecular phenotypes to trace the evolution of flagellar-based swimming. These efforts have resulted in a large body of evidence supporting a single evolutionary origin of the eukaryotic flagellum, an origin that dates back to before the diversification of modern eukaryotes. Actin-dependent crawling, in contrast, involves mutiple distinct molecular mechanisms, the evolution of which is just beginning to be explored.

We use Gaia DR2 systemic proper motions of 45 satellite galaxies to constrain the mass of the Milky Way using the scale free mass estimator of Watkins et al. (2010). We first determine the anisotropy parameter $\beta$, and the tracer satellites' radial density index $\gamma$ to be $\beta$=$-0.67^{+0.45}_{-0.62}$ and $\gamma=2.11\pm0.23$. When we exclude possible former satellites of the Large Magellanic Cloud, the anisotropy changes to $\beta$=$-0.21^{+0.37}_{-0.51}$. We find that the index of the Milky Way's gravitational potential $\alpha$, which is dependent on the mass itself, is the parameter with the largest impact on the mass determination. Via comparison with cosmological simulations of Milky Way-like galaxies, we carried out a detailed analysis of the estimation of the observational uncertainties and their impact on the mass estimator. We found that the mass estimator is biased when applied naively to the satellites of simulated Milky Way halos. Correcting for this bias, we obtain for our Galaxy a mass of $0.58^{+0.15}_{-0.14}\times10^{12}$M$_\odot$ within 64 kpc, as computed from the inner half of our observational sample, and $1.43^{+0.35}_{-0.32}\times10^{12}$M$_\odot$ within 273 kpc, from the full sample; this latter value extrapolates to a virial mass of $M_\mathrm{vir\,\Delta=97}$=$1.51^{+0.45}_{-0.40} \times 10^{12}M_{\odot}$ corresponding to a virial radius of R$_\mathrm{vir}$=$308\pm29$ kpc. This value of the Milky Way mass lies in-between other mass estimates reported in the literature, from various different methods., Comment: 17 pages, 10 figures, 2 tables. Accepted for publication in MNRAS

In the past few years, our understanding of immunological memory has evolved remarkably due to a growing body of new knowledge in innate immune memory and immunity. Immunological memory now encompasses both innate and adaptive immune memory. The hypo-reactive and hyper-reactive types of innate immune memory lead to a suppressed and enhanced innate immune protective outcome, respectively. The latter is also named trained innate immunity (TII). The emerging information on innate immune memory has not only shed new light on the mechanisms of host defense but is also revolutionizing our long-held view of vaccination and vaccine strategies. Our current review will examine recent progress and knowledge gaps in innate immune memory with a focus on tissue-resident Mϕs, particularly lung Mϕs, and their relationship to local antimicrobial innate immunity. We will also discuss the impact of innate immune memory and TII on our understanding of vaccine concept and strategies and the significance of respiratory mucosal route of vaccination against respiratory pathogens.

The introduction of 2,4-D–resistant soybean and cotton provided growers a new POST active ingredient to include in weed management programs. The technology raises concerns regarding potential 2,4-D off-target movement to sensitive vegetation, and spray droplet size is the primary management factor focused on to reduce spray particle drift. The objective of this study was to investigate the droplet size distribution, droplet velocity, and particle drift potential of glyphosate plus 2,4-D choline pre-mixture (Enlist Duo®) applications with two commonly used venturi nozzles in a low-speed wind tunnel. Applications with the TDXL11004 nozzle had larger DV0.1 (291 µm), DV0.5 (544 µm), and DV0.9 (825 µm) values compared with the AIXR11004 nozzle (250, 464, and 709 µm, respectively), and slower average droplet velocity (8.1 m s−1) compared with the AIXR11004 nozzle (9.1 m s−1). Nozzle type had no influence on drift deposition (P = 0.65), drift coverage (P = 0.84), and soybean biomass reduction (P = 0.76). Although the TDXL11004 nozzle had larger spray droplet size, the slower spray droplet velocity could have influenced the nozzle particle drift potential. As a result, both TDXL11004 and AIXR11004 nozzles had similar spray drift potential. Further studies are necessary to understand the impact of droplet velocity on drift potential at field scale and test how different tank solutions, sprayer configurations, and environmental conditions could influence the droplet size and velocity dynamics and consequent drift potential in pesticide applications.

Wheat head blast (WHB), caused by the fungus Magnaporthe oryzae pathotype triticum, is a devastating disease affecting South America and South Asia. Despite 30 years of intensive effort, the 2NVS translocation from Aegilops ventricosa contains the only useful source of resistance to WHB effective against M. oryzae triticum isolates. The objective of this study was to identify non-2NVS sources of resistance to WHB among elite cultivars, breeding lines, landraces, and wild-relative accessions. Over 780 accessions were evaluated under field and greenhouse conditions in Bolivia, greenhouse conditions in Brazil, and at two biosafety level-3 laboratories in the United States. The M. oryzae triticum isolates B-71 (2012), 008 (2015), and 16MoT001 (2016) were used for controlled experiments, while isolate 008 was used for field experiments. Resistant and susceptible checks were included in all experiments. Under field conditions, susceptible spreaders were inoculated at the tillering stage to guarantee sufficient inoculum. Disease incidence and severity were evaluated as the average rating for each 1-m-row plot. Under controlled conditions, heads were inoculated after full emergence and individually rated for percentage of diseased spikelets. The diagnostic marker Ventriup-LN2 was used to test for the presence of the 2NVS translocation. Four non-2NVS spring wheat International Maize and Wheat Improvement Center breeding lines (CM22, CM49, CM52, and CM61) and four wheat wild-relatives (A. tauschii TA10142, TA1624, TA1667, and TA10140) were identified as resistant (VS-based varieties. The low frequency of WHB resistance and the increase in aggressiveness of newer M. oryzae triticum isolates highlight the threat that the disease poses to wheat production worldwide and the urgent need to identify and characterize new resistance genes that can be used in breeding for durably resistant varieties.

The frog-killing fungus Batrachochytrium dendrobatidis (Bd) grows within the mucus-coated skin of amphibians. Each lifecycle, Bd transitions from its motile, dispersal form to its sessile growth form in a complex process called encystation. Although encystation is critical to Bd growth, whether and how this developmental transition is triggered by external signals was previously unknown. We discovered that exposure to amphibian mucus triggers rapid and reproducible encystation within minutes. This response can be recapitulated with purified mucin, the bulk component of mucus, but not by similarly-viscous methylcellulose or simple sugars. Mucin-induced encystation does not require gene expression, but does require surface adhesion, calcium signaling, and modulation of the actin cytoskeleton. Mucus-induced encystation may represent a key mechanism for synchronizing Bd development with arrival at the host.

BackgroundAdenovirus-vectored (Ad-vectored) vaccines are typically administered via i.m. injection to humans and are incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized, and its ability to induce mucosal immunity in humans is unknown. This phase Ib trial evaluated the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or i.m. injection.MethodsThirty-one healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb Solo Nebulizer or by i.m. injection. The study consisted of the low-dose (LD) aerosol, high-dose (HD) aerosol, and i.m. groups. The adverse events were assessed at various times after vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline, as well as at select time points after vaccination.ResultsThe nebulized aerosol droplets were5.39 μm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and i.m. injection were safe and well tolerated. Both aerosol doses, particularly LD, but not i.m., vaccination markedly induced airway tissue-resident memory CD4+ and CD8+ T cells of polyfunctionality. While as expected, i.m. vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages.ConclusionInhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens, including TB and COVID-19.Trial registrationClinicalTrial.gov, NCT02337270.FundingThe Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada funded this work.

Polycyclic conjugated non-benzenoid hydrocarbons are of great theoretical interest in connection with the question of the relationships between structure and aromaticity[1].

Polycyclisch konjugierte, nichtbenzoide Kohlenwasserstoffe beanspruchen im Zusammenhang mit der Frage nach den Beziehungen zwischen Konstitution und ,,aromatischem" Charakter theoretisches Interesse [1].

We measure homogeneous distances to M31 and 38 associated stellar systems ($-$16.8$\le M_V \le$ $-$6.0), using time-series observations of RR Lyrae stars taken as part of the Hubble Space Telescope Treasury Survey of M31 Satellites. From $>700$ orbits of new/archival ACS imaging, we identify $>4700$ RR Lyrae stars and determine their periods and mean magnitudes to a typical precision of 0.01 days and 0.04 mag. Based on Period-Wesenheit-Metallicity relationships consistent with the Gaia eDR3 distance scale, we uniformly measure heliocentric and M31-centric distances to a typical precision of $\sim20$ kpc (3%) and $\sim10$ kpc (8%), respectively. We revise the 3D structure of the M31 galactic ecosystem and: (i) confirm a highly anisotropic spatial distribution such that $\sim80$% of M31's satellites reside on the near side of M31; this feature is not easily explained by observational effects; (ii) affirm the thin (rms $7-23$ kpc) planar "arc" of satellites that comprises roughly half (15) of the galaxies within 300 kpc from M31; (iii) reassess physical proximity of notable associations such as the NGC 147/185 pair and M33/AND XXII; and (iv) illustrate challenges in tip-of-the-red-giant branch distances for galaxies with $M_V > -9.5$, which can be biased by up to 35%. We emphasize the importance of RR Lyrae for accurate distances to faint galaxies that should be discovered by upcoming facilities (e.g., Rubin Observatory). We provide updated luminosities and sizes for our sample. Our distances will serve as the basis for future investigation of the star formation and orbital histories of the entire known M31 satellite system., 32 pages, 15 figures, accepted for publication on ApJ

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a causative agent of chytridiomycosis, a skin disease associated with amphibian population declines around the world. Despite the major impact Bd is having on global ecosystems, much of Bd's basic biology remains unstudied. In addition to revealing mechanisms driving the spread of chytridiomycosis, studying Bd can shed light on the evolution of key fungal traits because chytrid fungi, including Bd, diverged before the radiation of the Dikaryotic fungi (multicellular fungi and yeast). Studying Bd in the laboratory is, therefore, of growing interest to a wide range of scientists, ranging from herpetologists and disease ecologists to molecular, cell, and evolutionary biologists. This protocol describes how to maintain developmentally synchronized liquid cultures of Bd for use in the laboratory, how to grow Bd on solid media, as well as cryopreservation and revival of frozen stocks. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Reviving cryopreserved Bd cultures Basic Protocol 2: Establishing synchronized liquid cultures of Bd Basic Protocol 3: Regular maintenance of synchronous Bd in liquid culture Alternate Protocol 1: Regular maintenance of asynchronous Bd in liquid culture Basic Protocol 4: Regular maintenance of synchronous Bd on solid medium Alternate Protocol 2: Starting a culture on solid medium from a liquid culture Basic Protocol 5: Cryopreservation of Bd.

Amoeboid cell types are fundamental to animal biology and broadly distributed across animal diversity, but their evolutionary origin is unclear. The closest living relatives of animals, the choanoflagellates, display a polarized cell architecture (with an apical flagellum encircled by microvilli) that resembles that of epithelial cells and suggests homology, but this architecture differs strikingly from the deformable phenotype of animal amoeboid cells, which instead evoke more distantly related eukaryotes, such as diverse amoebae. Here, we show that choanoflagellates subjected to confinement become amoeboid by retracting their flagella and activating myosin-based motility. This switch allows escape from confinement and is conserved across choanoflagellate diversity. The conservation of the amoeboid cell phenotype across animals and choanoflagellates, together with the conserved role of myosin, is consistent with homology of amoeboid motility in both lineages. We hypothesize that the differentiation between animal epithelial and crawling cells might have evolved from a stress-induced switch between flagellate and amoeboid forms in their single-celled ancestors.

The nuclear stellar disc (NSD) is a flattened high-density stellar structure that dominates the gravitational field of the Milky Way at Galactocentric radius 30pc≲R≲300pc⁠. We construct axisymmetric self-consistent equilibrium dynamical models of the NSD in which the distribution function is an analytic function of the action variables. We fit the models to the normalized kinematic distributions (line-of-sight velocities + VIRAC2 proper motions) of stars in the NSD survey of Fritz et al., taking the foreground contamination due to the Galactic Bar explicitly into account using an N-body model. The posterior marginalized probability distributions give a total mass of MNSD=10.5+1.1−1.0×108M⊙⁠, roughly exponential radial and vertical scale lengths of Rdisc=88.6+9.2−6.9pc and Hdisc=28.4+5.5−5.5pc⁠, respectively, and a velocity dispersion σ≃70kms−1 that decreases with radius. We find that the assumption that the NSD is axisymmetric provides a good representation of the data. We quantify contamination from the Galactic Bar in the sample, which is substantial in most observed fields. Our models provide the full 6D (position + velocity) distribution function of the NSD, which can be used to generate predictions for future surveys. We make the models publicly available as part of the software package AGAMA. © 2022 The Author(s) Published by Oxford University Press on behalf of Royal Astronomical Society., MCS acknowledges financial support from the European Research Council via the ERC Synergy Grant ‘ECOGAL – Understanding our Galactic ecosystem: from the disk of the Milky Way to the formation sites of stars and planets’ (grant 855130). JLS thanks the support of the Royal Society (URF\R1\191555). EV acknowledges support from STFC via the consolidated grant to the Institute of Astronomy. DM acknowledges support from project CATA FB210003. AMB acknowledges funding from the EU Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 895174. JM acknowledges support from the UK Science and Technology Facilities Council under grant number ST/S000488/I. MCS, NN, FN-L, and RSK gratefully acknowledge support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 138713538 - SFB 881 (‘The Milky Way System’, subprojects B1, B2, B8). FN-L acknowledges the sponsorship provided by the Federal Ministry for Education and Research of Germany through the Alexander von Humboldt Foundation. RS and BS acknowledge financial support from the State Agency for Research of the Spanish MCIU through the ‘Center of Excellence Severo Ochoa’ award for the Instituto de Astrofísica de Andalucía (SEV-2017-0709) and financial support from national project PGC2018-095049-B-C21 (MCIU/AEI/FEDER, UE). RSK acknowledges support from the Heidelberg cluster of excellence EXC 2181 (Project-ID 390900948) ‘STRUCTURES: A unifying approach to emergent phenomena in the physical world, mathematics, and complex data’ funded by the German Excellence Strategy. This work used computing resources provided by the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant INST 35/1134-1 FUGG. Data are stored at SDS@hd supported by the Ministry of Science, Research and the Arts Baden-Württemberg (MWK) and DFG through grant INST 35/1314-1 FUGG.

In hard‐winter wheat (Triticum aestivum L.) breeding, the evaluation of end‐use quality is expensive and time‐consuming, being relegated to the final stages of the breeding program after selection for many traits including disease resistance, agronomic performance, and grain yield. In this study, our objectives were to identify genetic variants underlying baking quality traits through genome‐wide association study (GWAS) and develop improved genomic selection (GS) models for the quality traits in hard‐winter wheat. Advanced breeding lines (n = 462) from 2015–2017 were genotyped using genotyping‐by‐sequencing (GBS) and evaluated for baking quality. Significant associations were detected for mixograph mixing time and bake mixing time, most of which were within or in tight linkage to glutenin and gliadin loci and could be suitable for marker‐assisted breeding. Candidate genes for newly associated loci are phosphate‐dependent decarboxylase and lipid transfer protein genes, which are believed to affect nitrogen metabolism and dough development, respectively. The use of GS can both shorten the breeding cycle time and significantly increase the number of lines that could be selected for quality traits, thus we evaluated various GS models for end‐use quality traits. As a baseline, univariate GS models had 0.25–0.55 prediction accuracy in cross‐validation and from 0 to 0.41 in forward prediction. By including secondary traits as additional predictor variables (univariate GS with covariates) or correlated response variables (multivariate GS), the prediction accuracies were increased relative to the univariate model using only genomic information. The improved genomic prediction models have great potential to further accelerate wheat breeding for end‐use quality.

Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed for expressing fluorescent protein fusions. Here, we describe a protocol for fixing, permeabilizing, and staining cells in a single step while imaging on a microscope. In contrast to traditional, multi-step fixing and staining protocols that take hours, the protocol outlined here achieves satisfactory staining within minutes. This approach takes advantage of well-characterized small molecules that stain specific subcellular structures, including nuclei, mitochondria, and actin networks. Direct visualization of the entire process allows for rapid optimization of cell fixation and staining, as well as straightforward identification of fixation artifacts. Moreover, live imaging prior to fixation reveals the dynamic history of cellular features, making it particularly useful for model systems without the capacity for expressing fluorescent protein fusions. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Fixing, permeabilizing, and staining mammalian cells in one step on the microscope.

SummaryBackgroundAdenoviral (Ad)-vectored vaccines are typically administered via intramuscular injection to humans, but this route of delivery is unable to induce respiratory mucosal immunity which requires respiratory mucosal route of vaccination. However, inhaled aerosol delivery of Ad-vectored vaccines has remained poorly characterized and its ability to induce respiratory mucosal immunity in humans is still unknown. The goal of our study was to evaluate and compare the safety and immunogenicity of a human serotype 5 Ad-based tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to healthy humans via inhaled aerosol or intramuscular injection.MethodsIn this open-labeled phase 1b trial, 31 healthy adults between 18 and 55 years of age with a history of BCG vaccination were enrolled at McMaster University Medical Centre, Hamilton, Ontario, Canada. AdHu5Ag85A was administered by a single-dose aerosol using the Aeroneb® Solo Vibrating Mesh Nebulizer or by intramuscular (IM) injection; 11 in the low dose (LD, 1×106 PFU) aerosol group, 11 in the high dose (HD, 2×106 PFU) aerosol group and 9 in the IM (1×108 PFU) group. The primary outcome was safety of a single administration of vaccine delivered to the respiratory tract by aerosol or by IM injection. The vaccine-related local and systemic adverse events were collected from participants from a self-completed diary for 14 days after vaccination and at scheduled follow-up visits. Routine laboratory biochemical and haematological tests were measured at 2, 4 and 12 weeks after vaccination and lung function was measured at 2, 4, 8 and 12 weeks after vaccination. The secondary outcome was comparison of immunogenicity among the different routes and aerosol dose groups. Immunogenicity to aerosol or IM vaccination was measured both in the peripheral blood and bronchoalveolar lavage samples by Luminex, and cell surface and intracellular cytokine immunostaining. Anti-AdHu5 antibodies and neutralization titers were determined before and after vaccination using ELISA and bioassay, respectively. This trial is registered with ClinicalTrial.gov, NCT02337270.ResultsThe aerosol droplets generated by Aeroneb® Solo Nebulizer were mostly InterpretationInhaled aerosol delivery of Ad-vectored vaccine is a safe, economical and superior way to elicit respiratory mucosal immunity. The results of this study encourage further development of aerosol vaccine strategies against not only TB but also other respiratory pathogens including COVID-19.FundingThe Canadian Institutes for Health Research and the Natural Sciences and Engineering Research Council of Canada.Research in contextEvidence before this studyWe searched PubMed for published research articles without language or date restrictions by using search terms “adenovirus”, “aerosol” and “clinical trial” or “virus”, “vaccine”, “aerosol”, and “clinical trial”. Our search results indicate that adenoviral-vectored vaccine has rarely been delivered via inhaled aerosol into the respiratory tract of healthy humans. Adenoviral-vectored TB or HIV vaccine was delivered via inhaled aerosol to non-human primates, providing support for its further development for human application. An aerosolized adenoviral vector expressing CFTR was tested as a gene replacement therapeutic in cystic fibrosis patients. Recently, although an adenoviral-vectored COVID-19 vaccine was delivered via aerosol to humans, there were no evidence presented that it induced local mucosal immunity. Given the increased recognition of its value, inhaled aerosol has been explored in humans with non-adenoviral, viral vaccines against respiratory infections of global importance including measles and TB. However, since different aerosol technologies were used in these studies and aerosol characteristics including delivery efficiency vary according to the viral platform, aerosol delivery technology and its efficiency in inducing respiratory mucosal immunity remain to be established for administering adenoviral-vectored vaccine to healthy humans.Added value of this studyThis represents the first study to demonstrate the characteristics of a single-dose aerosolized human serotype 5 adenoviral-vectored vaccine, and its safety and immunogenicity in BCG-vaccinated healthy humans. It is also the first to show the superiority of inhaled aerosol immunization with this type of vaccine platform, over intramuscular (IM) route of immunization, in inducing respiratory mucosal immunity. Robust adaptive immune memory responses were induced in the respiratory tract with an aerosol vaccine dose up to 100 times smaller than the dose for IM immunization. Besides local mucosal immunity induced by aerosol immunization, it also induces levels of systemic immunity comparable to those by IM immunization. Also for the first time, we show that contrast to IM immunization, aerosol immunization does not increase either local or systemic anti-adenoviral neutralizing antibodies.Implications of all the available evidenceCollectively our findings show the technical feasibility, safety and potency of needle/pain-free delivery of a recombinant adenoviral-vectored vaccine to the respiratory tract of healthy adults. This vaccine strategy differs from parenteral route of immunization in its potency to elicit much desired respiratory mucosal immunity consisting of trained macrophages and tissue-resident memory T cells. The study provides the proof of concept to endorse inhaled aerosol vaccine strategies against pulmonary TB. Of further importance, as a number of currently authorized COVID-19 vaccines are also adenoviral-vectored, our study offers important technological details and scientific rationale for the development of inhalable next-generation COVID-19 vaccines aiming to induce all-around protective respiratory mucosal immunity in humans.

The introgression from wild relatives have a great potential to broaden the availability of beneficial allelic diversity for crop improvement in breeding programs. Here, we assessed the impact of the introgression from 21 diverse accessions of Aegilops tauschii, the diploid ancestor of the wheat D genome, into 6 hard red winter wheat cultivars on yield and yield component traits. We used 5.2 million imputed D genome SNPs identified by the whole-genome sequencing of parental lines and the sequence-based genotyping of introgression population, including 351 BC1F3:5 lines. Phenotyping data collected from the irrigated and non-irrigated field trials revealed that up to 23% of the introgression lines (ILs) produce more grain than the parents and check cultivars. Based on 16 yield stability statistics, the yield of 12 ILs (3.4%) was stable across treatments, years, and locations; 5 of these lines were also high yielding lines, producing 9.8% more grain than the average yield of check cultivars. The most significant SNP- and haplotype-trait associations were identified on chromosome arms 2DS and 6DL for the spikelet number per spike (SNS), on chromosome arms 2DS, 3DS, 5DS, and 7DS for grain length (GL) and on chromosome arms 1DL, 2DS, 6DL, and 7DS for grain width (GW). The introgression of haplotypes from A. tauschii parents was associated with an increase in SNS, which was positively correlated with a heading date (HD), whereas the haplotypes from hexaploid wheat parents were associated with an increase in GW. We show that the haplotypes on 2DS associated with an increase in the spikelet number and HD are linked with multiple introgressed alleles of Ppd-D1 identified by the whole-genome sequencing of A. tauschii parents. Meanwhile, some introgressed haplotypes exhibited significant pleiotropic effects with the direction of effects on the yield component traits being largely consistent with the previously reported trade-offs, there were haplotype combinations associated with the positive trends in yield. The characterized repertoire of the introgressed haplotypes derived from A. tauschii accessions with the combined positive effects on yield and yield component traits in elite germplasm provides a valuable source of alleles for improving the productivity of winter wheat by optimizing the contribution of component traits to yield.

We present the color-magnitude diagrams and star formation histories (SFHs) of seven ultra-faint dwarf galaxies: Horologium 1, Hydra 2, Phoenix 2, Reticulum 2, Sagittarius 2, Triangulum 2, and Tucana 2, derived from high-precision Hubble Space Telescope photometry. We find that the SFH of each galaxy is consistent with them having created at least 80% of the stellar mass by $z\sim6$. For all galaxies, we find quenching times older than 11.5 Gyr ago, compatible with the scenario in which reionization suppresses the star formation of small dark matter halos. However, our analysis also reveals some differences in the SFHs of candidate Magellanic Cloud satellites, i.e., galaxies that are likely satellites of the Large Magellanic Cloud and that entered the Milky Way potential only recently. Indeed, Magellanic satellites show quenching times about 600 Myr more recent with respect to those of other Milky Way satellites, on average, even though the respective timings are still compatible within the errors. This finding is consistent with theoretical models that suggest that satellites' SFHs may depend on their host environment at early times, although we caution that within the error bars all galaxies in our sample are consistent with being quenched at a single epoch., 7 pages, 3 figures, 2 tables. Accepted for publication in ApJL

[Background] The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely., [Results] Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system., [Conclusions] In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen., EKH was supported by an AHFMR Fulltime Graduate Studentship and a Vanier Canada Graduate Scholarship. JBD is the Canada Research Chair in Evolutionary Cell Biology. Work in the Eliáš Lab is supported by CePaViP (CZ.02.1.01/0.0/0.0/16_019/0000759) and Czech Science Foundation project 18-18699S. This work was supported by CSIRO Land and Water (Australia).