Agrobacterium-mediated transfer of the Fusarium graminearum Tri6 gene into barley using mature seed-derived shoot tips as explants.

KEY MESSAGE: We transferred the Tri6 gene into the elite barley GemCraft via new transformation method through shoot organogenesis and identified the rearrangements of transgenes and phenotypic variations in the transgenic plants. Despite its agronomic and economic importance, barley transformation is still very challenging for many elite varieties. In this study, we used direct shoot organogenesis to transform the elite barley cultivar GemCraft with the RNAi constructs containing Tri6 gene of Fusarium graminearum, which causes fusarium head blight (FHB). We isolated 4432 shoot tips and co-cultured these explants with Agrobacterium tumefaciens. A total of 25 independent T0 transgenic plants were generated including 15 events for which transgene-specific PCR amplicons were observed. To further determine the presence of transgenes, the T1 progenies of all 15 T0 plants were analyzed, and the expected PCR products were obtained in 10 T1 lines. Droplet digital (dd) PCR analysis revealed various copy numbers of transgenes in the transgenic plants. We determined the insertion site of transgenes using long-read sequencing data and observed the rearrangements of transgenes. We found phenotypic variations in both T1 and T2 generation plants. FHB disease was evaluated under growth chamber conditions, but no significant differences in disease severity or deoxynivalenol accumulation were observed between two Tri6 transgenic lines and the wildtype. Our results demonstrate the feasibility of the shoot tip transformation and may open the door for applying this system for genetic improvement and gene function research in other barley genotypes.

Incidence and Predictors of Subsidence Using Modular, Tapered, Fluted Titanium Femoral Stems in Aseptic Revision Total Hip Arthroplasty.

BACKGROUND: Tapered, fluted titanium (TFT) femoral stems have become the gold standard in revision total hip arthroplasty (rTHA). However, there is a paucity of data on TFT stem subsidence rates following aseptic rTHA. Subsidence can lead to instability, mechanical failure, leg-length discrepancy, and may require revision surgery. This study evaluated the incidences and predictors of TFT subsidence in aseptic rTHA. METHODS: A total of 102 TFT femoral stems of 4 designs were retrospectively reviewed. Stem subsidence was measured on digital radiographs taken immediately after surgery and at standard clinical follow-up. Patient characteristics, risk factors for subsidence, revision etiologies, and implant characteristics were recorded. Patient-reported outcome measures were also evaluated for a subset of cases. RESULTS: Overall, 12% of stems subsided >1 cm, and subsidence was minimal (<3 mm) in ≥64% of cases. From immediate postoperative to 1-month radiographic follow-up, 79% of stems subsided a mean of 2.9 mm (range, 0.1 to 12 mm). Beyond 1 month, subsidence was minimal for ≥77% of cases. In multivariate analyses, women and less femoral implant canal fill were associated with greater subsidence (P ≤ .034). The TFT stem design was not associated with early subsidence (P = .816). There were no modular junction fractures. There were 2 fractures and 2 subsidence-related revisions for aseptic loosening that occurred postoperatively. CONCLUSIONS: The amount of subsidence in TFT stems was low and was detectable in the early (less than 1 year) postoperative period. Maximizing TFT stem fill within the femoral canal appears to reduce the risk of subsidence without increasing femoral fracture rates and should be the goal with implantation of these devices. LEVEL OF EVIDENCE: IV-Case Series, No Control Group.

An outbreak of SARS-CoV-2 with high mortality in mink (Neovison vison) on multiple Utah farms.

The breadth of animal hosts that are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may serve as reservoirs for continued viral transmission are not known entirely. In August 2020, an outbreak of SARS-CoV-2 occurred on five mink farms in Utah and was associated with high mink mortality (35-55% of adult mink) and rapid viral transmission between animals. The premise and clinical disease information, pathology, molecular characterization, and tissue distribution of virus within infected mink during the early phase of the outbreak are provided. Infection spread rapidly between independently housed animals and farms, and caused severe respiratory disease and death. Disease indicators were most notably sudden death, anorexia, and increased respiratory effort. Gross pathology examination revealed severe pulmonary congestion and edema. Microscopically there was pulmonary edema with moderate vasculitis, perivasculitis, and fibrinous interstitial pneumonia. Reverse transcriptase polymerase chain reaction (RT-PCR) of tissues collected at necropsy demonstrated the presence of SARS-CoV-2 viral RNA in multiple organs including nasal turbinates, lung, tracheobronchial lymph node, epithelial surfaces, and others. Localization of viral RNA by in situ hybridization revealed a more localized infection, particularly of the upper respiratory tract. Whole genome sequencing from multiple mink was consistent with published SARS-CoV-2 genomes with few polymorphisms. The Utah mink SARS-CoV-2 strains fell into Clade GH, which is unique among mink and other animal strains sequenced to date. While sharing the N501T mutation which is common in mink, the Utah strains did not share other spike RBD mutations Y453F and F486L found in nearly all mink from the United States. Mink in the outbreak reported herein had high levels of SARS-CoV-2 in the upper respiratory tract associated with symptomatic respiratory disease and death.

Genetic and physical localization of a major susceptibility gene to Pyrenophora teres f. maculata in barley.

KEY MESSAGE: Genetic characterization of a major spot form net blotch susceptibility locus to using linkage mapping to identify a candidate gene and user-friendly markers in barley. Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm), is an economically important foliar diseases in barley. Although various resistance loci have been identified, breeding for SFNB-resistant varieties has been hampered due to the complex virulence profile of Ptm populations. One resistance locus in the host may be effective against one specific isolate, but it may confer susceptibility to other isolates. A major susceptibility QTL on chromosome 7H, named Sptm1, was consistently identified in many studies. In the present study, we conduct fine mapping to localize Sptm1 with high resolution. A segregating population was developed from selected F2 progenies of the cross Tradition (S) × PI 67381 (R), in which the disease phenotype was determined by the Sptm1 locus alone. Disease phenotypes of critical recombinants were confirmed in the following two consecutive generations. Genetic mapping anchored the Sptm1 gene to an ⁓400 kb region on chromosome 7H. Gene prediction and annotation identified six protein-coding genes in the delimited Sptm1 region, and the gene encoding a putative cold-responsive protein kinase was selected as a strong candidate. Therefore, providing fine localization and candidate of Sptm1 for functional validation, our study will facilitate the understanding of susceptibility mechanism underlying the barley-Ptm interaction and offers a potential target for gene editing to develop valuable materials with broad-spectrum resistance to SFNB.

DNA Markers, Pathogenicity Test, and Multilocus Sequence Analysis to Differentiate and Characterize Cereal-Specific Xanthomonas translucens Strains.

Xanthomonas translucens contains a group of bacterial pathogens that are closely related and have been divided into several pathovars based on their host range. X. translucens pv. undulosa (Xtu) and X. translucens pv. translucens (Xtt) are two important pathovars that cause bacterial leaf streak disease on wheat and barley, respectively. In this study, DNA markers were developed to differentiate Xtu and Xtt and were then used to characterize a collection of X. translucens strains with diverse origins, followed by confirmation and characterization with pathogenicity tests and multilocus sequence analysis/typing (MLSA/MLST). We first developed cleaved amplified polymorphic sequence markers based on the single-nucleotide polymorphisms within a cereal pathovar-specific DNA sequence. In addition, two Xtt-specific markers, designated Xtt-XopM and Xtt-SP1, were developed from comparative genomics among the sequenced Xtt/Xtu genomes. Using the developed markers, a collection of X. translucens strains were successfully identified as Xtu or Xtt. Pathogenicity tests on wheat and barley plants and MLSA of four housekeeping genes validated the pathovar assignation of those strains. Furthermore, MLSA revealed distinct subclades within both Xtu and Xtt groups. Seven and three sequence types were identified from MLST for Xtu and Xtt strains, respectively. The establishment of efficient Xtt/Xtu differentiation methods and characterization of those strains will be useful in studying disease epidemiology and host-pathogen interactions and breeding programs when screening for sources of resistance for these two important bacterial pathogens.

Evaluation of sutureless anastomosis after ileostomy takedown using the self-forming magnet anastomosis system in a porcine model.

BACKGROUND: Current standard of care for creation of small bowel anastomoses after a loop ileostomy reversal includes the use of stapler devices and sutures. Compression anastomosis devices have been used for decades, aimed toward improved outcomes with a "staple free" & "suture free" anastomosis. The self-forming magnet (SFM) device is a type of compression anastomosis device used to safely and effectively create an end-to-end small bowel anastomosis without the localized inflammatory response seen with sutures or staples, as no foreign bodies are left behind. METHODS: A Good Laboratory Practice preclinical study using a porcine model to evaluate creating an in vivo anastomosis via magnetic compression between two segments of small bowel (jejunum or ileum) was performed. Magnetic anastomoses were compared to stapled and handsewn anastomoses. Six animals were used for the magnetic anastomosis and eight for the two control groups for a total of 14 subjects. RESULTS: Mean creation times were 17.1 min (SD 6.06) for the SFM group, 10.3 min (SD 6.55, CI 95%) for the stapled anastomosis group, and 28.3 min (SD 2.63, CI 95%) for the suture anastomosis group, with a statistically significant difference among groups (p < 0.0021). All evaluated SFM anastomosis, stapled anastomosis, and handsewn anastomosis underwent a burst test with a pressure of 1.3 PSI. All six magnets used for anastomoses were naturally expelled. The range of days to expel magnets was 10-17 days. Intestinal anastomoses using magnets had considerably less residual scarring and intestinal distortion than anastomoses done with either suture or staples. CONCLUSION: This preclinical study documents the safety and efficacy of creating end-to-end small bowel anastomoses after ileostomy takedown using a magnetic compression device. The result is an anastomosis free of foreign objects with less inflammation, scarring, distortion, and mural thickening than seen in sutured or stapled anastomoses.

Why Do Plant-Pathogenic Fungi Produce Mycotoxins? Potential Roles for Mycotoxins in the Plant Ecosystem.

For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host-pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and genders to discuss the diverse roles of mycotoxins in the lifestyles of filamentous fungi of the families Clavicipitaceae, Trichocomaceae (Eurotiales), and Nectriaceae (Hypocreales).

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species.

Protecting a solid-state spin from decoherence using dressed spin states.

We report experimental studies of dressing an electron spin in diamond with resonant and continuous microwave fields to protect the electron spin from magnetic fluctuations induced by the nuclear spin bath. We use optical coherent population trapping (CPT) to probe the energy level structure, optically induced spin transitions, and spin decoherence rates of the dressed spin states. Dressing an electron spin with resonant microwaves at a coupling rate near 1 MHz leads to a 50 times reduction in the linewidth of the spin transition underlying the CPT process, limited by transit-time broadening. Compared with dynamical decoupling, where effects of the bath are averaged out at specific times, the dressed spin state provides a continuous protection from decoherence.

Japanese Yew (Taxus) poisoning of wild ungulates in Utah during the winter of 2022-2023.

Taxus is a genus of coniferous shrubs and trees, commonly known as the yews, in the family Taxaceae. All species of yew contain taxine alkaloids, which are ascribed as the toxic principles. Anecdotally, free ranging ruminants such as antelope, deer, elk, and moose have been regarded as tolerant to yew. Herein several cases of intoxication of deer, elk, and moose by yew from the state of Utah in the winter of 2022-2023 are documented. Ingestion of yew was documented by three means among the poisoned cervids; plant fragments consistent with yew were visually observed in the rumen contents, chemical analysis, and subsequent detection of the taxines from rumen and liver contents, and identification of exact sequence variants identified as Taxus species from DNA metabarcoding. Undoubtedly, the record snowfall in Utah during the winter of 2022-2023 contributed to these poisonings.

Fungal biotechnology: From yesterday to tomorrow.

Fungi have been used to better the lives of everyday people and unravel the mysteries of higher eukaryotic organisms for decades. However, comparing progress and development stemming from fungal research to that of human, plant, and bacterial research, fungi remain largely understudied and underutilized. Recent commercial ventures have begun to gain popularity in society, providing a new surge of interest in fungi, mycelia, and potential new applications of these organisms to various aspects of research. Biotechnological advancements in fungal research cannot occur without intensive amounts of time, investments, and research tool development. In this review, we highlight past breakthroughs in fungal biotechnology, discuss requirements to advance fungal biotechnology even further, and touch on the horizon of new breakthroughs with the highest potential to positively impact both research and society.

Comparative early histologic healing quality of magnetic versus stapled small bowel anastomosis.

BACKGROUND: Successful anastomotic healing is critical to preventing complications after intestinal surgery. We aimed to compare the early healing of end-to-end small bowel anastomosis by self-forming magnets with surgical stapling in a porcine model. METHOD: Six Yorkshire pigs underwent 2 simultaneous small bowel anastomoses using a circular stapler and self-forming magnet technique. The primary outcome was healing quality, measured by 4 histologic features: inflammatory cell infiltration, collagen formation, grade of inflammation, and bacterial infiltration at the anastomosis. The samples were evaluated at days 1, 3, and 7. Gross evaluation of anastomotic integrity was a secondary outcome. RESULTS: The self-forming magnet group displayed significant differences at each time point. On day 1, the stapled group displayed dense inflammatory cell infiltration and extensively ulcerated intestinal layers with significant edema. The self-forming magnet group showed less inflammatory infiltrate, and all intestinal layers remained compressed in direct apposition. By day 3, the self-forming magnet group already exhibited neovascularization with scant bacterial colonies. By contrast, stapled anastomoses had large areas of inflammation separating collagen fibers with prevalent bacterial infiltrations. On day 7, self-forming magnet anastomoses were characterized by robust neovascularization, maturing granulation tissue, and mucosal re-epithelization without significant inflammation. Meanwhile, stapled samples had persisting dense inflammation, tissue cavities with hemorrhage, and immature fibrous tissue. Grossly, the self-forming magnet created a patent lumen without defect, whereas stapled anastomoses demonstrated focal areas of serosal separation. CONCLUSION: Bowel anastomosis by self-forming magnets is associated with superior early histologic healing metrics, including early seal generation through mechanical compression, decreased inflammation, early neovascularization, lower bacterial infiltration, and faster re-epithelization.

Public knowledge of the symptoms of myocardial infarction: a street survey in Birmingham, England.

BACKGROUND: Myocardial infarction (MI) is a leading cause of death in the UK. A good clinical outcome depends on rapid treatment following the onset of symptoms. A person's knowledge of typical symptoms determines how quickly they present to the medical services. OBJECTIVES: To investigate knowledge of MI symptoms among the general population and the relationship between age, gender and socio-economic status with knowledge. METHODS: Street survey of 302 participants in Birmingham, UK, using an interviewer-assisted questionnaire. RESULTS: Of seven symptoms accepted in the medical literature as typical of an MI, central chest pain was the most frequently identified (75% of the sample), followed by arm pain or numbness (40%), shortness of breath (35%), fainting or dizziness (21%) and sweating (21%). Feeling or being sick and neck or jaw pain were mentioned by 8.1% and 5.9%, respectively, while an atypical or inapplicable symptom, collapse (9.9%) was mentioned more often than these. Over half the sample knew only two or fewer MI symptoms. The mean number of typical symptoms identified was 2.2 (SD = 1.28). Respondents from professional occupations and those with previous experience of MI, whether direct or indirect, showed better awareness. CONCLUSIONS: The study demonstrated a paucity of knowledge of MI symptoms among the general public. Such findings provide a baseline to guide public health campaigns targeting awareness of MI.

Complete Genomes of Two Xanthomonas translucens pv. translucens Strains Isolated from Barley in North Dakota.

Xanthomonas translucens causes the disease bacterial leaf streak in several cereal crops and grasses. Here, we report the complete genome sequences of two isolates of X. translucens pv. translucens that were isolated from barley in an important cereal crop production region.

Detection of asinine gammaherpesviruses in association with pulmonary fibrosis in free-ranging donkeys.

A mortality event among recently captured feral donkeys (Equus asinus) occurred in south-central Utah in 2016. The deaths were sporadic, and clinical signs were indicative of respiratory disease, likely associated with an infectious etiology. Ten of 13 donkeys autopsied had moderate-to-severe interstitial fibrosing pneumonia, and one had pyogranulomatous pneumonia. Consensus PCRs directed toward the DNA polymerase and DNA packaging terminase subunit 1 for herpesviruses were performed followed by sequencing of the PCR amplicons and phylogenetic analysis. Asinine herpesvirus 4 (AsHV4) and 5 (AsHV5) were consistently identified in lung tissues of affected donkeys. No other herpesviruses were identified, and herpesviral DNA was not detected in lung tissues of 2 donkeys without evidence of respiratory disease. The detection of asinine gammaherpesviruses may have been associated with the lesions described. AsHV4 and AsHV5 have been reported in previous studies as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia and marked syncytial cell formation. Our findings suggest that the association of asinine gammaherpesviruses with respiratory conditions in equids deserves further attention.

OmniMapFree: a unified tool to visualise and explore sequenced genomes.

BACKGROUND: Acquiring and exploring whole genome sequence information for a species under investigation is now a routine experimental approach. On most genome browsers, typically, only the DNA sequence, EST support, motif search results, and GO annotations are displayed. However, for many species, a growing volume of additional experimental information is available but this is rarely searchable within the landscape of the entire genome. • RESULTS: We have developed a generic software which permits users to view a single genome in entirety either within its chromosome or supercontig context within a single window. This software permits the genome to be displayed at any scales and with any features. Different data types and data sets are displayed onto the genome, which have been acquired from other types of studies including classical genetics, forward and reverse genetics, transcriptomics, proteomics and improved annotation from alternative sources. In each display, different types of information can be overlapped, then retrieved in the desired combinations and scales and used in follow up analyses. The displays generated are of publication quality. • CONCLUSIONS: OmniMapFree provides a unified, versatile and easy-to-use software tool for studying a single genome in association with all the other datasets and data types available for the organism.

An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations.

In barley, the eukaryotic translation initiation factor 4E (eIF4E) gene situated on chromosome 3H is recognized as an important source of resistance to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In modern barley cultivars, two recessive eIF4E alleles, rym4 and rym5, confer different isolate-specific resistances. In this study, the sequence of eIF4E was analysed in 1090 barley landraces and noncurrent cultivars originating from 84 countries. An exceptionally high nucleotide diversity was evident in the coding sequence of eIF4E but not in either the adjacent MCT-1 gene or the sequence-related eIF(iso)4E gene situated on chromosome 1H. Surprisingly, all nucleotide polymorphisms detected in the coding sequence of eIF4E resulted in amino acid changes. A total of 47 eIF4E haplotypes were identified, and phylogenetic analysis using maximum likelihood provided evidence of strong positive selection acting on this barley gene. The majority of eIF4E haplotypes were found to be specific to distinct geographic regions. Furthermore, the eI4FE haplotype diversity (uh) was found to be considerably higher in East Asia, whereas SNP genotyping identified a comparatively low degree of genome-wide genetic diversity in 16 of 17 tested accessions (each carrying a different eIF4E haplotype) from this same region. In addition, selection statistic calculations using coalescent simulations showed evidence of non-neutral variation for eIF4E in several geographic regions, including East Asia, the region with a long history of the bymovirus-induced yellow mosaic disease. Together these findings suggest that eIF4E may play a role in barley adaptation to local habitats.

Analysis of genet size and local gene flow in the ectomycorrhizal basidiomycete Suillus spraguei (synonym S. pictus).

Several recent fine-scale genetic structure studies of ectomycorrhizal fungi have reported significant spatial clustering of genets with similar genotypes, supporting locally restricted gene flow. In this study we used genotype data from microsatellite markers and spatial autocorrelation analysis to examine local gene flow in Suillus spraguei at distances up to 2 km. Previously developed microsatellite markers for S. spraguei from Japan were unsuccessful at amplifying DNA isolated from sporocarps found in New York state, and other research suggested that both are disjunct species. Novel microsatellite markers therefore were developed with New York specimens. We identified nine polymorphic microsatellite loci and developed primer sets to amplify these regions. We tested the efficiency of the primer sets on 50 sporocarps collected from a natural Pinus strobus stand. The majority of the markers were in Hardy-Weinberg and linkage equilibrium. The location of all sampled sporocarps was recorded and used along with multilocus genotype data to create a genet map. The distance between sporocarps with the same multilocus genotype was small (≤ 7.65 m) and the majority of sporocarps collected were genetically unique, suggesting frequent spore establishment and sexual recombination on this site. Spatial autocorrelation analysis did not support clustering of similar genotypes, suggesting few restrictions to gene flow within this local population.