A systems genetics approach to deciphering the effect of dosage variation on leaf morphology in Populus.

Gene copy number variation is frequent in plant genomes of various species, but the impact of such gene dosage variation on morphological traits is poorly understood. We used a large population of Populus carrying genomically characterized insertions and deletions across the genome to systematically assay the effect of gene dosage variation on a suite of leaf morphology traits. A systems genetics approach was used to integrate insertion and deletion locations, leaf morphology phenotypes, gene expression, and transcriptional network data, to provide an overview of how gene dosage influences morphology. Dosage-sensitive genomic regions were identified that influenced individual or pleiotropic morphological traits. We also identified cis-expression quantitative trait loci (QTL) within these dosage QTL regions, a subset of which modulated trans-expression QTL as well. Integration of data types within a gene co-expression framework identified co-expressed gene modules that are dosage sensitive, enriched for dosage expression QTL, and associated with morphological traits. Functional description of these modules linked dosage-sensitive morphological variation to specific cellular processes, as well as candidate regulatory genes. Together, these results show that gene dosage variation can influence morphological variation through complex changes in gene expression, and suggest that frequently occurring gene dosage variation has the potential to likewise influence quantitative traits in nature.

Epidemiology of Invasive Candidiasis in Patients with Hematologic Malignancy on Antifungal Prophylaxis.

The landscape of invasive Candida infections in patients with hematologic malignancy has evolved due to the adoption of anti-fungal prophylaxis, advances in oncological therapies, and developments in antifungal therapies and diagnostics. Despite these scientific gains, the morbidity and mortality caused by these infections remain unchanged, highlighting the importance of an updated understanding of its epidemiology. Non-albicans Candida species are now the predominant cause of invasive candidiasis in patients with hematological malignancy. This epidemiological shift from Candida albicans to non-albicans Candida species is partially a consequence of selective pressure from extensive azole use. Further analysis of this trend suggests other contributing factors including immunocompromise caused by the underlying hematologic malignancy and the intensity of its associated treatments, oncological practices, and regional or institution specific variables. This review characterizes the changing distribution of Candida species in patients with hematologic malignancy, describes the causes driving this change, and discusses clinical considerations to optimize management in this high-risk patient population.

A comprehensive genomic scan reveals gene dosage balance impacts on quantitative traits in Populus trees.

Gene dosage variation and the associated changes in gene expression influence a wide variety of traits, ranging from cancer in humans to yield in plants. It is also expected to affect important traits of ecological and agronomic importance in forest trees, but this variation has not been systematically characterized or exploited. Here we performed a comprehensive scan of the Populus genome for dosage-sensitive loci affecting quantitative trait variation for spring and fall phenology and biomass production. The study population was a large collection of clonally propagated F1 hybrid lines of Populus that saturate the genome 10-fold with deletions and insertions (indels) of known sizes and positions. As a group, the phenotypic means of the indel lines consistently differed from control nonindel lines, with an overall negative effect of both insertions and deletions on all biomass-related traits but more diverse effects and an overall wider phenotypic distribution of the indel lines for the phenology-related traits. We also investigated the correlation between gene dosage at specific chromosomal locations and phenotype, to identify dosage quantitative trait loci (dQTL). Such dQTL were detected for most phenotypes examined, but stronger effect dQTL were identified for the phenology-related traits than for the biomass traits. Our genome-wide screen for dosage sensitivity in a higher eukaryote demonstrates the importance of global genomic balance and the impact of dosage on life history traits.

Genome-wide association study for salinity tolerance at the flowering stage in a panel of rice accessions from Thailand.

BACKGROUND: Salt stress, a major plant environmental stress, is a critical constraint for rice productivity. Dissecting the genetic loci controlling salt tolerance in rice for improving productivity, especially at the flowering stage, remains challenging. Here, we conducted a genome-wide association study (GWAS) of salt tolerance based on exome sequencing of the Thai rice accessions. RESULTS: Photosynthetic parameters and cell membrane stability under salt stress at the flowering stage; and yield-related traits of 104 Thai rice (Oryza sativa L.) accessions belonging to the indica subspecies were evaluated. The rice accessions were subjected to exome sequencing, resulting in 112,565 single nucleotide polymorphisms (SNPs) called with a minor allele frequency of at least 5%. LD decay analysis of the panel indicates that the average LD for SNPs at 20 kb distance from each other was 0.34 (r2), which decayed to its half value (~ 0.17) at around 80 kb. By GWAS performed using mixed linear model, two hundred loci containing 448 SNPs on exons were identified based on the salt susceptibility index of the net photosynthetic rate at day 6 after salt stress; and the number of panicles, filled grains and unfilled grains per plant. One hundred and forty six genes, which accounted for 73% of the identified loci, co-localized with the previously reported salt quantitative trait loci (QTLs). The top four regions that contained a high number of significant SNPs were found on chromosome 8, 12, 1 and 2. While many are novel, their annotation is consistent with potential involvement in plant salt tolerance and in related agronomic traits. These significant SNPs greatly help narrow down the region within these QTLs where the likely underlying candidate genes can be identified. CONCLUSIONS: Insight into the contribution of potential genes controlling salt tolerance from this GWAS provides further understanding of salt tolerance mechanisms of rice at the flowering stage, which can help improve yield productivity under salinity via gene cloning and genomic selection.

Production of a high-efficiency TILLING population through polyploidization.

Targeting Induced Local Lesions in Genomes (TILLING) provides a nontransgenic method for reverse genetics that is widely applicable, even in species where other functional resources are missing or expensive to build. The efficiency of TILLING, however, is greatly facilitated by high mutation density. Species vary in the number of mutations induced by comparable mutagenic treatments, suggesting that genetic background may affect the response. Allopolyploid species have often yielded higher mutation density than diploids. To examine the effect of ploidy, we autotetraploidized the Arabidopsis (Arabidopsis thaliana) ecotype Columbia, whose diploid has been used for TILLING extensively, and mutagenized it with 50 mm ethylmethane sulfonate. While the same treatment sterilized diploid Columbia, the tetraploid M1 plants produced good seed. To determine the mutation density, we searched 528 individuals for induced mutations in 15 genes for which few or no knockout alleles were previously available. We constructed tridimensional pools from the genomic DNA of M2 plants, amplified target DNA, and subjected them to Illumina sequencing. The results were analyzed with an improved version of the mutation detection software CAMBa that accepts any pooling scheme. This small population provided a rich resource with approximately 25 mutations per queried 1.5-kb fragment, including on average four severe missense and 1.3 truncation mutations. The overall mutation density of 19.4 mutations Mb(-1) is 4 times that achieved in the corresponding diploid accession, indicating that genomic redundancy engenders tolerance to high mutation density. Polyploidization of diploids will allow the production of small populations, such as less than 2,000, that provide allelic series from knockout to mild loss of function for virtually all genes.

Bring it on again: antimicrobial stewardship in transplant infectious diseases: updates and new challenges.

Advancement in solid organ transplantation and hematopoietic stem cell transplant continues to improve the health outcomes of patients and widens the number of eligible patients who can benefit from the medical progress. Preserving the effectiveness of antimicrobials remains crucial, as otherwise transplant surgeries would be unsafe due to surgical site infections, and the risk of sepsis with neutropenia would preclude stem cell transplant. In this review, we provide updates on three previously discussed stewardship challenges: febrile neutropenia, Clostridioides difficile infection, and asymptomatic bacteriuria. We also offer insight into four new stewardship challenges: the applicability of the "shorter is better" paradigm shift to antimicrobial duration; antibiotic allergy delabeling and desensitization; colonization with multidrug-resistant gram-negative organisms; and management of cytomegalovirus infections. Specifically, data are accumulating for "shorter is better" and antibiotic allergy delabeling in transplant patients, following successes in the general population. Unique to transplant patients are the impact of multidrug-resistant organism colonization on clinical decision-making of antibiotic prophylaxis in transplant procedure and the need for antiviral stewardship in cytomegalovirus. We highlighted the expansion of antimicrobial stewardship interventions as potential solutions for these challenges, as well as gaps in knowledge and opportunities for further research.

The Arabidopsis Hop1 homolog ASY1 mediates cross-over assurance and interference.

The chromosome axis plays a crucial role in meiotic recombination. Here, we study the function of ASY1, the Arabidopsis homolog of the yeast chromosome axis-associated component Hop1. Specifically, we characterized cross-over (CO) distribution in female and male meiosis by deep sequencing of the progeny of an allelic series of asy1 mutants. Combining data from nearly 1,000 individual plants, we find that reduced ASY1 functionality leads to genomic instability and sometimes drastic genomic rearrangements. We further observed that COs are less frequent and appear in more distal chromosomal regions in plants with no or reduced ASY1 functionality, consistent with previous analyses. However, our sequencing approach revealed that the reduction in CO number is not as dramatic as suggested by cytological analyses. Analysis of double mutants of asy1 with mutants with three other CO factors, MUS81, MSH4, and MSH5, as well as the determination of foci number of the CO regulator MLH1 demonstrates that the majority of the COs in asy1, similar to the situation in the wildtype (WT), largely belong to the class I, which are subject to interference. However, these COs are redistributed in asy1 mutants and typically appear much closer than in the WT. Hence, ASY1 plays a key role in CO interference that spaces COs along a chromosome. Conversely, since a large proportion of chromosomes do not receive any CO, we conclude that CO assurance, the process that ensures the obligatory assignment of one CO per chromosome, is also affected in asy1 mutants.

Transplantid.net: A Pilot Crowdsourced, Living, Online Library of Resources for the Teaching and Practice of Transplant Infectious Diseases.

The field of transplant infectious diseases is rapidly evolving, presenting a challenge for clinical practice and trainee education. Here we describe the construction of transplantid.net, a free online library, crowdsourced and continuously updated for the dual purpose of point-of-care evidence-based management and teaching.

Discovery of rare mutations in populations: TILLING by sequencing.

Discovery of rare mutations in populations requires methods, such as TILLING (for Targeting Induced Local Lesions in Genomes), for processing and analyzing many individuals in parallel. Previous TILLING protocols employed enzymatic or physical discrimination of heteroduplexed from homoduplexed target DNA. Using mutant populations of rice (Oryza sativa) and wheat (Triticum durum), we developed a method based on Illumina sequencing of target genes amplified from multidimensionally pooled templates representing 768 individuals per experiment. Parallel processing of sequencing libraries was aided by unique tracer sequences and barcodes allowing flexibility in the number and pooling arrangement of targeted genes, species, and pooling scheme. Sequencing reads were processed and aligned to the reference to identify possible single-nucleotide changes, which were then evaluated for frequency, sequencing quality, intersection pattern in pools, and statistical relevance to produce a Bayesian score with an associated confidence threshold. Discovery was robust both in rice and wheat using either bidimensional or tridimensional pooling schemes. The method compared favorably with other molecular and computational approaches, providing high sensitivity and specificity.

Bring it on: Top five antimicrobial stewardship challenges in transplant infectious diseases and practical strategies to address them.

Antimicrobial therapies are essential tools for transplant recipients who are at high risk for infectious complications. However, judicious use of antimicrobials is critical to preventing the development of antimicrobial resistance. Treatment of multidrug-resistant organisms is challenging and potentially leads to therapies with higher toxicities, intravenous access, and intensive drug monitoring for interactions. Antimicrobial stewardship programs are crucial in the prevention of antimicrobial resistance, though balancing these strategies with the need for early and frequent antibiotic therapy in these immunocompromised patients can be challenging. In this review, we summarize 5 frequently encountered transplant infectious disease stewardship challenges, and we suggest strategies to improve practices for each clinical syndrome. These 5 challenging areas are: asymptomatic bacteriuria in kidney transplant recipients, febrile neutropenia in hematopoietic stem cell transplantation, antifungal prophylaxis in liver and lung transplantation, treatment of left-ventricular assist device infections, and Clostridioides difficile infection in solid-organ and hematopoietic stem-cell transplant recipients. Common themes contributing to these challenges include limited data specific to transplant patients, shortcomings in diagnostic testing, and uncertainties in pharmacotherapy.

Norovirus disease among older adults.

Norovirus, a leading cause of gastroenteritis outbreaks worldwide, results in substantial direct and indirect healthcare costs. Adults older than 65 years of age bear a significant proportion of the disease burden, and the disease course in this population is often more severe and protracted. In this narrative review, we discuss the epidemiology of norovirus infection, mechanisms of pathogenesis, and transmission pertinent to outbreaks along with infection prevention and control efforts. We also describe the clinical manifestations of norovirus disease with a focus on individuals older than 65 years of age, diagnosis and available treatment options, and the challenges and progress within vaccine development.

Identification of Endogenous Peptides in Nasal Swab Transport Media used in MALDI-TOF-MS Based COVID-19 Screening.

Mass spectrometry (MS) based diagnostic detection of 2019 novel coronavirus infectious disease (COVID-19) has been postulated to be a useful alternative to classical PCR based diagnostics. These MS based approaches have the potential to be both rapid and sensitive and can be done on-site without requiring a dedicated laboratory or depending on constrained supply chains (i.e., reagents and consumables). Matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS has a long and established history of microorganism detection and systemic disease assessment. Previously, we have shown that automated machine learning (ML) enhanced MALDI-TOF-MS screening of nasal swabs can be both sensitive and specific for COVID-19 detection. The underlying molecules responsible for this detection are generally unknown nor are they required for this automated ML platform to detect COVID-19. However, the identification of these molecules is important for understanding both the mechanism of detection and potentially the biology of the underlying infection. Here, we used nanoscale liquid chromatography tandem MS to identify endogenous peptides found in nasal swab saline transport media to identify peptides in the same the mass over charge (m/z) values observed by the MALDI-TOF-MS method. With our peptidomics workflow, we demonstrate that we can identify endogenous peptides and endogenous protease cut sites. Further, we show that SARS-CoV-2 viral peptides were not readily detected and are highly unlikely to be responsible for the accuracy of MALDI based SARS-CoV-2 diagnostics. Further analysis with more samples will be needed to validate our findings, but the methodology proves to be promising.

Navigating Human Immunodeficiency Virus Screening Recommendations for People on Pre-Exposure Prophylaxis and the Need to Update Testing Algorithms.

Incident HIV infections occurring in people on PrEP may have delayed seroconversion. New CDC guidelines recommend the addition of HIV-1 viral load for screening for all on PrEP. We believe antigen/antibody screening should continue for tenofovir-based PrEP at this time.

Efficient construction of a linkage map and haplotypes for Mentha suaveolens using sequence capture.

The sustainability of many crops is hindered by the lack of genomic resources and a poor understanding of natural genetic diversity. Particularly, application of modern breeding requires high-density linkage maps that are integrated into a highly contiguous reference genome. Here, we present a rapid method for deriving haplotypes and developing linkage maps, and its application to Mentha suaveolens, one of the diploid progenitors of cultivated mints. Using sequence-capture via DNA hybridization to target single nucleotide polymorphisms (SNPs), we successfully genotyped ∼5000 SNPs within the genome of >400 individuals derived from a self cross. After stringent quality control, and identification of nonredundant SNPs, 1919 informative SNPs were retained for linkage map construction. The resulting linkage map defined a total genetic space of 942.17 cM divided among 12 linkage groups, ranging from 56.32 to 122.61 cM in length. The linkage map is in good agreement with pseudomolecules from our preliminary genome assembly, proving this resource effective for the correction and validation of the reference genome. We discuss the advantages of this method for the rapid creation of linkage maps.

Diploid mint (M. longifolia) can produce spearmint type oil with a high yield potential.

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native (Mentha Spicata L) and Scotch (M. × gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (-)-carvone and (-)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F2 population (32/160) produced from these two accessions. Field-testing of ten of these F2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.

A TILLING by sequencing approach to identify induced mutations in sunflower genes.

The Targeting Induced Local Lesions in Genomes (TILLING) technology is a reverse genetic strategy broadly applicable to every kind of genome and represents an attractive tool for functional genomic and agronomic applications. It consists of chemical random mutagenesis followed by high-throughput screening of point mutations in targeted genomic regions. Although multiple methods for mutation discovery in amplicons have been described, next-generation sequencing (NGS) is the tool of choice for mutation detection because it quickly allows for the analysis of a large number of amplicons. The aim of the present work was to screen a previously generated sunflower TILLING population and identify alterations in genes involved in several important and complex physiological processes. Twenty-one candidate sunflower genes were chosen as targets for the screening. The TILLING by sequencing strategy allowed us to identify multiple mutations in selected genes and we subsequently validated 16 mutations in 11 different genes through Sanger sequencing. In addition to addressing challenges posed by outcrossing, our detection and validation of mutations in multiple regulatory loci highlights the importance of this sunflower population as a genetic resource.

A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat.

BACKGROUND: Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use. RESULTS: We developed mutant populations of pasta and common wheat and organized them for TILLING. To simplify and decrease costs, we developed a non-denaturing polyacrylamide gel set-up that uses ethidium bromide to detect fragments generated by crude celery juice extract digestion of heteroduplexes. This detection method had similar sensitivity as traditional LI-COR screens, suggesting that it represents a valid alternative. We developed genome-specific primers to circumvent the presence of multiple homoeologous copies of our target genes. Each mutant library was characterized by TILLING multiple genes, revealing high mutation densities in both the hexaploid (~1/38 kb) and tetraploid (~1/51 kb) populations for 50% GC targets. These mutation frequencies predict that screening 1,536 lines for an effective target region of 1.3 kb with 50% GC content will result in ~52 hexaploid and ~39 tetraploid mutant alleles. This implies a high probability of obtaining knock-out alleles (P = 0.91 for hexaploid, P = 0.84 for tetraploid), in addition to multiple missense mutations. In total, we identified over 275 novel alleles in eleven targeted gene/genome combinations in hexaploid and tetraploid wheat and have validated the presence of a subset of them in our seed stock. CONCLUSION: We have generated reverse genetics TILLING resources for pasta and bread wheat and achieved a high mutation density in both populations. We also developed a modified screening method that will lower barriers to adopt this promising technology. We hope that the use of this reverse genetics resource will enable more researchers to pursue wheat functional genomics and provide novel allelic diversity for wheat improvement.

Strongyloides stercoralis Hyperinfection Syndrome as a Cause of Fatal Gastrointestinal Hemorrhage.

Strongyloides stercoralis is a small intestinal nematode that is widespread in regions with poor sanitation. We present a 57-year-old man from Colombia who was undergoing corticosteroid therapy for a meningioma who presented after neurosurgery with abdominal pain and a profound gastrointestinal (GI) bleed. The patient underwent an esophagogastroduodenoscopy (EGD), an attempted embolization, and an exploratory laparotomy to remove the necrosed duodenum. His pathology examination revealed Strongyloides infection of the duodenum, and he died of profound blood loss. This rare diagnosis displays the importance of screening patients at a high risk of Strongyloides infection before starting glucocorticoid therapy.

Tilling by sequencing.

TILLING is a method to find mutations in a gene of interest by scanning amplicons from a mutagenized population for sequence changes, commonly a single nucleotide. In the past 5 years, mutation detection by sequencing has become increasingly popular. This chapter details the experimental flow for TILLING-by-Sequencing, highlighting the critical steps involved in tridimensional pooling of genomic DNA templates, preparation of libraries for high-throughput sequencing, and bioinformatic processing of the sequence data.