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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.

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.

The COVID-19 pandemic has reduced access to solid organ transplantation, compounding organ shortages and waitlist mortality. A continued area of uncertainty is the safety of transplanting organs recovered from SARS-CoV-2 infected donors, as autopsies of patients who died with COVID-19 show that the virus can be found in extra-pulmonary organs1. Case reports and series on transplantation of these organs have been published 2, 3, but population-level data is lacking.We queried a national transplant database for recipients of organs recovered from donors recently infected by SARS-CoV-2. For organs with more than 50 cases, these were then propensity-score matched at a ratio of 1:10 to similar recipients of organs recovered from donors who tested negative for SARS-CoV-2 (controls). Data were extracted from the Scientific Registry of Transplant Recipients (SRTR - v2203 - updated March 2022), which collects detailed information on all solid organ transplants in the United States since 1986.Cases were defined as adult (≥ 18 years) recipients of organs recovered from deceased donors who tested positive for SARS-CoV-2 by nasopharyngeal or lower respiratory sample polymerase chain reaction or antigen assay within 7 days of organ transplantation. Multiple organ transplants were excluded.There were 775 kidney, 330 liver, 123 heart, 44 kidney-pancreas, 16 lung, 5 pancreas, and 3 small bowel transplants of organs recovered from 393 deceased donors recently infected by COVID-19. For kidney, liver, and heart transplants, Kaplan-Meier curves of both overall and graft survival at 90 days were similar between cases and controls.Our data shows that transplanting kidneys, livers, and hearts recovered from deceased donors recently infected by SARS-CoV-2 was not associated with increased recipient mortality or worse graft-survival. This should help transplant providers make decisions regarding acceptance of these organs, and counsel transplant candidates on the safety of receiving them. The limited number of kidney-pancreas, lung, pancreas, and intestinal cases precludes significant conclusions for these organs. Our data also strongly supports the notion that donors with recent COVID-19 infection should not be automatically excluded from the donor pool. The limited number of kidney-pancreas, lung, pancreas, and intestinal cases precludes significant conclusions for these organs.Limitations include lack of data on donor infection timeline and estimates of viral load (PCR cycle thresholds), description of donor COVID-19 symptomatology at organ procurement, donor or recipient vaccination or prior COVID-19 infection status, which are not tracked in the database. We did not have information regarding transmission of COVID-19 to transplant recipients. Future analysis of updated versions of the database should help address. Our data strongly support the notion that donors with recent COVID infection should not be automatically excluded from the donor pool. Prospective studies are needed to confirm our findings and provide insights on optimal post-transplant management of these recipients.

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.

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.

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.

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.

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.

Advances in DNA sequencing (i.e., next-generation sequencing, NGS) have greatly increased the power and efficiency of detecting rare mutations in large mutant populations. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach for identifying gene mutations resulting from chemical mutagenesis. In traditional TILLING, mutation discovery is accomplished through mismatch cleavage of mutant and wild-type DNA heteroduplexes using endonucleases. This is followed by Sanger sequencing to determine the specific sequence changes. TILLING by sequencing (TBS) uses NGS to facilitate the concurrent detection and sequence characterization of mutations, which allows researchers to prioritize mutants for further analyses. NGS increases the sensitivity of mutation detection and thus improves screening efficiency by allowing the pooling of more DNAs. Here we describe a protocol for TBS using rice as an example. First, DNA from a mutant population is quantified and combined in an overlapping pool design. Then, target genes are amplified from DNA pools and amplicons are combined to maximize throughput and increase likelihood of mutation detection during sequencing. Once sequence data is obtained, mutations are called using statistical approaches that weigh likelihood of rare mutations versus the probability of PCR and sequencing error.

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.

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.

The operating thermal power transfer mechanisms in a thermal-wave resonant cavity were explored theoretically and experimentally. Both steady-state ac (thermal-wave) and dc temperature rise were considered, and conduction and radiation heat transfer modes were found to co-exist in the cavity. By introducing controlled variable offset dc resistive heating superposed on the fixed-amplitude thermal-wave oscillation, it was also found that the thermal-diffusivity values of the intracavity gas can vary sensitively as a function of the dc temperature rise within a thin boundary layer adjacent to the cavity thermal source (a metallic Cr–Ni alloy strip). This resulted in the measurement of the temperature dependence of the thermal diffusivity of air. Furthermore, the observed dominance of thermal-wave radiation power transfer in the phase channel of the thermal-wave signal at large cavity lengths allowed the measurement of the absolute infrared emissivity of the thin Cr–Ni strip source material: e=0.091±0.004.

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.

Vicinal proton-proton NMR couplings have been used to estimate the changes in conformational equilibria for butanedioic acid in the progression of its diprotic to monoprotic to di-ionized forms as a function of pH, concentration, and nature of the counterion. The results support earlier conclusions that the K_1/K_2 ratio for butanedioic acid is little, if at all, affected by internal hydrogen bonding in the monoanion. Perhaps surprisingly, the conformational preferences are weak, despite the possibility of steric hindrance and strong stabilizations by hydrogen-bonding, induction, and/or electrostatic effects.

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.

Proton NMR spectra suggest that the conformation with trans vicinal hydrogens is favored for ephedrine as the free base in deuterium oxide, although in non-polar solvents, such as deuteriotrichloromethane, the gauche vicinal hydrogen conformations are favored, as has been reported previously. The conjugate acid has primarily gauche hydrogens. In contrast, the trans-hydrogen rotamers of pseudoephedrine dominate for both the non-protonated and protonated forms in deuterium oxide. Hydrogen bonding is unlikely to be as important as usually assumed in determining the conformational preferences of these substances.