Generating minimum set of gRNA to cover multiple targets in multiple genomes with MINORg.

MINORg is an offline gRNA design tool that generates the smallest possible combination of gRNA capable of covering all desired targets in multiple non-reference genomes. As interest in pangenomic research grows, so does the workload required for large screens in multiple individuals. MINORg aims to lessen this workload by capitalising on sequence homology to favour multi-target gRNA while simultaneously screening multiple genetic backgrounds in order to generate reusable gRNA panels. We demonstrated the practical application of MINORg by knocking out 11 homologous genes tandemly arrayed in a multi-gene cluster in two Arabidopsis thaliana lineages using three gRNA output by MINORg. We also described a new PCR-free modular cloning system for multiplexing gRNA, and used it to knockout three tandemly arrayed genes in another multi-gene cluster with gRNA designed by MINORg. Source code is freely available at https://github.com/rlrq/MINORg.

Evaluating Clinical Prediction Rules for Bacteremia Detection in the Emergency Department: A Retrospective Review.

BACKGROUND: Bacteremia is a major cause of morbidity. Blood cultures are the gold standard for diagnosing bacteremia. OBJECTIVE: To compare previously published clinical decision rules for predicting a true positive blood culture (bacteremia) in the emergency department. METHODS: Retrospective analysis of medical records of patients who had a blood culture performed in a tertiary hospital emergency department in 2020 (12 months). Positive blood cultures were compared with randomly selected negative blood cultures (1:4 ratio). Blood cultures were analyzed per patient presentation. Clinical data from patient presentations were extracted and appraised against the modified-Shapiro (mShapiro) rule and systemic inflammatory response syndrome (SIRS) criteria to calculate diagnostic accuracy to detect bacteremia. RESULTS: During the study period, 3870 blood cultures were taken from 2921 patients: 476 (12.3%) cultures were positive for bacterial growth, from 421 individual patient presentations (10 excluded as incomplete data). Of included patients, 338 were true positives and 73 contaminates, these were compared with 1446 patients with negative blood culture presentations. Evaluating mShapiro's rule and SIRS criteria to detect bacteremia vs. no bacteremia (negative + contaminated cultures) had a sensitivity of 94.4% (95% confidence interval [CI] 91.4-96.4%) and 84.9% (95% CI 80.7-88.3%), respectively, and a specificity of 37.9% (95% CI 35.5-40.1%) and 33.8% (95% CI 31.5-36.3%), respectively. Both had a high negative predictive value for bacteremia of 96.8% (95% CI 95.1-98.0) and 91.0% (95% CI 88.3-93.1) for mShapiro's rule and SIRS criteria, respectively. CONCLUSIONS: In this cohort, mShapiro's rule performed better than the SIRS criteria at predicting bacteremia.

A Truncated Singleton NLR Causes Hybrid Necrosis in Arabidopsis thaliana.

Hybrid necrosis in plants arises from conflict between divergent alleles of immunity genes contributed by different parents, resulting in autoimmunity. We investigate a severe hybrid necrosis case in Arabidopsis thaliana, where the hybrid does not develop past the cotyledon stage and dies 3 weeks after sowing. Massive transcriptional changes take place in the hybrid, including the upregulation of most NLR (nucleotide-binding site leucine-rich repeat) disease-resistance genes. This is due to an incompatible interaction between the singleton TIR-NLR gene DANGEROUS MIX 10 (DM10), which was recently relocated from a larger NLR cluster, and an unlinked locus, DANGEROUS MIX 11 (DM11). There are multiple DM10 allelic variants in the global A. thaliana population, several of which have premature stop codons. One of these, which has a truncated LRR-PL (leucine-rich repeat [LRR]-post-LRR) region, corresponds to the DM10 risk allele. The DM10 locus and the adjacent genomic region in the risk allele carriers are highly differentiated from those in the nonrisk carriers in the global A. thaliana population, suggesting that this allele became geographically widespread only relatively recently. The DM11 risk allele is much rarer and found only in two accessions from southwestern Spain-a region from which the DM10 risk haplotype is absent-indicating that the ranges of DM10 and DM11 risk alleles may be nonoverlapping.

Antithymocyte Globulin at Clinically Relevant Concentrations Kills Leukemic Blasts.

In contrast to cyclosporine or methotrexate, rabbit antithymocyte globulin (ATG) used for graft-versus-host disease (GVHD) prophylaxis with myeloablative conditioning does not increase the risk of relapse after hematopoietic cell transplantation. The reason for this is unknown. We hypothesized that ATG at concentrations achieved with our standard ATG dose of 4.5 mg/kg exerts antileukemic activity. We measured ATG-induced killing of leukemic blasts via complement-dependent cytotoxicity (CDC) and via complement-independent cytotoxicity (CIC) in marrow or blood from 36 patients with newly diagnosed acute leukemia. The median percentage of blasts killed by CDC was 0.3% at 1 mg/L ATG, 2.8% at 10 mg/L ATG, 12.6% at 25 mg/L ATG, and 42.2% at 50 mg/L ATG. The median percentage of blasts killed by CIC after a 4-hour incubation with ATG was 1.9% at 1 mg/L ATG, 7.15% at 10 mg/L ATG, 12.1% at 25 mg/L ATG, and 13.9% at 50 mg/L ATG. CIC appeared to represent a direct induction of apoptosis by ATG. There was a high variability in the sensitivity of the blasts to ATG; at 50 mg/L, the percentage of blasts killed ranged from 2.6% to 97.2% via CDC and from 1.4% to 69.9% via CIC. In conclusion, ATG at clinically relevant concentrations kills leukemic blasts in vitro. Some acute leukemias are highly sensitive to ATG, whereas others are relatively resistant. This finding could lead to personalized administration of ATG.

Patient Perspectives of Living with Coeliac Disease and Accessing Dietetic Services in Rural Australia: A Qualitative Study.

Adapting to living with coeliac disease requires individuals to learn about and follow a strict gluten-free diet. Utilising a qualitative inductive approach, this study aimed to explore the perspectives of adults diagnosed with coeliac disease who have accessed dietetic services in a rural outpatient setting. A purposive sample of adults with coeliac disease who had accessed dietetic services from two rural dietetic outpatient clinics were recruited. Semi-structured interviews were conducted by telephone. Data were thematically analysed. Six participants were recruited and interviewed. Three key themes emerged: (i) optimising individualised support and services, (ii) adapting to a gluten-free diet in a rural context, and (iii) managing a gluten-free diet within the context of interpersonal relationships. Key issues identified in the rural context were access to specialist services and the increased cost of gluten-free food in more remote areas. The findings of this study have highlighted the difficulties associated with coeliac disease management and how dietetic consultation has the potential to influence confidence in management and improve lifestyle outcomes. Further qualitative research is required to expand on the findings of this study and inform future dietetic practice that meets the expectations and individual needs of people with coeliac disease in rural settings.

Variation Patterns of NLR Clusters in Arabidopsis thaliana Genomes.

The nucleotide-binding domain and leucine-rich repeat (NLR) gene family is highly expanded in the plant lineage with extensive sequence and structure polymorphisms. To survey the landscape of NLR expansion, we mined the published long-read data generated by the resistance gene enrichment sequencing of 64 diverse Arabidopsis thaliana accessions. We found that the hot spots of massive multi-gene NLR cluster expansion did not typically span the whole cluster; instead, they were restricted to a handful of, or only one, dominant radiation(s). All sequences in such a radiation were distinct from other genes in the cluster but not from each other in the clade, making it difficult to assign trustworthy reference-based orthologies when multiple reference genes were present in the radiation. Consequently, NLR genes can be broadly divided into two types: radiating or high-fidelity, where high-fidelity genes are well conserved and well separated from other clades. A similar distinction could be made for NLR clusters, depending on whether cluster size was determined primarily by extensive radiation or the presence of numerous high-fidelity genes. We also identified groups of well-conserved NLR clades that were missing from the Columbia-0 reference genome. This suggests that the classification of NLRs using gene IDs from a single reference accession can rarely capture all major paralogs in a cluster accurately and representatively and that a reference-agnostic perspective is required to properly characterize these additional variations. Finally, we present a quantitative visualization method for differentiating these situations in a given clade of interest.