Detection of Podosphaera macularis in Air Samples by Quantitative PCR.

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay utilizes primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit (LSU) of the nuclear ribosomal rDNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.

Genetic Variation in miR-27a Is Associated with Fluoropyrimidine-Associated Toxicity in Patients with Dihydropyrimidine Dehydrogenase Variants after Genotype-Guided Dose Reduction.

Dihydropyrimidine dehydrogenase (DPYD) is the rate-limiting enzyme involved in the metabolism of fluoropyrimidine-based chemotherapy. However, single-nucleotide variants (SNVs) in DPYD only partially explain fluoropyrimidine-induced toxicity. The expression of DPYD has previously been shown to be regulated by microRNA-27a (miR-27a) and a common miR-27a SNV (rs895819) has been associated with an increased risk of toxicity in patients harboring a DPYD variant who received standard fluoropyrimidine dosing. We investigated if the miR-27a rs895819 SNV was associated with toxicity in DPYD wildtype patients and carriers of DPYD variants who received a reduced dose. The regulation of DPYD using miR-27a was investigated in HepG2 cells utilizing a miR-27a mimic. miR-27a overexpression decreased DPYD mRNA expression compared to control cells (p < 0.0001). In a cohort of patients that received pre-emptive DPYD genotyping, 45 patients had a DPYD variant and 180 were wildtype. Patients heterozygous for rs895819 had an increased risk of toxicity, which was seen in both patients who were wildtype for DPYD variants (OR (95%CI) = 1.99 (1.00-3.99)) and DPYD variant carriers (OR (95%CI) = 8.10 (1.16-86.21)). Therefore, miR-27a rs895819 may be a clinically relevant predictor of fluoropyrimidine-associated toxicities. Furthermore, toxicity was more profound in DPYD variant carriers, even after DPYD genotype-guided dose reduction. This suggests that patients may benefit from miR-27a genotyping to guide fluoropyrimidine dosing.

RETROFIT: Reference-free deconvolution of cell-type mixtures in spatial transcriptomics.

Spatial transcriptomics (ST) profiles gene expression in intact tissues. However, ST data measured at each spatial location may represent gene expression of multiple cell types, making it difficult to identify cell-type-specific transcriptional variation across spatial contexts. Existing cell-type deconvolutions of ST data often require single-cell transcriptomic references, which can be limited by availability, completeness and platform effect of such references. We present RETROFIT, a reference-free Bayesian method that produces sparse and interpretable solutions to deconvolve cell types underlying each location independent of single-cell transcriptomic references. Results from synthetic and real ST datasets acquired by Slide-seq and Visium platforms demonstrate that RETROFIT outperforms existing reference-based and reference-free methods in estimating cell-type composition and reconstructing gene expression. Applying RETROFIT to human intestinal development ST data reveals spatiotemporal patterns of cellular composition and transcriptional specificity. RETROFIT is available at https://bioconductor.org/packages/release/bioc/html/retrofit.html.

DPYD Exon 4 Deletion Associated with Fluoropyrimidine Toxicity and Importance of Copy Number Variation.

Fluoropyrimidine chemotherapy is associated with interpatient variability in toxicity. A major contributor to unpredictable and severe toxicity relates to single nucleotide variation (SNV) in dihydropyrimidine dehydrogenase (DPYD), the rate-limiting fluoropyrimidine metabolizing enzyme. In addition to SNVs, a study of Finnish patients suggested that a DPYD exon 4 deletion was observed in their population. To better understand the potential generalizability of such findings, we investigated the presence of this exon 4 deletion in our Canadian patient population, using a TaqMan assay. We selected 125 patients who experienced severe fluoropyrimidine-associated toxicity, and 125 matched controls. One patient in the severe toxicity group harbored a haploid DPYD exon 4 deletion, and required a 35% dose reduction after their first fluoropyrimidine treatment cycle due to toxicity and required an additional 30% dose reduction before tolerating treatment. The predicted allele frequency was 0.2% in our cohort, much lower than the 2.4% previously reported. We also carried out a literature review of copy number variation (CNV) in the DPYD gene, beyond fluoropyrimidine toxicity and show that various types of CNV in DPYD are present in the population. Taken together, our findings suggest that CNV in DPYD may be an underappreciated determinant of DPYD-mediated fluoropyrimidine toxicity.

Impact of organic anion transporting polypeptide, P-glycoprotein, and breast cancer resistance protein transporters on observed tamoxifen and endoxifen concentration and adverse effects.

OBJECTIVE: Drug transporters are important determinants of drug disposition and response. Tamoxifen is an antiestrogen for breast cancer therapy known for adverse drug reactions (ADRs). In this study, the involvement of OATP transporters in tamoxifen and endoxifen transport was studied in vitro while the impact of single nucleotide variation (SNV) in OATP and efflux transporters P-glycoprotein ( ABCB1 ) and Breast Cancer Resistance Protein ( ABCG2 ) on ADRs during tamoxifen therapy were assessed. METHODS: Patients receiving tamoxifen for breast cancer, who were CYP2D6 normal metabolizers were enrolled ( n = 296). Patients completed a survey that captured ADRs and a blood sample was collected. Tamoxifen and endoxifen plasma concentration were measured, while DNA was genotyped for SNVs in ABCB1, ABCG2, SLCO1A2, SLCO1B1 , and SLCO2B1 . HEK293T cells were used to determine the extent of OATP-mediated transport of tamoxifen and endoxifen. RESULTS: Common SNVs of ABCB1, ABCG2, SLCO1A2 , and SLCO1B1 were not associated with tamoxifen or endoxifen concentration. However, tamoxifen concentration was significantly higher in carriers of SLCO2B1 c.935G>A (129.8 ng/mL) compared to wildtype (114.9 ng/mL; P = 0.036). Interestingly, subjects who carried SLCO1A2 c.38A>G reported significantly less dizziness ( P = 0.016). In-vitro analysis demonstrated increased cellular accumulation of tamoxifen in cells overexpressing OATP1A2 and 1B1, but endoxifen uptake was not effected in OATP overexpressing cells. CONCLUSIONS: We showed that OATP1A2 , a transporter known to be expressed at the blood-brain barrier, is capable of tamoxifen transport. Additionally, OATP1A2 c.38A>G was associated with reduced ADRs. Taken together, our findings suggest genetic variation in OATP transporters may be an important predictor of tamoxifen ADRs.

Developing a watershed screening tool to estimate relative contribution of phosphorus to guide management planning.

To support the development of clean water plans, as required by the federal Clean Water Act 303(d) program, the New York State Department of Environmental Conservation (DEC) developed the Loading Estimator of Nutrient Sources (LENS) tool. DEC has prioritized clean water planning for fresh waterbodies experiencing negative impacts due to excessive phosphorus levels. LENS, an Excel based tool, combines several simple steady state models into a single screening tool that may be used to estimate the relative contribution of phosphorus sources within a watershed, waterbody response, and recovery potential of a waterbody. To validate that LENS results are reasonable approximations, LENS loading estimates were statistically compared with loading estimates from more complex watershed models that were used to develop existing clean water plans using simple linear regressions. For this comparison, DEC selected a variety of completed models that have modeled watershed phosphorus loads with different land use compositions and loading from both point and nonpoint sources. This analysis shows that LENS performs reasonably well at estimating the relative loading from a range of source sectors, though cannot replace more robust watershed models. DEC has used LENS to prioritize waterbodies for clean water plans and to guide management actions in watersheds where data is lacking to support more complex modeling efforts. Future expansions of LENS may include modifying the tool to estimate other pollutants (i.e. nitrogen), add the ability to account for internal loading of nutrients within waterbodies; and estimate the contribution of nutrients from groundwater.

High oncostatin M predicts lack of clinical remission for patients with inflammatory bowel disease on tumor necrosis factor α antagonists.

The interleukin-6 family cytokine, oncostatin-M (OSM) has been associated with response to tumor necrosis factor-α antagonists (anti-TNFs) in small cohorts of patients with inflammatory bowel disease (IBD). We aimed to evaluate the association between plasma OSM concentrations and response to anti-TNFs (infliximab and adalimumab) in both ulcerative colitis (UC) and Crohn's disease (CD). A retrospective cohort study was conducted in patients with IBD with a history of anti-TNF exposure. Blood samples, collected prior to anti-TNF exposure, were analyzed by enzyme-linked immunosorbent assay for the presence and quantity of OSM. Clinical remission was assessed at 1-year post anti-TNF exposure in addition to the occurrence of surgery, hospitalization, corticosteroid use, and adverse drug events. Lastly the threshold OSM plasma concentration associated with anti-TNF non-response was assessed by receiver operator characteristic (ROC) curve analysis. Patients with IBD (CD, n = 82; UC, n = 40) were assessed. In both UC and CD, mean pre-treatment OSM concentrations were significantly lower in those who achieved clinical remission at 1-year (p < 0.0001). A threshold plasma OSM concentration of 168.7 pg/ml and 233.6 pg/ml respectively separated those who achieved clinical remission at 1-year on an anti-TNF from those who did not in CD and UC respectively (CD: area under the receiver operator characteristic curve, AUROC = 0.880, 95% CI 0.79-0.96; UC: AUROC = 0.938, 95% CI 0.87-1.00). High OSM concentrations were associated with anti-TNF discontinuation and use of rescue steroids in CD and UC. High pre-treatment OSM concentrations identify IBD patients at-risk of anti-TNF non-response at 1-year as well as other deleterious clinical outcomes.

Novel bioassay to assess antibiotic effects of fungal endophytes on aphids.

Perennial ryegrass is an important feed base for the dairy and livestock industries around the world. It is often infected with mutualistic fungal endophytes that confer protection to the plant against biotic and abiotic stresses. Bioassays that test their antibiotic effect on invertebrates are varied and range from excised leaves to whole plants. The aim of this study was to design and validate a "high-throughput" in-planta bioassay using 7-day-old seedlings confined in small cups, allowing for rapid assessments of aphid life history to be made while maintaining high replication and treatment numbers. Antibiosis was evaluated on the foliar and the root aphid species; Diuraphis noxia (Mordvilko) and Aploneura lentisci (Passerini) feeding on a range of perennial ryegrass-Epichloë festucae var. Lolii endophyte symbiota. As expected, both D. noxia and A. lentisci reared on endophyte-infected plants showed negatively affected life history traits by comparison to non-infected plants. Both species exhibited the highest mortality at the nymphal stage with an average total mortality across all endophyte treatments of 91% and 89% for D. noxia and A. lentisci respectively. Fecundity decreased significantly on all endophyte treatments with an average total reduction of 18% and 16% for D. noxia and A. lentisci respectively by comparison to non-infected plants. Overall, the bioassay proved to be a rapid method of evaluating the insecticidal activity of perennial ryegrass-endophyte symbiota on aphids (nymph mortality could be assessed in as little as 24 and 48 hours for D. noxia and A. lentisci respectively). This rapid and simple approach can be used to benchmark novel grass-endophyte symbiota on a range of aphid species that feed on leaves of plants, however we would caution that it may not be suitable for the assessment of root-feeding aphids, as this species exhibited relatively high mortality on the control as well.

Case report: atenolol overdose successfully treated with hemodialysis.

Owing to the drug's favorable hydrophilic and pharmacokinetic characteristics, a number of case reports have demonstrated effective treatment of atenolol overdose with hemodialysis. However, the efficiency of atenolol clearance throughout hemodialysis treatments has not previously been examined. In this report, a patient with impaired renal function was successfully treated with two 5-hour intermittent high-flux high-efficiency hemodialysis therapies after atenolol overdose. Serial atenolol levels were measured during his hemodialysis treatments. We observed an over 50% plasma atenolol concentration reduction after each 5-hour hemodialysis therapy. Hemodialysis therapy is an effective treatment for atenolol overdose, especially in patients with impaired renal function.