A cluster of acute thebaine poisonings from non-food grade poppy seeds in the Australian food supply.

INTRODUCTION: Poppy seed tea is used for its opioid effects and contains multiple opium alkaloids, including morphine, codeine, papaverine, and thebaine. Animal studies indicate thebaine has strychnine-like properties, but there is limited literature describing human thebaine poisoning. We describe a cluster of acute thebaine poisoning in people ingesting tea made using poppy seeds with high thebaine content that entered the Australian food supply chain. METHODS: This is an observational study of patients poisoned after drinking poppy seed tea. Cases were identified by three prospective toxicovigilance systems: the Emerging Drug Network of Australia collaboration, the New South Wales Prescription, Recreational and Illicit Substance Evaluation program, and the Emerging Drugs Network of Australia Victoria study. We report characteristics of clinical toxicity in cases with reported ingestion of poppy seed tea and analytical confirmation of thebaine exposure. RESULTS: Forty cases presenting with multi-system toxicity following poppy seed tea ingestion were identified across seven Australian states/territories from November 2022 to January 2023. Blood testing in 23 cases confirmed high thebaine concentrations. All 23 were male (median age 35, range 16-71 years). All patients experienced muscle spasms. Rigidity was described in nine, convulsions in six, while rhabdomyolysis, acute kidney injury, and metabolic acidosis occurred in five patients. There were two cardiac arrests. The thebaine median admission blood concentration was 1.6 mg/L, with a range of 0.1-5.6 mg/L, and was the dominant opium alkaloid in all samples. Convulsions, acute kidney injury, metabolic acidosis, and cardiac arrest were associated with increasing median thebaine concentrations. Four patients were managed in the Intensive Care Unit, with two receiving continuous kidney replacement therapy (one also received intermittent haemodialysis) for kidney injury. There was one death. CONCLUSIONS: Thebaine toxicity, like strychnine poisoning, resulted in neuromuscular excitation characterized by muscle spasm, rigidity, and convulsions. Severe toxicity, including acute kidney injury, metabolic acidosis, and cardiac arrest, appears dose-dependent.

Quantitative genetic analysis reveals potential to breed for improved white clover growth in symbiosis with nitrogen-fixing Rhizobium bacteria.

White clover (Trifolium repens) is integral to mixed pastures in New Zealand and temperate agriculture globally. It provides quality feed and a sustainable source of plant-available nitrogen (N) via N-fixation through symbiosis with soil-dwelling Rhizobium bacteria. Improvement of N-fixation in white clover is a route to enhancing sustainability of temperate pasture production. Focussing on seedling growth critical for crop establishment and performance, a population of 120 half-sibling white clover families was assessed with either N-supplementation or N-fixation via inoculation with a commercial Rhizobium strain (TA1). Quantitative genetic analysis identified significant (p < 0.05) family additive genetic variance for Shoot and Root Dry Matter (DM) and Symbiotic Potential (SP), and Root to Shoot ratio. Estimated narrow-sense heritabilities for above-ground symbiotic traits were moderate (0.24-0.33), and the strong (r ≥ 0.97) genetic correlation between Shoot and Root DM indicated strong pleiotropy or close linkage. The moderate (r = 0.47) phenotypic correlation between Shoot DM under symbiosis vs. under N-supplementation suggested plant growth with mineral-N was not a strong predictor of symbiotic performance. At 5% among-family selection pressure, predicted genetic gains per selection cycle of 19 and 17% for symbiotic traits Shoot DM and Shoot SP, respectively, highlighted opportunities for improved early seedling establishment and growth under symbiosis. Single and multi-trait selection methods, including a Smith-Hazel index focussing on an ideotype of high Shoot DM and Shoot SP, showed commonality of top-ranked families among traits. This study provides a platform for proof-of-concept crosses to breed for enhanced seedling growth under Rhizobium symbiosis and is informative for other legume crops.

High-resolution dose-response screening using droplet-based microfluidics.

A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC(50) values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC(50) of 27 ± 0.83 µM.

A competitive co-cultivation assay for cancer drug specificity evaluation.

The identification of compounds that specifically inhibit or kill cancer cells without affecting cells from healthy tissues is very challenging but very important for reducing the side effects of current cancer therapies. Hence, there is an urgent need for improved assays allowing the selectivity of a given compound to be monitored directly. The authors present an assay system based on the competitive co-cultivation of an excess of cancer cells with a small fraction of noncancer human indicator cells generating a fluorescence signal. In the absence of a specific anticancer compound, the cancer cells outgrow the indicator cells and abolish the fluorescence signal. In contrast, the presence of specific anticancer drugs (such as Tyrphostin-AG1478 or PLX4720) results in the selective growth of the indicator cells, giving rise to a strong fluorescence signal. Furthermore, the authors show that the nonspecific cytotoxic compound sodium azide kills both cancer and noncancer cells, and no fluorescence signal is obtained. Hence, this assay system favors the selection of compounds that specifically target cancer cells and decreases the probability of selecting nonspecific cytotoxic molecules. Z factors of up to 0.85 were obtained, indicating an excellent assay that can be used for high-throughput screening.

An automated two-phase microfluidic system for kinetic analyses and the screening of compound libraries.

Droplet-based microfluidic systems allow biological and chemical reactions to be performed on a drastically decreased scale. However, interfacing the outside world with such systems and generating high numbers of microdroplets of distinct chemical composition remain challenging. We describe here an automated system in which arrays of chemically distinct plugs are generated from microtiter plates. Each array can be split into multiple small-volume copies, thus allowing several screens of the same library. The system is fully compatible with further on-chip manipulation(s) and allows monitoring of individual plugs over time (e.g. for recording reaction kinetics). Hence the technology eliminates several bottlenecks of current droplet-based microfluidic systems and should open the way for (bio-)chemical and cell-based screens.

A competition-based assay for the screening of species-specific antibiotics.

OBJECTIVES: To develop a high throughput screening-compatible assay for the selection of species-specific antibiotics that do not harm human cells. METHODS: Staphylococcus aureus and human reporter cells continuously generating a fluorescence signal were competitively co-cultivated. The fluorescence signals were determined in the presence and absence of the specific antibiotic streptomycin and the toxic compound sodium azide. The results were compared with a standard cfu assay. RESULTS: In the absence of an effective antibiotic, S. aureus outgrew the human reporter cells and thus abolished the fluorescence signal. Conversely, the addition of streptomycin resulted in the growth of the reporter cells and a strong fluorescence signal. When sodium azide was added instead of streptomycin, only a very low background signal was obtained indicating toxicity and damage to the human reporter cells. The assay proved to be highly reliable (Z-factor >0.9) and high fluorescence signals correctly correlated with the efficient inhibition of S. aureus, as determined in comparative cfu assays. CONCLUSIONS: In contrast to conventional cfu assays, the co-cultivation system allows the effects of a drug candidate on pathogens and human cells to be monitored simultaneously. Cytotoxic compounds can, therefore, be quickly ruled out during a primary screen. The nature of the screen also enables effective antibiotics to be identified without engineering the target pathogen to yield a fluorescence signal.

Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents.

Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.