Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Effects of norethisterone acetate on the performance of female racing Greyhounds.

A comparison between the performance of spayed female racing Greyhounds and those suppressed with norethisterone acetate (NTA) was made. Previous work by the author has shown that the racing performance of spayed bitches is the same as that of entire bitches in anoestrus, i.e. spaying is just a permanent anoestrus. The aim was to assess any performance difference between suppression and anoestrus, and thus to determine the effect of norethisterone acetate on race performance. The study was designed as a retrospective case-control. Raceform data was obtained for female racing Greyhounds which had raced, and which were either spayed or suppressed with norethisterone acetate. Analysis showed that suppressed bitches run on average 0.049 to 0.061 s slower over 480 m (centralised models). Since endogenous progesterone (P4) has been linked with reduced race performance, it is logical that progesterone analogues like NTA should have a similar effect. It is likely that the depression in performance is dose-related, but not quantifiable with the current dataset.

Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle.

Vinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds.

Risk of iatrogenic damage to the sciatic nerve in dairy cattle.

Sciatic nerve damage in cattle could lead to pain, paralysis and lameness, creating welfare and productivity issues. Damage following intramuscular injection is highlighted in some clinical texts although there is no indication of prevalence. This cross-sectional study investigated the anatomical size and position of the sciatic nerve in four dairy cattle type cadavers. To evaluate the risk of iatrogenic damage, 54 participants placed needles in the dorsal gluteal region of two cadavers each, as per their routine intramuscular injection method. The distance from needle tip to the sciatic nerve was estimated for each. Sixty-nine per cent of injections were placed within 5 cm of the sciatic nerve. Direct nerve puncture occurred in the cadaver in poorest body condition. A short questionnaire revealed that 70 per cent of the participants regularly used the dorsal gluteal region for intramuscular injection and 50 per cent commonly used a volume of ≥20 mL at a single site. It is concluded that the sciatic nerve is at considerable risk of iatrogenic damage. The neck should be promoted as the optimum site for intramuscular injection and improved guidelines are required if the gluteal region is to remain in common use. A lateral site, between the tuber coxae and tuber ischium, is recommended.

An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

The effect of spaying on the racing performance of female greyhounds.

The effect of spaying (ovariohysterectomy) on racing greyhound performance was analysed. Using data from a longitudinal observational study design, random effects models were used to compare the racing performance before and after spaying. Greyhounds were matched by distance and date of racing, and then both spayed and entire bitches were compared for each stadium which had spayed greyhounds on the racing strength. The racing performance of entire females was selected from the anoestral period (from 90 days post-oestrus to the next oestrus). Spaying had no real effect on performance, with a mean difference of +0.066 seconds (90% CI: -0.004, +0.135 s) over 480 metres. The effect of time since spaying had no effect (90% CI: -0.0002 to +0.0001 s/day). Spaying of racing greyhound bitches produced no overall change in performance, and had no apparent long term effect on performance. These results are discussed within the context of the current information on the advantages and disadvantages of spaying at a young age.

The effect of dioestrus on the racing performance of Greyhounds.

The degree of performance change during the dioestrus of racing Greyhounds has long been the subject of debate. Assessments have previously been on a qualitative basis. The analyses in this paper are unique and produce a quantitative assessment of the change in performance during dioestrus. By accessing a large dataset, race form and oestrous data were analysed using a longitudinal observational study design. The performance changes in dioestrus were modelled with a series of multilevel linear regressions revealing a pattern such that the performance loss varied from 0.031 to 0.733 s (90% confidence interval) between 41 and 56 days since oestrus over 450 m, returning to the baseline (anoestral) performance level after about 80-100 days. The changes in performance formed a temporal match with the changes in serum progesterone concentration noted by other workers. Whilst serum hormone concentrations were not measured, it is suggested that the performance changes are linked with the changes in serum progesterone concentrations rather than prolactin. It is therefore proposed that a minimum time off of 70 days could be set, which would capture the majority of females with a genuine change in performance, and with a caution regarding the use of entire female Greyhounds for performance studies.