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The equine bloodworm, Strongylus vulgaris, is a highly pathogenic parasite causing potentially fatal vascular and intestinal damage. Parasites express and release microRNAs (miRNAs) for internal regulation and to modulate host immunity. The complete set of miRNAs expressed by S. vulgaris (the S. vulgaris miRNAome) remains unannotated and the aim of this study was to annotate the miRNAome of L4 and L5 stages of S. vulgaris, and to examine differences in miRNA abundance between larval stages and sexes. Furthermore, we aimed to determine if miRNAs were detectable in excretory/secretory products (ESPs) from larvae and in arterial tissue from their predilection site, the cranial mesenteric artery (CMA). Larvae were collected from naturally infected foals, and categorized by sex and stage. A subset of larvae was snap-frozen, while those remaining were incubated and the (ESPs) collected. Arterial tissue samples were collected from the CMA. Small RNA sequencing, followed by a custom bioinformatic pipeline, was used for annotation. We identified 142 S. vulgaris miRNAs in larvae and 136 in ESPs. Significant differences in miRNA abundance were observed between larvae and ESPs, and between L5 females (L5Fs) and L5 males (L5Ms), L4s and L5Fs, and L4s and L5Ms. No differences were found between L4s and L5s overall. In ESPs, several miRNAs were differentially abundant across all groups. Validation through quantitative real-time PCR (qPCR) detected selected miRNAs and their differential abundance in larvae and ESPs. One parasite-derived miRNA was detected in some of the horse arterial tissue samples but at very low levels. This study provided the first annotation of the S. vulgaris miRNAome. Most of the annotated larval miRNAs were also detectable in ESPs, and differences in miRNA abundance between sexes were found for larvae, and between sexes and stages for ESPs. Parasite-derived miRNAs were, however, not consistently detectable in the surrounding host arterial tissue., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The 99mTc complex of NC100692 is being evaluated as a diagnostic agent for imaging of angiogenesis. We here report in vivo studies performed with NC100692 and 14C-labelled NC100692 using liquid chromatography coupled to either an ion-trap mass spectrometer or a radiochemical detector. Following injection of 14C-labelled NC100692, only the parent compound and no metabolites was observed in rat blood, whereas no parent compound and only 1 metabolite was observed in urine. Analysis of rat urine samples with liquid chromatography with mass spectrometric detection following administration of NC100692 verified the absence of the parent compound and showed the presence of 2 metabolites. The structures of the 2 metabolites were identified based on mass spectra and accurate mass determinations. The major metabolite was identified as the molecule obtained following hydrolytic cleavage at the end of the C-terminal amino acid of NC100692. The minor metabolite was identified as that obtained following removal of phenylalanine within the cyclic structure of the major metabolite.

The enzymatic dephosphorylation of the magnetic resonance imaging contrast agent Teslascan was studied in in vitro experiments with acid phosphatase (prostatic, from human semen) and alkaline phosphatase (from human placenta). The active component, MnDPDP (manganese (II)-N,N'-dipyridoxylethylenediamine-N,N'-diacetate-5,5'-bis(phosphate), was dephosphorylated by both enzymes to the monophosphate MnDPMP and the totally dephosphorylated compound MnPLED. The corresponding zinc compound, ZnDPDP (which is a result of in vivo metabolism), was also dephosphorylated by both enzymes to ZnDPMP and ZnPLED. In separate experiments, both enzymes dephosphorylated MnDPMP and ZnDPMP. With the same amount of enzyme units, alkaline phosphatase was almost four times more active than acid phosphatase in dephosphorylating MnDPDP and ZnDPDP with only minor differences whether the substrate contained Mn or Zn. A similar difference in enzymatic activity was seen with the monophosphates, MnDPMP and ZnDPMP. This, taken together with the approximately 50 times higher activity of alkaline phosphatase than acid phosphatase in serum shows that alkaline phosphatase is responsible for most of the dephosphorylation of MnDPDP and its metabolites in vivo.

The binding to human serum proteins of MnDPDP (manganese(II) dipyridoxyl diphosphate), the active component of the magnetic resonance imaging contrast medium mangafodipir trisodium injection (Teslascan) was studied in ultrafiltration experiments. Sera from three males and three females were incubated with 86 microM [14C]MnDPDP for 60 min at room temperature (20-23 degrees C), followed by centrifugation through filters with a cut-off of 30 kDa. Analysis of the filtrates and the initial incubation mixtures for manganese, by ICP-AES, and for DPDP and its dephosphorylated metabolites DPMP (dipyridoxyl monophosphate) and PLED (dipyridoxyl ethylenediamine diacetate) by liquid scintillation counting, showed a clear difference in protein binding of manganese and the ligands under these conditions. Only 2.2 +/- 1.8% (mean +/- S.E.; n = 6) of DPDP, DPMP and PLED were bound to protein, whereas 26.9 +/- 2.9% (mean +/- S.E.; n = 6) of manganese was bound to protein. No binding of DPDP, DPMP or PLED to blood cells was observed when whole blood, containing either heparin or EDTA as anticoagulant, was spiked with [14C]MnDPDP and the cell-free fraction and the lysed cell fraction analysed by liquid scintillation counting. The extent of protein binding of manganese corresponded well with results from an in vitro metabolism study, in which MnDPDP was added to heparinized human whole blood, showing that approximately 25% of DPDP, DPMP or PLED were not bound to manganese. The in vitro metabolism study revealed that transmetallation with zinc was nearly complete within 1 min, and that dephosphorylation is a sequential process going from DPDP to the monophosphate DPMP, and then to the fully dephosphorylated compound PLED.

Manganese(II) dipyridoxyl diphosphate (MnDPDP) is the active component of mangafodipir trisodium injection (Teslascan), a new contrast medium for magnetic resonance imaging. A high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of MnDPDP and its five major metabolites in human plasma, i.e. the dephosphorylation products MnDPMP (manganese(II) dipyridoxyl monophosphate) and MnPLED (manganese(II) dipyridoxyl ethylenediamine diacetate) and the corresponding substances obtained after transmetallation with zinc (ZnDPDP, ZnDPMP and ZnPLED). Heparinized blood samples from patients receiving mangafodipir trisodium injection were immediately mixed with solid trisodium phosphate dodecahydrate to obtain pH 10.0 +/- 0.2 in order to inhibit further in vitro dephosphorylation and transmetallation. The plasma thus obtained was ultrafiltrated prior to HPLC analysis. The chromatographic separation was obtained using a mixed-bed resin with both anion exchange and reversed-phase functions (OmniPac PAX-500) using isocratic elution and UV detection at 310 nm. With an injection volume of 50 microliters, the limit of quantitation (LOQ) values were 0.8-2.3 microM for the Mn compounds and 0.1-0.8 microM for the Zn compounds. The between-run accuracy of spiked plasma samples was in the range 97.5-106.7% with a precision in the range 3.1-9.0%. The best fit calibration curves were obtained using non-linear regression according to the equation Y = A + BXM in the concentration range from LOQ to 100 microM. Long-term storage of spiked plasma samples for three months at -20 degrees C demonstrated the required stability with recovery values within 85-115% of MnDPDP and its five metabolites.