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Larger dogs may be at greater risk of prednisolone side effects, yet there is limited research about how bodyweight affects prednisolone pharmacokinetics in dogs. To describe the relationship between prednisolone dose, bodyweight, body surface area (BSA) and prednisolone area under the curve (AUC) in dogs receiving prednisolone for medical reasons. 25 client owned dogs receiving prednisolone for medical reasons. Observational population pharmacokinetic study. Liquid chromatography tandem mass spectrometry was used for plasma prednisolone quantification. Data analysis was conducted in a two-stage approach using non-compartmental modelling. A Bayesian non-linear regression model described the relationship between AUC over 8 hours ([Formula: see text]ng·min/mL), bodyweight and prednisolone dose. From the allometric scaling model of the form AUC8h = A · BW B, the scaling exponent [Formula: see text] was.83 (90% credible interval (CrI):.60-1.06) and the coefficient [Formula: see text] was 22.8 (90% CrI: 11.8-43.4). This model suggests that equivalent exposure would be obtained using an intermediate strategy between BSA and bodyweight dosing, but the total evidence provided was relatively weak. Evidence was obtained regarding the nonlinear relationship between prednisolone pharmacokinetics and bodyweight in dogs; however, this model is currently too imprecise for clinical dose determination.

Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5′- O -sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5- O -sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial, dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple aminoacyl-tRNA synthetases (aaRSs), including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition.

Porphyromonas gingivalis and Tannerella forsythia , two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies. Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis , the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2- N -seryl, 3- N -acetylglucuronamide in P. gingivalis and 2- N -glycyl, 3- N -acetylmannuronic acid in T. forsythia . The structures indicated the involvement of the Wbp pathway to produce 2,3-di- N -acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis . The wbpS gene was identified as PGN_1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalis vimA mutant which lacks A-LPS was successfully complemented by the T. forsythia vimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia . We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway. IMPORTANCE Porphyromonas gingivalis and Tannerella forsythia , two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies.

The effects of varying intensities of Australian Synchrotron source terahertz (THz) radiation on pheochromocytoma (PC 12) neuronal cells were investigated. PC 12 cells were exposed to THz radiation at beam incident power intensities of 0.25 W m−2 (low intensity, LI), 0.5 W m−2 (medium intensity, MI) and 1 W m−2 (high intensity, HI) for 10 min. After exposure, the morphological and physiological status of the cells was evaluated using scanning electron microscopy (SEM) and confocal laser scanning microscopy. SEM imaging revealed that, after exposure to LI THz radiation, the cells exhibited membrane protrusions (blebs) measuring 70–120 nm in diameter. In contrast, cells exposed to HI THz radiation demonstrated increased uptake of FITC–dextran and nanospheres. Analysis of single‐cell populations counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) showed a decrease in the proportion of DAPI‐positive cells, with approximately 90, 80 and 50% remaining positive after exposure to LI, MI and HI THz radiation, respectively. However, only a slight increase in the proportion of dead cells was observed at varying THz intensities. Proteomic analysis of the cell changes following exposure to LI and HI THz irradiation indicated that THz radiation activated the CaN complex and upregulated genes involved in ribosome biogenesis and DNA damage repair. PC 12 neuron‐like cells were exposed to terahertz (THz) radiation of variable beam incident power intensity from 0.25 to 1.0 W m−2. THz irradiation activated the CaN complex, ribosome biogenesis and DNA damage reparation.

Background Nippostrongylus brasiliensis —a nematode of rodents—is commonly used as a model to study the immunobiology of parasitic nematodes. It is a member of the Strongylida—a large order of socioeconomically important parasitic nematodes of animals. Lipids are known to play essential roles in nematode biology, influencing cellular membranes, energy storage and/or signalling. Methods The present investigation provides a comprehensive, untargeted lipidomic analysis of four developmental stages/sexes (i.e. egg, L3, adult female and adult male stages) of N. brasiliensis utilising liquid chromatography coupled to mass spectrometry. Results We identified 464 lipid species representing 18 lipid classes and revealed distinct stage-specific changes in lipid composition throughout nematode development. Triacylglycerols (TGs) dominated the lipid profile in the egg stage, suggesting a key role for them in energy storage at this early developmental stage. As N. brasiliensis develops, there was a conspicuous transition toward membrane-associated lipids, including glycerophospholipids (e.g. PE and PC) and ether-linked lipids, particularly in adult stages, indicating a shift toward host adaptation and membrane stabilisation. Conclusions We provide a comprehensive insight into the lipid composition and abundance of key free-living and parasitic stages of N. brasiliensis . This study provides lipidomic resources to underpin the detailed exploration of lipid biology in this model parasitic nematode. Graphical Abstract

Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber’s pole worm, and in other socioeconomically important parasitic nematodes. The “hit-to-target” workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.

Due to their sessile nature, plants rely on root systems to mediate many biotic and abiotic cues. To overcome these challenges, the root proteome is shaped to specific responses. Proteome-wide reprogramming events are magnified in meristems due to their active protein production. Using meristems as a test system, here, we study the major rewiring that plants undergo during cold acclimation. We performed tandem mass tag-based bottom-up quantitative proteomics of two consecutive segments of barley seminal root apexes subjected to suboptimal temperatures. After comparing changes in total and ribosomal protein (RP) fraction-enriched contents with shifts in individual protein abundances, we report ribosome accumulation accompanied by an intricate translational reprogramming in the distal apex zone. Reprogramming ranges from increases in ribosome biogenesis to protein folding factors and suggests roles for cold-specific RP paralogs. Ribosome biogenesis is the largest cellular investment; thus, the vast accumulation of ribosomes and specific translation-related proteins during cold acclimation could imply a divergent ribosomal population that would lead to a proteome shift across the root. Consequently, beyond the translational reprogramming, we report a proteome rewiring. First, triggered protein accumulation includes spliceosome activity in the root tip and a ubiquitous upregulation of glutathione production and S -glutathionylation (S-GSH) assemblage machineries in both root zones. Second, triggered protein depletion includes intrinsically enriched proteins in the tip-adjacent zone, which comprise the plant immune system. In summary, ribosome and translation-related protein accumulation happens concomitantly to a proteome reprogramming in barley root meristems during cold acclimation. The cold-accumulated proteome is functionally implicated in feedbacking transcript to protein translation at both ends and could guide cold acclimation.

Background and Methods: Using a nanoparticle-based enrichment (Proteonano) methodology on human plasma samples, we achieved a substantial increase in identified proteins from ~700 to >5000 proteins compared to neat plasma digest. In a small-scale pilot test, we applied this methodology to a small cohort of plasma samples from pancreatic cancer (PC) patients with different disease stages: (I) primary tumor and (II) metastases, and compared them with healthy controls. Most identified proteins are within the Human Plasma Proteome Project (HPPP) database, and more than 300 proteins are on the list of FDA-approved drug targets. Results: We observed a large and significant increase in ribosomal proteins in the plasma of patients with metastatic PC. ADH1C and ADH1B, both members of the alcohol dehydrogenase family, were particularly upregulated in patients with liver metastasis. Fifteen other predicted secreted and/or cell surface–associated proteins with known cancer associations are also significantly altered and would otherwise go undetected in neat, digested plasma. Conclusions: The significant increase in proteome depth allows a strong foundation for future large-scale experimental and comparative analysis. Lastly, similar conclusions could be reached from comparing different mass spectrometers (Orbitrap Astral and Orbitrap Ascend) and columns (depth and throughput) setups on the same dataset, although the depth approach on the newer Orbitrap Astral instrumentations can reveal additional insights in the plasma proteome.

Abstract Motivation A large number of experimental and bioinformatic parameters must be set to identify and quantify peptides in mass spectrometry experiments and each of these will impact the results. An ability to simulate raw data with known contents would allow researchers to rapidly explore the effects of varying experimental parameters and systematically investigate downstream processing software. A range of data simulators are available for established data-dependent acquisition methodologies, but these do not extend to the rapidly developing field of data-independent acquisition (DIA) strategies. Results Here, we present Synthedia—a software package to simulate DIA liquid chromatography-mass spectrometry for bottom-up proteomics experiments. Synthedia can generate datasets with known peptide precursor ions and fragments and allows for the customization of a wide variety of chromatographic and mass spectrometry parameters. Availability and implementation Synthedia is freely available via the internet and can be used through a graphical website (https://synthedia.org/) or locally via the command line (https://github.com/mgleeming/synthedia/). Supplementary information Supplementary data are available at Bioinformatics Advances online.

Protonated nitroarginine, [R NO2 + H] + , which contains the nitroguanidine ‘explosophore,’ undergoes homolytic N – N nitro-imine bond cleavage to expel NO 2 • and form a radical cation of arginine in high yield (100 % relative abundance) upon low-energy collision-induced dissociation (CID). Other ionization states of nitroarginine, including [R NO2 - H] – , and a fixed-charge derivative of nitroarginine do not expel NO 2 • (<1 %), but instead dissociate via heterolytic bond cleavage with abundant losses of small molecules (N 2 O and H 2 N 2 O 2 ) from the nitroguanidine group. The effects of proton mobility on the CID reactions of nitroarginine containing peptides was investigated for peptide derivatives of leucine enkephalin, including XYGGFLR NO2 , X = D, G, K, and R, by examining the different protonation states: [M – H] – ; [M + H] + ; and [M + 2H] 2+ . For [M + H] + containing the less basic N -terminal residues (X = D, G) and all [M + 2H] 2+ , mobile proton fragmentation reactions that result in peptide sequence ions dominate. In contrast, for peptides containing the basic N -terminal residues (R and K), the CID spectra of both the [M – H] – and [M + H] + are dominated by the losses of small even-electron neutrals from the nitroarginine side-chain. The fraction of nitroguanidine directed fragmentation of the nitroarginine side chain that results in bond homolysis to form [XYGGFLR] +• by expulsion of NO 2 • increases by more than 10 times as the protonation state changes from [M – H] – (<10 %) to [M + 2H] 2+ ( ca. 90 %) and by about four times as the acidity of the [M + H] + N -terminal residue increases from R (19.0 %) to D (76.5 %). These results indicate that protonated peptides containing nitroarginine can undergo non-canonical mobile proton triggered radical fragmentation. Figure ᅟ