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The objective was to determine the concentration of total Se and the proportion of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in postmortem tissues of beef cattle offered diets containing graded additions of selenized enriched yeast (SY; Saccharomyces cerevisiae CNCM 1-3060) or sodium selenite (SS). Oxidative stability and tissue glutathione peroxidase (GSH-Px) activity of edible muscle tissue were assessed 10 d postmortem. Thirty-two beef cattle were offered, for a period of 112 d, a total mixed ration that had been supplemented with SY (0, 0.15, or 0.35 mg of Se/kg of DM) or SS (0.15 mg of Se/kg of DM). At enrollment (0 d) and at 28, 56, 84, and 112 d following enrollment, blood samples were taken for Se and Se species determination, as well as whole blood GSH-Px activity. At the end of the study beef cattle were killed and samples of heart, liver, kidney, and skeletal muscle (LM and psoas major) were retained for Se and Se species determination. Tissue GSH-Px activity and thiobarbituric acid reactive substances were determined in skeletal muscle tissue (LM only). The incorporation into the diet of ascending concentrations of Se as SY increased whole blood total Se and the proportion of total Se comprised as SeMet, as well as GSH-Px activity. There was also a dose-dependent response to the graded addition of SY on total Se and proportion of total Se as SeMet in all tissues and GSH-Px activity in skeletal muscle tissue. Furthermore, total Se concentration of whole blood and tissues was greater in those animals offered SY when compared with those receiving a comparable dose of SS, indicating an improvement in Se availability and tissue Se retention. Likewise, GSH-Px activity in whole blood and LM was greater in those animals offered SY when compared with those receiving a comparable dose of SS. However, these increases in tissue total Se and GSH-Px activity appeared to have little or no effect in meat oxidative stability. Key words: beef, selenium, selenocysteine, selenomethionine, thiobarbituric acid reactive substance

The N and energy utilization of diets with different legume seeds was analysed in lactating goats. Four isonitrogenous and isoenergetic diets were used and in each case 30% of the protein was supplied by one of four different legume seeds: lupins (L), faba beans (FB), bitter vetch (BV) and vetch (V). A group of eight Granadina goats, allocated to a replicated 4 × 4 Latin Square, were used. The N and energy utilization of the different diets may be considered adequate; milk N/intake N was approximately 0.20, milk N/N balance was approximately 0.50 and milk energy/metabolizable energy was approximately 0.30. Under similar N and energy intakes, faecal and urinary excretion of N and the quantities in milk did not vary significantly. However, the partition of the N balance between milk production and deposit within the body varied depending on the diet. Although this N available to the metabolism was the highest for the FB diet, the quantity of this directed to milk was the lowest. Finally, the faecal and urinary excretion with respect to the quantity directed to milk production did not vary significantly among the different diets. [ABSTRACT FROM AUTHOR]

The ADAT2/ADAT3 complex catalyzes the adenosine to inosine modification at the wobble position of eukaryotic tRNAs. Mutations in ADAT3 , the catalytically inactive subunit of the ADAT2/ADAT3 complex, have been identified in patients presenting with severe neurodevelopmental disorders (NDDs). Yet, the physiological function of ADAT2/ADAT3 complex during brain development remains totally unknown. Here we showed that maintaining a proper level of ADAT2/ADAT3 catalytic activity is required for correct radial migration of projection neurons in the developing mouse cortex. In addition, we not only reported 20 new NDD patients carrying biallelic variants in ADAT3 but also deeply characterized the impact of those variants on ADAT2/ADAT3 structure, biochemical properties, enzymatic activity and tRNAs editing and abundance. We demonstrated that all the identified variants alter both the abundance and the activity of the complex leading to a significant decrease of I 34 with direct consequence on their steady-state. Using in vivo complementation assays, we correlated the severity of the migration phenotype with the degree of the loss of function caused by the variants. Altogether, our results indicate a critical role of ADAT2/ADAT3 during cortical development and provide cellular and molecular insights into the pathogenicity of ADAT3-related neurodevelopmental disorder.

The NAD + -dependent lysine deacylase sirtuin 2 (Sirt2) is involved in multiple pathological conditions such as cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purposes. Furthermore, the orthologue from Schistosoma mansoni ( Sm Sirt2) has been considered for the potential treatment of the neglected tropical disease schistosomiasis. We previously identified a 1,2,4-oxadiazole-based scaffold from the screening of the "Kinetobox" library as a dual inhibitor of human Sirt2 (hSirt2) and Sm Sirt2. Herein, we describe the structure-activity studies on 1,2,4-oxadiazole-based analogues, which are potent inhibitors of human Sirt2 deacetylation. As proposed by docking studies, a substrate-competitive and cofactor-noncompetitive binding mode of inhibition could be determined in vitro via binding assays and kinetic analysis and further confirmed by a crystal structure of an oxadiazole inhibitor in complex with hSirt2. Optimized analogues reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.

Background: The red macroalgae Asparagopsis is an effective methanogenesis inhibitor due to the presence of halogenated methane (CH 4 ) analogues, primarily bromoform (CHBr 3 ). This study aimed to investigate the degradation process of CHBr 3 from A. taxiformis in the rumen and whether this process is diet-dependent. An in vitro batch culture system was used according to a 2 × 2 factorial design, assessing two A. taxiformis inclusion rates [0 (CTL) and 2% DM diet (AT)] and two diets [high-concentrate (HC) and high-forage diet (HF)]. Incubations lasted for 72 h and samples of headspace and fermentation liquid were taken at 0, 0.5, 1, 3, 6, 8, 12, 16, 24, 48 and 72 h to assess the pattern of degradation of CHBr 3 into dibromomethane (CH 2 Br 2 ) and fermentation parameters. Additionally, an in vitro experiment with pure cultures of seven methanogens strains (Methanobrevibacter smithii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanosarcina barkeri, Methanobrevibacter millerae, Methanothermobacter wolfei and Methanobacterium mobile) was conducted to test the effects of increasing concentrations of CHBr 3 (0.4, 2, 10 and 50 µmol/L)., Results: The addition of AT significantly decreased CH 4 production (P = 0.002) and the acetate:propionate ratio (P = 0.003) during a 72-h incubation. The concentrations of CHBr 3 showed a rapid decrease with nearly 90% degraded within the first 3 h of incubation. On the contrary, CH 2 Br 2 concentration quickly increased during the first 6 h and then gradually decreased towards the end of the incubation. Neither CHBr 3 degradation nor CH 2 Br 2 synthesis were affected by the type of diet used as substrate, suggesting that the fermentation rate is not a driving factor involved in CHBr 3 degradation. The in vitro culture of methanogens showed a dose-response effect of CHBr 3 by inhibiting the growth of M. smithii, M. ruminantium, M. stadtmanae, M. barkeri, M. millerae, M. wolfei, and M. mobile., Conclusions: The present work demonstrated that CHBr 3 from A. taxiformis is quickly degraded to CH 2 Br 2 in the rumen and that the fermentation rate promoted by different diets is not a driving factor involved in CHBr 3 degradation., (© 2023. Chinese Association of Animal Science and Veterinary Medicine.)

Schistosomiasis is a prevalent yet neglected tropical parasitic disease caused by the Schistosoma genus of blood flukes. Praziquantel is the only currently available treatment, hence drug resistance poses a major threat. Recently, histone deacetylase 8 (HDAC8) selective inhibitors have been proposed as a viable treatment for schistosomiasis. Herein, we report the phenotypic screening of a focused library of small molecules of varying HDAC isozyme-inhibition profiles, including eight HDAC8 inhibitors with >10-fold selectivity in comparable functional inhibition assays and IC 50 values against HDAC8<100 nM. HDAC8-selective inhibitors showed the lowest potency against Schistosoma mansoni newly transformed schistosomula (NTS). Pan-HDAC inhibitors MMH258, MMH259, and MMH373, as assessed by functional inhibition assays, with minimal or no-observed hHDAC8 and SmHDAC8 activities, were active against both NTS (MMH258, IC 50 =1.5 μM; MMH259, IC 50 =2.3 μM) and adult S. mansoni (MMH258, IC 50 =2.1 μM; MMH373, IC 50 =3.4 μM). Our results indicate that neither hHDAC8 nor SmHDAC8 activity were directly correlated to their NTS and adult S. mansoni activities., (© 2022 Wiley-VCH GmbH.)

Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks. Here, we show that the zinc-dependent histone deacetylases (HDACs) of the protozoan parasite Trypanosoma cruzi are key regulators that have significantly diverged from their human counterparts. Depletion of T. cruzi class I HDACs tcDAC1 and tcDAC2 compromises cell-cycle progression and division, leading to cell death. Notably, tcDAC2 displays a deacetylase activity essential to the parasite and shows major structural differences with human HDACs. Specifically, tcDAC2 harbors a modular active site with a unique subpocket targeted by inhibitors showing substantial anti-parasitic effects in cellulo and in vivo. Thus, the targeting of the many atypical HDACs in pathogens can enable anti-parasitic selective chemical impairment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The only drug currently available for treatment of the neglected disease Schistosomiasis is Praziquantel, and the possible emergence of resistance makes research on novel therapeutic agents necessary and urgent. To this end, the targeting of Schistosoma mansoni epigenetic enzymes, which regulate the parasitic life cycle, emerged as a promising approach. Due to the strong effects of human sirtuin inhibitors on parasite survival and reproduction, Schistosoma sirtuins were postulated as potential therapeutic targets. In vitro testing of synthetic substrates of S. mansoni sirtuin 2 ( Sm Sirt2) and kinetic experiments on a myristoylated peptide demonstrated lysine long-chain deacylation as an intrinsic Sm Sirt2 activity in addition to its known deacetylase activity for the first time. Focused in vitro screening of the GSK Kinetobox library and structure-activity relationships of identified hits led to the first Sm Sirt2 inhibitors with activity in the low micromolar range. Several Sm Sirt2 inhibitors showed potency against both larval schistosomes (viability) and adult worms (pairing, egg laying) in culture without general toxicity to human cancer cells.

Although the potential of plants extracts to improve feed efficiency and animal productivity and decrease methane emissions by enteric fermentation has been shown, the information available is often contradictory which has been attributed to differences in the complex mixture of bioactive compounds and their interactions. Understanding the degree to which structural features in a compound may affect the biological activity of an extract is essential. We hypothesised that relative small variations in the structure of a compound can have a significant influence on the ability of the derivatives to alter fermentation in the rumen. Nine compounds were synthetized from the natural alkaloid haemanthamine and tested in vitro for their effects on rumen protozoa and fermentation parameters. Our results showed that simple esterifications of haemanthamine or its derivative dihydrohaemanthamine with acetate, butyrate, pivalate or hexanoate led to compounds that differed in their effects on rumen fermentation.

Metal-dependent histone deacetylases (HDACs) are key epigenetic regulators that represent promising therapeutic targets for the treatment of numerous human diseases. Yet the currently FDA-approved HDAC inhibitors nonspecifically target at least several of the 11 structurally similar but functionally different HDAC isozymes, which hampers their broad usage in clinical settings. Selective inhibitors targeting single HDAC isozymes are being developed, but precise understanding in molecular terms of their selectivity remains sparse. Here, we show that HDAC8-selective inhibitors adopt a L-shaped conformation required for their binding to a HDAC8-specific pocket formed by HDAC8 catalytic tyrosine and HDAC8 L1 and L6 loops. In other HDAC isozymes, a L1-L6 lock sterically prevents L-shaped inhibitor binding. Shielding of the HDAC8-specific pocket by protein engineering decreases potency of HDAC8-selective inhibitors and affects catalytic activity. Collectively, our results unravel key HDAC8 active site structural and functional determinants important for the design of next-generation chemical probes and epigenetic drugs.

The antiprotozoal effect of saponins is transitory, as when saponins are deglycosylated to sapogenins by rumen microorganisms they become inactive. We hypothesised that the combination of saponins with glycosidase-inhibiting iminosugars might potentially increase the effectiveness of saponins over time by preventing their deglycosylation in the rumen. Alternatively, modifying the structure of the saponins by substituting the sugar moiety with other small polar residues might maintain their activity as the sugar substitute would not be enzymatically cleaved. The aim of this in vitro study was to evaluate the acute antiprotozoal effect and the stability of this effect over a 24 h incubation period using ivy saponins, a stevia extract rich in iminosugars, ivy saponins with stevia extract, and a chemically modified ivy saponin, hederagenin bis-succinate (HBS). The effects on fermentation parameters and rumen bacterial communities were also studied. Ivy saponins with stevia and HBS had a greater antiprotozoal effect than ivy saponins, and this effect was maintained after 24 h of incubation (P<0.001). The combination of ivy and stevia extracts was more effective in shifting the fermentation pattern towards higher propionate (+39%) and lower butyrate (-32%) and lower ammonia concentration (-64%) than the extracts incubated separately. HBS caused a decrease in butyrate (-45%) and an increase in propionate (+43%) molar proportions. However, the decrease in ammonia concentration (-42%) observed in the presence of HBS was less than that caused by ivy saponins, either alone or with stevia. Whereas HBS and stevia impacted on bacterial population in terms of community structure, only HBS had an effect in terms of biodiversity (P<0.05). It was concluded that ivy saponins with stevia and the modified saponin HBS had a strong antiprotozoal effect, although they differed in their effects on fermentation parameters and bacteria communities. Ivy saponins combined with an iminosugar-rich stevia extract and/or HBS should be evaluated to determine their antiprotozoal effect in vivo.

The aim of this work was to evaluate the effect of feeding management during the first month of life (natural with the mother, NAT, or artificial with milk replacer, ART) on the rumen microbial colonization and the host innate immune response. Thirty pregnant goats carrying two fetuses were used. At birth one kid was taken immediately away from the doe and fed milk replacer (ART) while the other remained with the mother (NAT). Kids from groups received colostrum during first 2 days of life. Groups of four kids (from ART and NAT experimental groups) were slaughtered at 1, 3, 7, 14, 21 and 28 days of life. On the sampling day, after slaughtering, the rumen content was sampled and epithelial rumen tissue was collected. Pyrosequencing analyses of the bacterial community structure on samples collected at 3, 7, 14 and 28 days showed that both systems promoted significantly different colonization patterns (P = 0.001). Diversity indices increased with age and were higher in NAT feeding system. Lower mRNA abundance was detected in TLR2, TLR8 and TLR10 in days 3 and 5 compared to the other days (7, 14, 21 and 28). Only TLR5 showed a significantly different level of expression according to the feeding system, presenting higher mRNA abundances in ART kids. PGLYRP1 showed significantly higher abundance levels in days 3, 5 and 7, and then experienced a decline independently of the feeding system. These observations confirmed a highly diverse microbial colonisation from the first day of life in the undeveloped rumen, and show that the colonization pattern substantially differs between pre-ruminants reared under natural or artificial milk feeding systems. However, the rumen epithelial immune development does not differentially respond to distinct microbial colonization patterns.

The antiprotozoal effect of saponins is transitory, as when saponins are deglycosylated to the sapogenin by rumen microorganisms they become inactive. We postulated that the substitution of the sugar moiety of the saponin with small polar residues would produce sapogen-like analogs which might be resistant to degradation in the rumen as they would not be enzymatically cleaved, allowing the antiprotozoal effect to persist over time. In this study, we used an acute assay based on the ability of protozoa to break down [ 14 C] leucine-labeled Streptococcus bovis and a longer term assay based on protozoal motility over 24 h to evaluate both the antiprotozoal effect and the stability of this effect with fifteen hederagenin bis -esters esterified with two identical groups, and five cholesterol and cholic acid based derivatives carrying one to three succinate residues. The acute antiprotozoal effect of hederagenin derivatives was more pronounced than that of cholesterol and cholic acid derivatives. Modifications in the structure of hederagenin, cholesterol, and cholic acid derivatives resulted in compounds with different biological activities in terms of acute effect and stability, although those which were highly toxic to protozoa were not always the most stable over time. Most of the hederagenin bis -esters, and in particular hederagenin bis -succinate (TSB24), hederagenin bis -betainate dichloride (TSB37) and hederagenin bis -adipate (TSB47) had a persistent effect against rumen protozoa in vitro , shifting the fermentation pattern toward higher propionate and lower butyrate. These chemically modified triterpenes could potentially be used in ruminant diets as an effective defaunation agent to, ultimately, increase nitrogen utilization, decrease methane emissions, and enhance animal production. Further trials in vivo or in long term rumen simulators are now needed to confirm the in vitro observations presented.

Background: Eight natural products from animal, unicellular algae, brown seaweed and plant origins were chosen according to their theoretical antimicrobial activity: Diatomaceous earths (DE), insoluble chitosan (ICHI), soluble chitosan (CHI), seaweed meal (SWM), Ascophyllum nodosum (ASC), Laminaria digitata (LAM), neem oil (NOIL) and an ivy fruit extract rich in saponins (IVY). Dose-response incubations were conducted to determine their effect on rumen fermentation pattern and gas production, while their anti-protozoal activity was tested using (14) C-labelled bacteria., Results: DE, SWM, NOIL and ICHI had very small effects on rumen function when used at inclusion rate up to 2 g L(-1) . ASC had anti-protozoal effects (up to -23%) promoting a decrease in gas production and methanogenesis (-15%). LAM increased VFA production (+7%) and shifted from butyrate to acetate. CHI also shifted fermentation towards propionate production and lower methane (-23%) and protozoal activity (-56%). IVY decreased protozoal activity (-39%) and ammonia concentration (-56%), as well as increased feed fermentation (+11% VFA concentration) and shifted from acetate to propionate production., Conclusions: ASC, LAM, CHI and IVY showed promising potential in vitro as feed additives to improve rumen function, thus more research is needed to investigate their mode of action in the rumen microbial ecosystem. © 2015 Society of Chemical Industry., (© 2015 Society of Chemical Industry.)

First described in 1843, Rumen protozoa with their striking appearance were assumed to be important for the welfare of their host. However, despite contributing up to 50% of the bio-mass in the rumen, the role of protozoa in rumen microbial ecosystem remains unclear. Phylogenetic analysis of 18S rDNA libraries generated from the rumen of cattle, sheep, and goats has revealed an unexpected diversity of ciliated protozoa although variation in gene copy number between species makes it difficult to obtain absolute quantification. Despite repeated attempts it has proven impossible to maintain rumen protozoa in axenic culture. Thus it has been difficult to establish conclusively a role of ciliate protozoa in rumen fiber degradation. The development of techniques to clone and express ciliate genes in λ phage, together with bioinformatic indices to confirm the ciliate origin of the genes has allowed the isolation and characterization of fibrolytic genes from rumen protozoa. Elimination of the ciliate protozoa increases microbial protein supply by up to 30% and reduces methane production by up to 11%. Our recent findings suggest that holotrich protozoa play a disproportionate role in supporting methanogenesis whilst the small Entodinium are responsible for much of the bacterial protein turnover. As yet no method to control protozoa in the rumen that is safe and practically applicable has been developed, however a range of plant extract capable of controlling if not completely eliminating rumen protozoa have been described.