The work reported in the Research Communication investigated in vitro rumen gas kinetics and fermentation profile as well as in vivo performance of lactating ewes fed corn silage (CS), sunflower silage (SFS) and their 50 : 50 mixture (CS-SFS). For the in vivo experiment, nine early-lactation Suffolk × Texel ewes were grouped in a replicated 3 × 3 Latin square design of three 21-d periods. Treatments were based on ad libitum CS, SFS, and CS-SFS supplemented with concentrate at 48 g/kg LW0.75. In vitro results showed that the CS had the highest dry matter degraded substrate and microbial crude protein production followed by CS-SFS. The in vivo data showed that animals fed on CS had higher digestibility of dry matter and organic matter than CS-SFS, while SFS were intermediate. Nitrogen (N) intake, fecal N excretion, and urine N excretion were similar between groups, however, milk N excretion was lower in SFS than CS. Milk yield was higher for CS and CS-SFS than SFS group, however, SFS-fed ewes had higher milk fat content than either CS or CS-SFS (all differences reported here were significant, P < 0.05 or better). Overall, CS-SFS could be used as dietary roughage for dairy ewes without deleterious effects on nutrient intake, N-balance and milk yield whilst potentially offering a more sustainable alternative to CS.

Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum. Herein, we investigated its in vitro and in vivo activity against L. amazonensis. CL5564 was 6.5-fold (P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg−1, intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg−1, while the combination (10 + 40 mg kg−1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

The experiments reported in this research paper address the effects of replacing ground corn (GC) with full-fat corn germ (FFCG) on nutrient intake and digestibility, nitrogen utilization efficiency, performance, and predicted methane production in dairy cows fed cactus cladodes and sugarcane. We hypothesized that the inclusion of FFCG in the diet would not alter the performance of lactating cows but would reduce the predicted methane production in vivo. Ten multiparous Holstein cows at 90 ± 10 d of lactation and yielding 24.2 ± 3.5 kg milk/d were assigned to dietary treatments consisting of different levels of replacement of GC by FFCG (0; 25; 50; 75 and 100% of diet dry matter) in a replicated 5 × 5 Latin square design with 21-d periods. Methane production was predicted using an automated gas in vitro production system. Except for ether extract intake, which increased, the intake of all nutrients decreased linearly with the replacement of GC by FFCG. The digestibility of dry matter, organic matter and neutral detergent fiber reduced, whereas the digestibility of ether extract increased linearly with FFCG. There were no changes in the digestibility of crude protein. The nitrogen intake and daily excretion in urine and feces decreased, while nitrogen use efficiency increased linearly. There was no significant effect of diets on nitrogen balance or microbial protein synthesis and efficiency. The yield of protein, lactose and total solids in milk showed a quadratic behavior. On the other hand, milk fat yield and energy-corrected milk yield decreased linearly with the replacement of GC by FFCG. No effect on pH or ammonia nitrogen was observed. The production of methane (CH4, g/kg DM) and total CH4 (g/d), and CH4 intensity decreased linearly with the replacement of GC by FFCG. In conclusion, FFCG has been shown to be an effective source of fat to reduce methane production in dairy cows, partially supporting our initial hypothesis. However, as it decreases milk fat production, it is not recommended to replace more than 50% of GC by FFCG for lactating cows fed cactus cladodes and sugarcane.

Glioblastoma (GBM) is the most frequent adult malignant brain tumour and despite different therapeutic efforts, the median overall survival still ranges from 14 to 18 months. Thus, new therapeutic strategies are urgently needed. However, the identification of cancer-specific targets is particularly challenging in GBM, due to the high heterogeneity of this tumour in terms of histopathological, molecular, genetic and epigenetic features. Telomerase reactivation is a hallmark of malignant glioma. An activating mutation of the hTERT gene, encoding for the active subunit of telomerase, is one of the molecular criteria to establish a diagnosis of GBM, IDH-wildtype, in the 2021 WHO classification of central nervous system tumours. Telomerase inhibition therefore represents, at least theoretically, a promising strategy for GBM therapy: pharmacological compounds, as well as direct gene expression modulation therapies, have been successfully employed in in vitro and in vivo settings. Unfortunately, the clinical applications of telomerase inhibition in GBM are currently scarce. The aim of the present systematic review is to provide an up-to-date report on the studies investigating telomerase inhibition as a therapeutic strategy for malignant glioma in order to foster the future translational and clinical research on this topic.

In vitro rearing of honey bee larvae is ideal for bioassay studies; no honey bee stable cell lines are available. Inconsistency of internal development staging of reared larvae and a susceptibility to contamination are common problems encountered. Standardized protocols on rearing larvae in vitro to make the larvae growth and development more similar to that of natural colonies are necessary to ensure the accuracy of experimental results and promote honey bee research as a model organism. Here, we concluded that when larval fasting weight was >160 mg, the time point of gut emptying can be defined as the critical point separating the larval and prepupal stages. In this way, we can conduct precise studies on the prepupal stage, such as organ remodeling during metamorphosis. Simultaneously, we further verified that recombinant AccApidaecin in genetic engineered bacteria added to the larval diet upregulated antibacterial peptide gene expression, and did not stimulate the stress response in larvae, nor did it affect the pupation rate or eclosion rate. This demonstrated that feeding recombinant AccApidaecin can enhance the individual antibacterial ability at the molecular level.

Chagas disease (CD) is a neglected tropical disease caused by the intracellular protozoan Trypanosoma cruzi that remains a serious public health issue affecting more than 6 million people worldwide. The available treatment includes 2 nitro derivatives, benznidazole (BZ) and nifurtimox, that lack in efficacy in the later chronic phase and when administered against the several naturally resistant parasite strains and present several side-effects, demanding new therapeutic options. One strategy is based on repurposing by testing drugs already used for other illness that may share similar targets. In this context, our previous data on imatinib (IMB) and derivatives motivated the screening of 8 new IMB analogues. Our findings showed that all except 1 were active against bloodstream trypomastigotes reaching drug concentration capable of inducing a 50% of parasite lysis (EC50) values < 12 μm after 2 h while BZ was inactive. After 24 h, all derivatives were more potent than BZ, exhibiting EC50 values 1.5–5.5 times lower. Against intracellular forms, 7 out of 8 derivatives presented high activity, with EC50 values ≤ BZ. LS2/89 stood out as one of the most promising, reaching EC90 values of 1.68 and 4.9 μm on intracellular and trypomastigote forms, respectively, with the best selectivity index (>60) towards the proliferative forms. Physicochemical parameters as well as the absorption, distribution, metabolism, excretion and toxicity properties were predicted to be acceptable and with good chance of a favourable oral bioavailability. The promising results motivate further studies such as in vivo and combinatory assays aiming to contribute for a novel safer and effective therapy for CD.

Taenia solium is the aetiological agent of cysticercosis, a zoonosis that causes severe health and economic losses across Latin America, Africa and Asia. The most serious manifestation of the disease is neurocysticercosis, which occurs when the larval stage (cysticercus) establishes in the central nervous system. Using Taenia crassiceps as an experimental model organism for the study of cysticercosis, we aimed to identify the in vitro conditions necessary to allow parasite development at the short- and long terms. First, cysticerci were incubated for 15 days in different media and parasite densities. The number of buddings and cysticerci diameter were measured to evaluate asexual multiplication and parasite growth, respectively. Vitality was determined by trypan blue staining and morphology analysis. As a result, high cysticerci density and medium containing FBS and the excretion/secretion (E/S) products of feeder cells induced parasite survival, growth and multiplication. Then, the long-term (5 weeks) incubation of the parasites in co-culture with feeder cells was evaluated. Consequently, the mammalian cell lines induced a significant increase in total parasite volume while axenic cultures did not show any statistically significant change over time. In this study, the proper conditions to maintain T. crassiceps in vitro are described for the first time in a simpler and more controlled setting other than experimental infections. In addition, it was shown that cysticerci growth, survival and asexual multiplication depend on a complex network of secreted factors from both parasite and host.

Fasciola gigantica, the causative agent of tropical fasciolosis, is a food-borne zoonotic trematode that affects around 80% livestock of Bangladesh. Triclabendazole (TCBZ), nitroxynil (NTON) and oxyclozanide (OCZN) are frequently used against fascioliasis; however, the current status of potency of these flukicides was unknown. In this study, in vitro efficacy of TCBZ, NTON and OCZN at various concentrations on F. gigantica has been evaluated by relative motility (RM), morphological distortions of apical cone through an inverted microscope, architectural and ultra-structural changes through histopathological and scanning electron microscopy (SEM). It is observed that TCBZ, NTON and OCZN at higher concentrations significantly (P < 0.05) reduced RM of the flukes compared to untreated control. NTON at 150 μg mL−1 was the most potent to reduce the motility within 4 h whereas TCBZ and OCZN were much delayed. Histopathological changes showed swollen, extensive cracking, numerous vacuoles and splitting of the tegument surrounding the spines; spine dislodged from its socket in treated flukes compared to untreated worms. Histopathological changes were more conspicuous at higher doses of TCBZ, NTON and OCZN. SEM has shown the disruption of the apical cone, apart from swelling of the tegument on the ventral surface corrugation and disruption of the ventral apical cone. All these changes indicate that NTON is the most potent in killing flukes in vitro among the tested flukicides and suggest the presence of TCBZ-resistant fluke populations in Bangladesh. It is imperative to explore the in vivo effects of these flukicides and subsequently their molecular mechanisms.

We review key findings of one the most cited papers in the 75-year history of BJN. We then identify important consequent developments, as well as opportunities to use analytical and molecular biology advances to maximise conversion of non-protein nitrogen into microbial protein.

Micropropagation techniques allow producing large numbers of clones of genetically identical plants. However, there is evidence of disorders in internal structures due to sophisticated in vitro conditions. Such variations are responsible for the mortality of plantlets in the field and cause huge loss to the tissue culture industry. Anatomical evaluation at different growth conditions allows for understanding structural repair of in vitro raised plantlets. Therefore, the present study was aimed to identify the structural changes that occurred in micropropagated plants of Vitex negundo under heterotrophic, photomixotrophic, and photoautotrophic conditions. To achieve this, structural variations were analyzed in the plantlets obtained from in vitro, greenhouse and field transferred stages using light microscopy. Underdeveloped dermal tissues, palisade cells, intercellular spaces, mechanical tissues, vascular bundles, and ground tissues were observed with the plants growing under in vitro conditions. The self-repairing of structural disorders and transitions in vegetative anatomy was observed during hardening under the greenhouse environment. Field transferred plantlets were characterized by well-developed internal anatomy. These findings showed that the micropropagated plantlets of V. negundo were well-adapted through a series of self-repairing the in vitro induced structural abnormalities at the subsequent stages of plant development.

In vitro culture of the embryo is a useful method to treat infertility that shows embryo potential for selecting the best one to transfer and successfully implantation. However, embryo development in vitro is affected by oxidative stresses such as reactive oxygen species that may damage embryo development. Antioxidants are molecules found in fruits, vegetables, and fish that play an important role in reducing oxidative processes. In the natural environment, there is a physiological antioxidant system that protects embryos against oxidative damage. This antioxidant system does not exist in vitro. Antioxidants act as free radical scavengers and protect cells or repair damage done by free radicals. Various studies have shown that adding antioxidants into embryo culture medium improves embryo development in vitro. This review article emphasizes different aspects of various antioxidants, including types, functions and mechanisms, on the growth improvement of different species of embryos in vitro.

The supplementation of ruminant diets with exogenous cellulolytic enzymes can improve their digestibility and feeding value. The objective of this study was to determine the effect of supplementing roughage (rice straw) and concentrate with inoculants containing four fungal strains (Pleurotus ostreatus, Phanerochaete chrysosporium, Trichoderma reesei and Trichoderma viride) and four bacterial strains (Paenibacillus polymyxa, Bacillus megaterium, Bacillus circulans and Bacillus subtilis), given separately or as a mixture, as a source of exogenous cellulolytic enzymes, on basic rumen parameters in vitro, including digestibility and methane production. A batch culture trial was used to select the best supplements, and a long-term rumen simulation technique (RUSITEC) was used to evaluate the effects of P. chrysosporium, B. subtilis, and a 1 : 1 mixture of these two on dietary component digestibility and fermentation parameters. In the batch culture evaluation, there were significant increases in the organic matter (OM) digestibility, the total gas production expressed as ml/g of dry matter, the OM, the neutral detergent fibre (NDF) and the acid detergent fibre (ADF) of the supplemented rations, as compared to the control, excluding the rations supplemented with T. viride and B. circulans. In the RUSITEC, the ration supplemented with mixed inoculants showed significantly higher digestibility of crude protein, ether extract, NDF and ADF than did the ration supplemented with the P. chrysosporium and B. subtilis inoculants. It can be concluded that the simultaneous use of fungal and bacterial exogenous cellulases on rice straw roughage improves its digestibility, without negative effects on other rumen parameters.

In previous studies, we have identified several families of 5-nitroindazole derivatives as promising antichagasic prototypes. Among them, 1-(2-aminoethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one, (hydrochloride) and 1-(2-acetoxyethyl)-2-benzyl-5-nitro-1,2-dihydro-3H-indazol-3-one (compounds 16 and 24, respectively) have recently shown outstanding activity in vitro over the drug-sensitive Trypanosoma cruzi CL strain (DTU TcVI). Here, we explored the activity of these derivatives against the moderately drug-resistant Y strain (DTU TcII), in vitro and in vivo. The outcomes confirmed their activity over replicative forms, showing IC50 values of 0.49 (16) and 5.75 μm (24) towards epimastigotes, 0.41 (16) and 1.17 μm (24) against intracellular amastigotes. These results, supported by the lack of toxicity on cardiac cells, led to better selectivities than benznidazole (BZ). Otherwise, they were not as active as BZ in vitro against the non-replicative form of the parasite, i.e. bloodstream trypomastigotes. In vivo, acute toxicity assays revealed the absence of toxic events when administered to mice. Moreover, different therapeutic schemes pointed to their capability for decreasing the parasitaemia of T. cruzi Y acute infected mice, reaching up to 60% of reduction at the peak day as monotherapy (16), 79.24 and 91.11% when 16 and 24 were co-administered with BZ. These combined therapies had also a positive impact over the mortality, yielding survivals of 83.33 and 66.67%, respectively, while untreated animals reached a cumulative mortality of 100%. These findings confirm the 5-nitroindazole scaffold as a putative prototype for developing novel drugs potentially applicable to the treatment of Chagas disease and introduce their suitability to act in combination with the reference drug.

The process of embryonic development is crucial and radically influences preimplantation embryo competence. It involves oocyte maturation, fertilization, cell division and blastulation and is characterized by different key phases that have major influences on embryo quality. Each stage of the process of preimplantation embryonic development is led by important signalling pathways that include very many regulatory molecules, such as primary and secondary messengers. Many studies, both in vivo and in vitro, have shown the importance of the contribution of reactive oxygen species (ROS) as important second messengers in embryo development. ROS may originate from embryo metabolism and/or oocyte/embryo surroundings, and their effect on embryonic development is highly variable, depending on the needs of the embryo at each stage of development and on their environment (in vivo or under in vitro culture conditions). Other studies have also shown the deleterious effects of ROS in embryo development, when cellular tissue production overwhelms antioxidant production, leading to oxidative stress. This stress is known to be the cause of many cellular alterations, such as protein, lipid, and DNA damage. Considering that the same ROS level can have a deleterious effect on the fertilizing oocyte or embryo at certain stages, and a positive effect at another stage of the development process, further studies need to be carried out to determine the rate of ROS that benefits the embryo and from what rate it starts to be harmful, this measured at each key phase of embryonic development.

Paroxetine is a highly potent and selective inhibitor of serotonin reuptake, with in vitro potency greater than that of fluoxetine, fluvoxamine, and sertraline. It has little affinity for a wide variety of neurotransmitter receptors, including catecholaminergic or histaminergic systems, in marked contrast to the tricyclic antidepressants. Paroxetine undergoes first-pass metabolism that is partially saturable to give metabolites that are pharmacologically inactive in vivo, unlike those of fluoxetine or sertraline.

Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 μm (10 μg mL−1) and IVM at 1.1 μm (1 μg mL−1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

The effects of silicon incorporation on the in vitro and in vivo properties of magnesium phosphate (MgP) bioceramics were studied. Samples were prepared by conventional solid state synthesis method. Scanning electron microscopy and micro-computed tomography (µ-CT) analysis showed that Si doping reduces degradability of MgP. In vitro studies have shown that MG63 cells can attach and proliferate on MgP samples. Live/dead imaging showed that MgP–0.5Si sample had highest cell proliferation, which was also quantitatively confirmed by alamar blue assay. In vivo biocompatibility of MgP ceramics was assessed after implantation in rabbit model. Detailed µ-CT analysis showed new bone tissue formation around the implant after 30 and 90 days. MgP–0.5Si ceramics had 84% bone regeneration compared with 56% for pure MgP ceramics, as confirmed by oxytetracycline labeling. Our finding suggests that Si doping can alter physicochemical properties of MgP ceramics and promotes osseointegration, which can be a useful choice for bone tissue engineering.

Nitroethane (NE), 2-nitroethanol (NEOH) and 2-nitro-1-propanol (NPOH) were investigated in order to determine their inhibitory effects on in vitro ruminal fermentation and methane (CH4) production of a hay-rich substrate (alfalfa hay: maize meal = 4:1, w/w). The rumen liquor collected from cannulated Holstein dairy cows was incubated at 39 °C for 72 h. The addition of NE, NEOH and NPOH slowed down the fermentation process and notably decreased molar CH4 proportion by 96.8, 96.4 and 35.0%, respectively. The abundance of total methanogen and methanogens from the order Methanobacteriales were all decreased with NE, NEOH and NPOH supplementation. Meanwhile, the nitrocompound addition reduced mcrA gene expression, coenzyme F420 and F430 contents. The correlation analysis showed that CH4 production was correlated positively with the population abundance of total methanogens, Methanobacteriales, mcrA gene expression, coenzyme contents of F420 and F430. The nitrocompound addition decreased acetate concentration and increased propionate and butyrate concentrations in the culture fluid. In summary, both NE and NEOH addition presented nearly the same inhibitory effectiveness on in vitro CH4 production; they were more effective than NPOH. The results of the current study provide evidence that NE, NEOH and NPOH can dramatically decrease methanogen population, mcrA gene expression and the coenzyme content of F420 and F430 in ruminal methanogenesis.