Performing AI 55 or 65 h after progesterone device removal did not differ in a 7-d progesterone-based protocol for timed-AI in Nellore suckled cows.

This study aimed to evaluate the reproductive performance of Nellore suckled cows inseminated 55 (n = 304) or 65 (n = 296) h after progesterone (P4) removal in a 7-d protocol. The protocol consisted of the insertion of a device with 2 g of P4 and 2 mg of estradiol benzoate on d 0, with the device remaining in the cows for 7 d. Cows in the 55-h treatment had the P4 device removed in the morning, while cows in the 65-h treatment had the P4 device removed in the afternoon. At P4 removal, cows received intramuscularly 300 IU of eCG, 0.6 mg of estradiol cypionate and 0.52 mg cloprostenol sodium. Artificial insemination was performed according to treatments (55 vs. 65 h after P4 removal). The results of the study showed that the estrus detection rate (69% vs 65%) and pregnancy per AI (P/AI; 49% vs 49%) did no differ in cows inseminated 55 or 65 h after P4 removal, respectively. Furthermore, ovulation rate, the diameter of the largest follicle at the time of AI, and P4 concentration after AI were not affected by treatments. The probability of P/AI was not affected by parity, BCS, age, diameter of largest follicle at AI, days postpartum, BW and time to AI. This study suggests that performing AI from 55 to 65 h after the P4 removal in the 7-d-P4 protocol did not affect the reproductive performance in Nellore cows, and opens the possibility for producers to take more time to perform AI of cows in the field without affecting P/AI.

Single-cell transcriptomics reveals immune suppression and cell states predictive of patient outcomes in rhabdomyosarcoma.

Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin that is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we use single-cell mRNA sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche reveals evidence of an immunosuppressive microenvironment. We also identify a putative interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a potential cause of tumour-induced T-cell dysfunction. In malignant RMS cells, we define transcriptional programs reflective of normal myogenic differentiation and show that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the less aggressive fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Processing methods of flint corn and protein supplement in forage-free diets for feedlot lambs.

Diets without forage increase the productivity in less time and favor greater practicality and better quality carcass. The corn grain is mostly used associated with pellets but processing these ingredients can bring benefits. The present study evaluated the effect of diets without forage based on whole or coarse ground corn associated with pelleted or ground protein supplement on performance, eating behavior, carcass characteristics, and ruminal morphology of feedlot lambs. Thirty-five Dorper × Santa Inês lambs were used, with 23.85 ± 3.88 kg of initial body weight and 88 ± 9 days old. The experimental design was in a randomized complete block, defined by body weight and age. The experimental diets were control (CONT), containing 90% of concentrate and 10% of forage (coastcross hay), and 4 diets without forage: WC+P, 70% whole flint corn and 30% pelleted protein supplement; WC+G, 70% whole flint corn and 30% ground protein supplement; GC+P, 70% ground flint corn and 30% pelleted protein supplement; and GC+G, 70% ground flint corn and 30% ground protein supplement. There was an interaction between treatments and experimental periods for DMI in kg/day (P = 0.01) and g/kg of BW0.75 (P < 0.01; Table 3). For the DMI in kg/day, no significant differences were observed between the treatments in any of the experimental periods. However, for DMI expressed in g/kg of BW0.75, the animals fed WC+P had lower DMI than the animals on the CONT (P < 0.01) only in the first period. The ADG, FBW, and FE were not affected by the treatments. Compared to CONT, forage-free diets decreased ingestion time (min/day) and rumination and chewing (min/day and min/g of dry matter). There was no effect of treatments for any of the carcass traits evaluated. The diets did not cause lesions suggestive of ruminitis. Forage-free diets containing whole or ground corn associated with pelleted or ground protein supplement can be used successfully for feedlot lambs; they provide proper performance and carcass characteristics, without harming the animal's health.

Effects of injectable and intravaginal progesterone on ewes' reproductive performance at breeding season beginning.

Progesterone (P4) is a steroid hormone that has a regulatory role in the female reproductive system. Studies on the effects of injectable progesterone on ewes are scarce, mainly related to their reproductive responses in the breeding season. This study aimed to compare reproductive performance and serum P4 concentration using injectable or intravaginal P4 in ewes. Two hundred and forty and eight Santa Inês x Dorper ewes (BW; 52.67 ± 11.76 kg; mean ± SE), body condition score (BCS; 2.5 ± 0.8; scale of 1-5), were distributed in four treatments: (i) Control: without administration of P4; (ii) CIDR: intravaginal implantation of 330 mg of P4 for 7 days; (iii) 1P4: 15 mg of P4 intramuscular (IM); and (iv) 2P4I: 30 mg of P4 IM. The first 18 days of breeding season were considered the synchronization period. Except for ewes in the control group, all other ewes received 263 µg IM of cloprostenol sodium for lysis of eventual CL at 24 h before the P4 treatment. After the synchronization period, all ewes were kept together with males for extra 28 days in the breeding season. At the beginning of breeding season, 90% of the ewes had serum P4 concentration less than 1 ng/mL. The estrus rate was greater (P < 0.01) in ewes on the CIDR treatment, with similar estrus rate among the other treatments. The P4 implant was able to keep blood P4 concentration greater than 1 ng/mL in ewes that received an implant of P4 during the 7 days. After implant removal, there was a great increase in the estrus manifestation on ewes in the CIDR treatment, leading to an increase in pregnancy rate at the beginning of breeding season. The current study demonstrated that ewes that received an intramuscular injection of 15 or 30 mg of P4 had similar reproductive performance than ewes that did not receive any P4 intramuscular injection. However, when ewes were implanted with P4 (CIDR), these animals had an increase in estrus manifestation, leading to greater pregnancy earlier during the breeding season.

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes.

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.

Contextualization of causal regulatory networks from toxicogenomics data applied to drug-induced liver injury.

In recent years, network-based methods have become an attractive analytical approach for toxicogenomics studies. They can capture not only the global changes of regulatory gene networks but also the relationships between their components. Among them, a causal reasoning approach depicts the mechanisms of regulation that connect upstream regulators in signaling networks to their downstream gene targets. In this work, we applied CARNIVAL, a causal network contextualisation tool, to infer upstream signaling networks deregulated in drug-induced liver injury (DILI) from gene expression microarray data from the TG-GATEs database. We focussed on six compounds that induce observable histopathologies linked to DILI from repeated dosing experiments in rats. We compared responses in vitro and in vivo to identify potential cross-platform concordances in rats as well as network preservations between rat and human. Our results showed similarities of enriched pathways and network motifs between compounds. These pathways and motifs induced the same pathology in rats but not in humans. In particular, the causal interactions "LCK activates SOCS3, which in turn inhibits TFDP1" was commonly identified as a regulatory path among the fibrosis-inducing compounds. This potential pathology-inducing regulation illustrates the value of our approach to generate hypotheses that can be further validated experimentally.

New insights into the mechanisms underlying 5-fluorouracil-induced intestinal toxicity based on transcriptomic and metabolomic responses in human intestinal organoids.

5-Fluorouracil (5-FU) is a widely used chemotherapeutical that induces acute toxicity in the small and large intestine of patients. Symptoms can be severe and lead to the interruption of cancer treatments. However, there is limited understanding of the molecular mechanisms underlying 5-FU-induced intestinal toxicity. In this study, well-established 3D organoid models of human colon and small intestine (SI) were used to characterize 5-FU transcriptomic and metabolomic responses. Clinically relevant 5-FU concentrations for in vitro testing in organoids were established using physiologically based pharmacokinetic simulation of dosing regimens recommended for cancer patients, resulting in exposures to 10, 100 and 1000 µM. After treatment, different measurements were performed: cell viability and apoptosis; image analysis of cell morphological changes; RNA sequencing; and metabolome analysis of supernatant from organoids cultures. Based on analysis of the differentially expressed genes, the most prominent molecular pathways affected by 5-FU included cell cycle, p53 signalling, mitochondrial ATP synthesis and apoptosis. Short time-series expression miner demonstrated tissue-specific mechanisms affected by 5-FU, namely biosynthesis and transport of small molecules, and mRNA translation for colon; cell signalling mediated by Rho GTPases and fork-head box transcription factors for SI. Metabolomic analysis showed that in addition to the effects on TCA cycle and oxidative stress in both organoids, tissue-specific metabolic alterations were also induced by 5-FU. Multi-omics integration identified transcription factor E2F1, a regulator of cell cycle and apoptosis, as the best key node across all samples. These results provide new insights into 5-FU toxicity mechanisms and underline the relevance of human organoid models in the safety assessment in drug development.

Effect of bleaching agents on hardness, surface roughness and color parameters of dental enamel.

BACKGROUND: In this study was evaluated the effect of carbamide peroxide (CP) and hydrogen peroxide (HP) in different concentrations on hardness, roughness, and color parameters (color change - ΔE, lightness - ΔL, and yellow-blue axis - Δb) of bovine teeth. MATERIAL AND METHODS: Fifty square dental blocks (7 x 7 x 2 mm) were submitted to initial readings of Knoop hardness, surface roughness (Ra), and color parameters. Specimens were divided into 5 groups (n = 10): control group was kept in artificial saliva during the experimental period; CP 20% was bleached for 2 h daily for 14 days, HP 9.5% was bleached for 30 min daily for 14 days, HP 38% the bleaching gel was applied for 15 min, gel was removed and it was reapplied for two more times, the bleaching session was repeated for another 2 times every 7 days, totaling three sessions, and in CP 45% three sessions of 30 min each were performed of 7 in 7 days. All groups after the bleaching procedures had the bleaching gel removed, washed, and kept in artificial saliva. At the end of bleaching treatment, the hardness, roughness, and color parameters (ΔE, ΔL, and Δb) were re-evaluated. Data were analyzed by ANOVA, Tukey, and Dunnett tests (α = 0.05). RESULTS: Bleaching promoted a reduction in hardness, the CP 45% showed the lowest hardness and the CP 20% the highest, the HP 9.5% and HP 38% showed intermediate values of hardness. Bleaching agents did not affect the roughness. CP 20% and HP 38% promoted the highest values of ΔE and higher reduction of yellowish tone of tooth. Lightness increase after bleaching treatment for all groups. CONCLUSIONS: All the bleaching agents tested showed effectiveness, but with reduced hardness. Key words:Carbamide peroxide, hydrogen peroxide, tooth whitening, hardness, roughness.

Proteomics analysis of zebrafish larvae exposed to 3,4-dichloroaniline using the fish embryo acute toxicity test.

The zebrafish (Danio rerio) is a small teleost fish that is becoming increasingly popular in laboratories worldwide and several attributes have also placed the zebrafish under the spotlight of (eco)toxicological studies. Since the 1990s, international organizations such as ISO and OECD have published guidelines for the use of zebrafish in ecotoxicological assessment of environmental toxicants such as the Fish Embryo Acute Toxicity (FET) test, OECD n° 236 guideline. This protocol uses 3,4-dichloroaniline (DCA), an aniline pesticide whose toxicity to fish species at early life stages is well known, as a positive control. Despite its use, little is known about its molecular mechanisms, especially in the context of the FET test. Therefore, this study aimed to investigate such changes in zebrafish larvae exposed to DCA (4 mg/L) for 96 hours using gel-free proteomics. Twenty-four proteins detected in both groups were identified as significantly affected by DCA exposure, and, when considering group-specific entities, 48 proteins were exclusive to DCA (group-specific proteins) while 248 were only detected in the control group. Proteins modulated by DCA treatment were found to be involved in metabolic processes, especially lipids and hormone metabolism (eg, Apoa1 and Apoa1b and vitelogenins), as well as proteins important for developmental processes and organogenesis (eg, Myhc4, Acta2, Sncb, and Marcksb). The results presented here may therefore provide a better understanding of the relationships between molecular changes and phenotype in zebrafish larvae treated with DCA, the reference compound of the FET test.

Trypsin inhibitor from Leucaena leucocephala seeds delays and disrupts the development of Aedes aegypti, a multiple-disease vector.

BACKGROUND: Nowadays, the Aedes aegypti mosquito represents a serious public health issue in view of the large outbreaks of the arboviral diseases zika, dengue, chikungunya and yellow fever. This holometabolous insect has midgut digestive enzymes that are trypsin- and chymotrypsin-like proteins. Protease inhibitors are able to bind to proteolytic enzymes and promote a blockage in digestion and nutrition, leading to death. Thus, we investigated the effect of trypsin inhibitor of Leucaena leucocephala (LTI) seeds on egg hatching, larval development and digestive midgut proteases. RESULTS: LTI was obtained by trichloroacetic acid precipitation followed by a single chromatography step on anhydrous trypsin sepharose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed a single protein band with a molecular mass close to 20 kDa. After exposure of Ae. aegypti eggs to LTI (0.3 mg mL-1 ), egg hatching was reduced (50%). LTI did not show acute toxicity on newly hatched larvae incubated under the same conditions, but after 10 days of exposure a high mortality rate (86%) was observed and the surviving larvae had a 25% delay in development. LTI was able to inhibit in vitro the midgut enzymatic activity (70%), and when larvae were incubated with LTI solution we observed an inhibition of 56%. CONCLUSIONS: LTI is a promising new tool to control critical points of Ae. aegypti development. © 2016 Society of Chemical Industry.

Further insecticidal activities of essential oils from Lippia sidoides and Croton species against Aedes aegypti L.

This study assessed new insecticidal activities of essential oils from Lippia sidoides and Croton species (Croton zehntneri, Croton nepetaefolius, Croton argyrophylloides, and Croton sonderianus) against Aedes aegypti mosquito. In addition, the acute toxicity upon mice was determined. All essential oils showed inhibition of egg hatching, with IC50 values ranging from 66.4 to 143.2 µg mL(-1), larvicidal activity with LC50 ranging from 25.5 to 94.6 µg mL(-1), and pupicidal action with PC50 ranging from 276.8 to over 500 µg mL(-1). Only L. sidoides, C. zehntneri, and C. argyrophylloides essential oils were able to inhibit the oviposition of female gravid mosquitoes with OD50 values of 35.3, 45.3, and 45.8 µg mL(-1), respectively. Oral acute toxicity in mice showed that C. sonderianus and C. argyrophylloides oils are nontoxic (LD50 > 6,000 mg.kg(-1)) while C. nepetaefolius, C. zehntneri, and L. sidoides oils are moderately toxic (LD50 3,840; 3,464, and 2,624 mg.kg(-1), respectively). The results indicate that these oils are promising sources of bioactive compounds, showing low or no toxicity to mammals.