Proteomic profile of vitrified in vitro-produced bovine embryos (Bos taurus indicus).

BACKGROUND: The proteomic profile of cryopreserved in vitro produced bovine embryos is little known but can provide insights on the successful application of cryo procedures in support of animal breeding. OBJECTIVE: To identify embryonic proteins and biomarkers related to improved cryotolerance of vitrified in vitro produced bovine embryos. MATERIALS AND METHODS: Proteins were isolated from embryo pools (n = 25 embryos per replicate) and analyzed using the nanoLC - MS/MS system. Further, the UniProtKB database (Uniprot -http://www.uniprot.org/) was used for protein identification. Proteins were classified based on their molecular mass, isoelectric point, and enzymatic activity. Post-translational modification predictions and functional gene ontology analysis were performed as well. Finally, a protein-protein interaction network was created to shed light on the embryo interactome. RESULTS: Based on the MS/MS approach, 66 proteins were identified from vitrified Bos taurus embryos. The retrieved proteins were presumably annotated, which allowed a description of the qualitative and functional aspects of the embryo proteome after the vitrification process. CONCLUSION: These findings allowed us to conclude that in vitro-produced vitrified embryos expressed proteins that underlie biological processes related to reproduction, stress and lipid metabolic process, which are essential to maintain embryo viability. doi.org/10.54680/fr22410110512.

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

Proteomic response of Moniliophthora perniciosa exposed to pathogenesis-related protein-10 from Theobroma cacao.

TcPR-10, a member of the pathogenesis-related protein 10 family, was identified in EST library of interactions between Theobroma cacao and Moniliophthora perniciosa. TcPR-10 has been shown to have antifungal and ribonuclease activities in vitro. This study aimed to identify proteins that are differentially expressed in M. perniciosa in response to TcPR-10 through a proteomic analysis. The fungal hyphae were subjected to one of four treatments: control treatment or 30-, 60- or 120-min treatment with the TcPR-10 protein. Two-dimensional maps revealed 191 differentially expressed proteins, 55 of which were identified by mass spectrometry. The proteins identified in all treatments were divided into the following classes: cell metabolism, stress response, zinc binding, phosphorylation mechanism, transport, autophagy, DNA repair, and oxidoreductases. The predominant class was stress-response proteins (29%), such as heat shock proteins; these proteins exhibited the highest expression levels relative to the control treatment and are known to trigger defense mechanisms against cytotoxic drugs as well as TcPR-10. Oxidoreductases (25%) were overexpressed in the control and in 30-min treatments but exhibited reduced expression at 120 min. These proteins are involved in the repair of damage caused by oxidative stress due to the contact with TcPR- 10. Consistent with the antifungal activity of TcPR-10, several proteins identified were related to detoxification, autophagy or were involved in mechanisms for maintaining fungal homeostasis, such as ergosterol biosynthesis. These results show that the sensitivity of the fungus to TcPR-10 involves several biochemical routes, clarifying the possible modes of action of this antifungal protein.

Distinct carbapenems susceptibility profiles in isogenic isolates of Klebsiella pneumoniae presenting Ompk36 disruption and expression of down-regulated blaKPC-2.

The isolation of multidrug-resistant Klebsiella pneumoniae in hospitals is a major public health threat, increasing patient hospitalization costs, morbidity and mortality. Therefore, this work investigated the resistance mechanisms that produced different carbapenems susceptibility profiles in two isogenic strains of K. pneumoniae isolated from the same patient in a public hospital in Recife, Pernambuco. The genes that encode the main porins in K. pneumoniae, ompK35 and ompK36, and several beta-lactamase genes were analyzed. The expression of these genes was evaluated by quantitative real time PCR (polymerase chain reaction) with reverse transcriptase (RT-qPCR). SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) was performed to analyze the outer membrane proteins. The analysis of the ompK36 genetic environment disclosed an IS903 insertion sequence disrupting this gene in the ertapenem resistant isolate (KPN133). The blaKPC-2 gene showed down-regulated expression in both isolates. Our findings show that changes in porins, especially OmpK36, are more determinant to carbapenems susceptibility profile of bacterial isolates than variations in blaKPC gene expression.

Effects of nutrient restriction and subsequent realimentation in pregnant beef cows: Maternal endocrine profile, umbilical hemodynamics, and mammary gland development and hemodynamics.

Our hypothesis was that maternal nutrient restriction would negatively impact the endocrine and metabolic status of the pregnant cow, therefore influencing the mammary gland in preparation for lactation. We further hypothesized that earlier timing of realimentation could prevent negative impacts of nutrient restriction. The objectives were to investigate the influence of nutrient restriction and realimentation during early to late gestation on endocrine profile, umbilical hemodynamics, and mammary gland development and hemodynamics in pregnant beef cows. In Experiment 1, on d 30 of pregnancy cows (initial BW = 667.5 ± 13.4 kg, BCS = 6.2 ± 0.1) were randomly assigned to one of 3 treatments: 1) 100% NRC requirements from d 30 to 254 of gestation (CCC; n = 6); 2) 60% NRC from d 30 to 85, thereafter being re-alimented to 100% NRC to d 254 (RCC; n = 5); 3) or receive 60% NRC from d 30 to 140, thereafter being re-alimented to 100% NRC to d 254 (RRC; n = 6). Cows were returned to a common outdoor facility for calving thereafter and were fed ad libitum. In Experiment 2, on d 30 of pregnancy, cows (initial BW = 620.5 ± 11.3 kg, BCS = 5.1 ± 0.1) were randomly assigned to dietary treatments including: control (CON; 100% NRC; n = 18) and nutrient restriction (RES; 60% NRC; n = 30). On d 85 of pregnancy, cows were either slaughtered (CON, n = 6 and RES, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12) treatments, or were realimented to control (RC; n = 11). On d 140 of pregnancy, cows were either slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5), or were realimented to control (RRC, n = 6). On d 254 of pregnancy, all remaining cows were slaughtered (CCC, n = 6; RCC, n = 5; RRC, n = 6). Mammary hemodynamics and endocrine profile were measured. Serum urea nitrogen, NEFA, as well as fetal parameters were measured in Experiment 1; whereas in Experiment 2, mammary gland development was recorded. In Experiment 1, RRC cows had lower dry matter intake (P = 0.001) and consequently lower BW change (P = 0.06). However, maternal nutrition did not alter mammary hemodynamics, hormonal patterns, and fetal characteristics (P > 0.11). In Experiment 2, CCC cows had increased (P = 0.02) mammary gland blood flow ipsilateral to the gravid horn as well as greater (P = 0.02) mammary gland fat on d 254. Nevertheless, plane of nutrition did not alter hormonal concentrations nor mammary gland characteristics (P > 0.15). These data indicate that nutrient restriction did not alter mammary hemodynamics nor endocrine profile throughout gestation.

Puberty attainment and reproductive performance of yearling Bos indicus-influenced heifers after two sequential treatments with progesterone.

Number of pubertal heifers at time of breeding season initiation is a primary determinant to pregnancy success during the breeding season. It was hypothesized that pre-breeding progesterone (P4) supplementation (induction) would increase the number of heifers pubertal at the time of imposing estrous synchronization treatment regimens and P/AI. Yearling, Bos indicus-influenced (n = 577) or Bos indicus (n = 174) heifers were or were not treated with P4 (CIDR and Non-CIDR, respectively) for 10 d starting on D-23 (D0 = TAI). Presence of a CL on D-33 or D-23 was considered to indicate heifers were pubertal. On D-13, there was a PGF analogue administered. On D-9, there was treatment with GnRH analogue, 6d-CIDR and PGF. There were inseminations based on estrus (D-2 to D0) or TAI on D0 for non-estrous animals. There were 5.2 % and 62.9 % purebred and crossbred heifers pubertal, respectively. Proportion of prepubertal crossbred than purebred heifers with CL on D-3 was greater as a result of imposing the pubertal induction regimen (P < 0.05 and P> 0.10, respectively). Regardless of puberty status, proportion of heifers in estrus prior to AI in the CIDR group was similar to the heifers of the Non-CIDR group for crossbreds and purebreds. Similarly, P/AI of CIDR group was similar to the Non-CIDR group for crossbreds and purebreds. In summary, imposing the pubertal induction regimen hastened attainment of puberty in yearling crossbred, but not purebred heifers. Puberty induction did not affect estrous response, neither fertility after imposing an estrous synchronization treatment regimen.

Population dynamics of Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) on Host Plants.

The stink bug Dichelops melacanthus (Dallas) has become one of the major pests of corn and wheat in Brasil, mainly after a shift from the conventional tillage system to the no tillage cultivation system. This fact may be due to the simultaneous occurrence of second planting corn with wheat cultivation, and the presence of wild hosts. This study aimed to evaluate the population dynamics of D. melacanthus on wild hosts adjacent to areas cultivated with corn, wheat, and soybean during the season and off-season of soybean cultivation. Weekly surveys were conducted in the region of Londrina, PR, Brasil from the beginning of July 2007 up to the end of June 2008 using the square meter method. Corn (Zea mays), soybean (Glycine max), tropical spiderwort (Commelina benghalensis), hairy indigo (Indigofera hirsuta), crotalaria (Crotalaria pallida), wheat (Triticum aestivum), and signal grass (Brachiaria decumbens) were identified as hosts of D. melacanthus. Signal grass was the host in which stink bug adults were found in higher numbers, while nymphs and adults were consistently collected on tropical spiderwort. Although nymphs completed their development on tropical spiderwort seeds, this host was found less suitable than soybean seeds.

Feeding activity, salivary amylase activity, and superficial damage to soybean seed by adult Edessa meditabunda (F.) and Euschistus heros (F.) (Hemiptera: Pentatomidae).

Greenhouse and laboratory studies were conducted to evaluate feeding activity and superficial damage to soybean seed by the brown-winged stink bug, Edessa meditabunda (F.), and the Neotropical brown stink bug, Euschistus heros (F.). Soybean plants (cv. BRS 282), at R6 stage of development were used. Thirty pairs of each species were used individually for 48 h. Two daily observations (9:00 AM and 3:00 PM) were taken to record the number of bugs (feeding/resting) on plant parts. Harvested seeds imbibed in tetrazolium solution were photographed for measurement of the damaged surface. Adult E. meditabunda significantly preferred soybean stems (19.7 bugs) to pods (2.7). Feeding/resting was similar at 9:00 AM (mean number of 28.0 bugs) and 3:00 PM (24.3). Euschistus heros equally fed/stayed on stems (7.3 bugs) and pods (6.9), although most bugs (12.3) remained on the cage net; feeding/resting on all plant structures amounted to 13.7 bugs at 9:00 AM and 17.7 bugs at 3:00 PM. Amylase activity was greater for E. heros (41.61 ± 0.89 U/mg) and almost none for E. meditabunda (2.35 ± 0.14 U/mg). The superficial damage to seeds was significantly greater for E. meditabunda (22. 9 mm(2)) compared to E. heros (12.5 mm(2)). However, E. meditabunda caused less shrinkage of the seed tegument, while E. heros damage was deeper and seeds showed reduction in size.

Survivorship and egg production of phytophagous pentatomids in laboratory rearing.

Survivorship and reproductive performance of the pentatomids Euschistus heros (F.) (EH), Nezara viridula (L.) (NV), and Dichelops melacanthus (Dallas) (DM) were tested in the laboratory. A mixture of natural foods (pods of green beans, Phaseolus vulgaris, raw shelled peanuts, Arachis hypogaea, and fruits of privet, Ligustrum lucidum, and 50 pairs/box (25 x 20 x 20 cm) were used, observed for 30 days, and replicated three times. Thirty days after emergence, mean female survivorship was 91% (EH), 60% (NV), and 30% (DM). More egg masses were deposited during 11-20 days after emergence, with mean number of 45.1 (EH), 5.3 (NV), and 11.8 (DM). These values were smaller during the first 10 days (25.5, 2.1, and 4.7) and last 10 days (21-30 days) (39.4, 3.9, and 4.9), respectively. Mean maximum number of eggs/day was 489 (EH) on day 29, 474 (NV) on day 11, and 153 (DM) on day 14. Mean monthly fecundity (egg masses/box) was 985 (EH), 92 (NV), and 193 (DM), and mean number of eggs/box was 8,480; 5,147, and 2,042.7, respectively.

An invasive pentatomid pest in Argentina: neotropical brown stink bug, Euschistus heros (F.) (Hemiptera: Pentatomidae).

This is the first record on the invasion of the Neotropical brown stink bug, Euschistus heros (F.) in Paraná County (latitude 31º 51' 9.6" S, longitude 60º 32' 11.2" W), Entre Ríos province, Argentina. Five adults were intercepted in 2009/2010, one in crop residues and four on soybean fields. The expanding range in the distribution of E. heros in the Southern Hemisphere is believed to be due to the increased commercial trade among countries, increase in the area cultivated with soybean, and the adoption of no-tillage cropping systems.