Use of trichostatin A alters the expression of HDAC3 and KAT2 and improves in vitro development of bovine embryos cloned using less methylated mesenchymal stem cells

The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC‐AF), adipose tissue (MSC‐AT) and fibroblasts (FIB‐control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT‐PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC‐AF were less hydroxymethylated for both animals. MSC‐AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC‐AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC‐AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.     

Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A,B, and J

Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus(ALV)subgroups using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A(TVA) and generate chicken cells resistant to infection by this virus.Results: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely,overexpression of the wild-type TVA receptor(wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na~+/H~+ exchange 1(chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively.Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.     

Hemolytic anemia,spherocytosis, and thrombocytopenia associated with honey bee envenomation in a dog

This case report describes a massive honey bee envenomation in a 14‐month‐old male Belgian Malinois dog from St. Kitts, West Indies. Acute and delayed onsets of hemolytic anemia, echinocytosis, spherocytosis, thrombocytopenia, hemoglobinemia, and hemoglobinuria developed following envenomation. The dog recovered after treatment with glucocorticoids and supportive therapy. Spherocytosis, hemolysis, and thrombocytopenia in patients with massive bee envenomation are likely due to the direct toxic effects of the primary components of bee venom, melittin and phospholipase A₂ (PLA₂). Mellitin causes hemolysis by forming large pores in erythrocytes resulting in leakage of hemoglobin and also causes spectrin stiffening and resultant echinocyte and spherocyte formation. Melittin also stimulates PLA₂, a hydrolase that causes echinocytosis and spherocytosis, in vivo and in vitro, and mitochondrial breakdown in platelets. However, delayed manifestations could be attributed to immune‐mediated mechanisms from the generation of antibodies against damaged erythrocytes and platelet membrane proteins.     

Classification of soils supporting mangrove plantation in Kuwait

The State of Kuwait has initiated an ambitious national program to establish greenery in 20000 ha of open spaces in inland and coastal areas. Afforestation of intertidal zones with mangrove plants is being considered as a viable option for greening coastal areas. In view of this fact, experimental plantations were established using two ecotypes of Avicennia marina (Bahrain and UAE) and one ecotype of Avicennia germinans (Florida). The study consisted of two 10-year old plantations and two 2-year old plantations. Seedlings established successfully and grew vigorously on some of the locations within the site. Therefore, studies were conducted during 2002-2003 to describe and classify soils that supported maximum mangrove establishment and growth under the harsh arid coastal conditions of Kuwait. For this purpose, eight soil profiles (four each representing areas where seedlings established successfully and grew vigorously and where all seedlings died in the initial stages of establishment) were investigated and classified according to the soil taxonomy. The main soil types observed in the study site were: Typic Aquisalids, Typic/sodic Aquicambids and Calcic Aquisalids. Mangrove growth performance in relation to the landform, soil type and properties of the soil at various depths is discussed in the paper. The plant growth appeared to be the better on the typic Aquisalid soil than that on the Typic/sodic Aquicambid soils. The relative proportions of the coarse sand fraction, occurrence of the anaerobic layer in the profile and the surface layer salinity influenced both the establishment and growth of mangrove plants.     

Effect of Artificial Insemination Protocol and Dose of Frozen/Thawed Stallion Semen on Pregnancy Results in Mares

Deep intra‐uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra‐uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen–thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi‐flexible Minitube pipette was compared with a newly designed insemination device with a more flexible telescopic insemination catheter (Ghent device). The semi‐flexible Minitube pipette performed better than the newly designed insemination device with respect to pregnancy outcome (p = 0.008). The superiority of deep horn insemination over uterine body insemination was reflected by the better pregnancy rates obtained after deep insemination using the same low doses (30.6% better pregnancy rates) (p = 0.0123).     

Potential for greenhouse gas emissions from soil carbon stock following biofuel cultivation on degraded lands

Consequent to the interest in converting degraded lands for cultivation of biofuel crops, concerns have been expressed about greenhouse gas (GHG) emissions resulting from changes in soil‐carbon (C) stock following land conversions. A literature‐based study was undertaken for estimating the magnitude of emission of GHGs, particularly carbon dioxide (CO₂), following an assessment of the extent and causes of land degradation and the nature of CO₂ emission from soils. The study estimated the potential for CO₂ emission resulting from changes in soil‐carbon stock following land conversions, using oil palm (Elaeis guineensis Jacq.) as a case study. The analysis indicated that, overall, the magnitude of CO₂ emission resulting from changes in soil C stock per se following opening up of degraded land would be low compared with other potential sources of CO₂ emission. However, lack of data on critical aspects such as baseline soil C status was a limitation of the study. Soil respiration is the single best measure of GHG emission from soils. Fixation of C in additional biomass will compensate, over time, for C loss through soil respiration following a change in land use or land management, unless such changes involve conversion of existing large C stocks. Therefore, any net CO₂ emission from soils resulting from changes in soil C stock following opening up of degraded land is likely to be a short‐term phenomenon. The estimations used in the study are based on various assumptions, which need to be validated by experimental field data. Copyright © 2010 John Wiley & Sons, Ltd.     

Production of ascorbyl palmitate by surfactant-coated lipase in organic media

The surface of a lipase from Burkholderia cepacia was coated with a nonionic surfactant, propylene glycol monostearate, and was used as a biocatalyst in the production of ascorbic acid in tert-butyl alcohol. The influence of various factors such as the type of surfactant, the pH of the buffer used for coating, the amount of surfactant in the coating, the organic solvent, and the temperature and molar ratio of the substrates used in the reaction on the conversion of ascorbyl palmitate were studied. After 24 h of reaction at 50 degrees C, a conversion of 47% was obtained using an ascorbic acid to palmitic acid molar ratio of 1:6. The native lipase showed only 6% conversion.     

Amelioration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas)

Much attention has been focused on food that may be beneficial in preventing diet-induced body fat accumulation and possibly reduce the risk of diabetes and heart disease. Cornelian cherries (Cornus mas) are used in the preparation of beverages in Europe and also to treat diabetes-related disorders in Asia. In this study, the most abundant bioactive compounds in C. mas fruits, the anthocyanins and ursolic acid, were purified, and their ability to ameliorate obesity and insulin resistance in C57BL/6 mice fed a high-fat diet was evaluated. Mice were initially fed a high-fat diet for 4 weeks and then switched to a high-fat diet containing anthocyanins (1 g/kg of high-fat diet) and ursolic acid (500 mg/kg of high-fat diet) for an additional 8 weeks. The high-fat diet induced glucose intolerance, and this was prevented by anthocyanins and ursolic acid. The anthocyanin-treated mice showed a 24% decrease in weight gain. These mice also showed decreased lipid accumulation in the liver, including a significant decrease in liver triacylglycerol concentration. Anthocyanin and ursolic acid treated mice exhibited extremely elevated insulin levels. Both treatments, however, showed preserved islet architecture and insulin staining. Overall, these data suggest that anthocyanins and ursolic acid purified from C. mas fruits have biological activities that improve certain metabolic parameters associated with diets high in saturated fats and obesity.     

Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclooxygenase enzyme, and lipid peroxidation

The antioxidant ferulic and caffeic acid phenolics are ubiquitous in plants and abundant in fruits and vegetables. We have synthesized a series of ferulic and caffeic acid esters and tested for tumor cell proliferation, cyclooxygenase enzymes (COX-1 and -2) and lipid peroxidation inhibitory activities in vitro. In the tumor cell proliferation assay, some of these esters showed excellent growth inhibition of colon cancer cells. Among the phenolics esters assayed, compounds 10 (C12-caffeate), 11 (C16-caffeate), 21 (C8-ferulate), and 23 (C12-ferulate) showed strong growth inhibition with IC50 values of 16.55, 13.46, 18.67, and 7.57 microg/mL in a breast cancer cell line; 9.65, 7.45, 17.05, and 4.35 microg/ mL in a lung cancer cell line; 5.78, 3.5, 4.29, and 2.46 microg/mL in a colon cancer cell line; 12.04, 12.21, 14.63, and 8.09 microg/ mL in a central nervous system cancer cell line; and 8.62, 7.76, 11.0, and 5.37 in a gastric cancer cell line. In COX enzyme inhibitory assays, ferulic and caffeic acid esters significantly inhibited both COX-1 and COX-2 enzymes. Caffeates 5-10 (C4-C12), inhibited COX-1 enzyme between 50% and 90% and COX-2 enzyme by about 70%, whereas ferulates 15-21 (C3-C8) inhibited COX-1 and COX-2 enzymes by 85-95% 25 microg/mL. Long-chain caffeates 11-14 (C16-C22) and short-chain ferulates 15-20 (C3-C5) were the most active in lipid peroxidation inhibition and showed 60-70% activity at 5 microg/mL concentration.     

Molecular characterization and identification of markers associated with yield traits in mulberry using ISSR markers

Mulberry (Morus indica L.) is an important tree crop being exploited for feeding the silk‐producing insect Bombyx mori L. In order to identify parents suitable for breeding to raise high‐yielding varieties for the non‐traditional areas of Kerala, India and also to identify markers associated with leaf yield attributing traits, the present study was undertaken with 44 mulberry genotypes. Variability on morpho‐biometric traits and molecular markers, generated with 12 selected ISSR primers, was estimated. Significant differences between genotypes were observed for all the traits. The dendrogram generated with morpho‐biometric characters clustered the genotypes into three distinct groups and one isolate, while the same using Inter simple sequence repeat (ISSR) markers clustered the genotypes into five groups and six isolates. The greater resolving power of the ISSR markers was evident form the dendrograms. Using step‐wise multiple regression analysis, a number of markers associated with number of branches, total shoot length, leaf weight, internodal distance, leaf chlorophyll, protein, leaf moisture percentage were identified. These markers could be of much use in Marker assisted selection (MAS) breeding programmes in mulberry, especially when no genetic information in terms of linkage maps and Quantitative Trait Locis (QTLs) is available a plant with high heterozygosity and a long juvenile period.