Safety Evaluation of Neo Transgenic Pigs by Studying Changes in Gut Microbiota Using High-Throughput Sequencing Technology

The neo (neomycin phosphotransferase) gene is widely used as a selection marker in the production of genetically engineered animals and plants. Recent attention has been focused on safety concerns regarding neo transgene expression. In this study, neo transgenic and non-transgenic piglets were randomly assigned into Group A and Group B to evaluate effects of neo transgene by studying changes in gut microbiota using high-throughput sequencing. Group A pigs were fed a standard diet supplemented with antibiotic neomycin; Group B pigs were fed a standard diet. We examined horizontal transfer of exogenous neo gene using multiplex PCR; and investigated if the presence of secreted NPT II (neo expression product) in the intestine could lead to some protection against neomycin in transgenic pigs by monitoring different patterns of changes in gut microbiota in Group A animals. The unintended effects of neo transgene on gut microbiota were studied in Group B animals. Horizontal gene transfer was not detected in gut microbiota of any transgenic pigs. In Group A, a significant difference was observed between transgenic pigs and non-transgenic pigs in pattern of changes in Proteobacteria populations in fecal samples during and post neomycin feeding. In Group B, there were significant differences in the relative abundance of phyla Firmicutes, Bacteroidetes and Proteobacteria, and genera Lactobacillus and Escherichia-Shigella-Hafnia between transgenic pigs and non-transgenic pigs. We speculate that the secretion of NPT II from transgenic tissues/cells into gut microbiota results in the inhibition of neomycin activity and the different patterns of changes in bacterial populations. Furthermore, the neo gene also leads to unintended effects on gut microbiota in transgenic pigs that were fed with basic diet (not supplemented with neomycin). Thus, our data in this study caution that wide use of the neo transgene in genetically engineered animals should be carefully considered and fully assessed.     

Elicited Teucrium chamaedrys cell cultures produce high amounts of teucrioside,but not the hepatotoxic neo-clerodane diterpenoids

Teucrium chamaedrys, one of the most common and investigated species of the genus Teucrium, has been used for centuries in traditional medicine for many purposes. Its phytochemical components comprise, among others, phenylethanoid glycosides (PGs) and neo-clerodane diterpenoids. Several reports have demonstrated a wide range of beneficial biological and pharmacological activities of the phenylethanoid components, while the diterpenes were shown to be strongly hepatotoxic. In this work, in vitro cultures were established from leaf explants of T. chamaedrys. Both solid (callus) and liquid (cell suspension) cultures maintained the capacity to produce PGs, with teucrioside (TS) representing the most abundant one. Cell suspensions had a lower TS content than that found in leaf extracts, but higher than that of calli. An NMR-based metabolomics approach was used to compare the product profile of intact plants vs. cell suspension cultures, and results showed that neo-clerodane diterpenes, present in the intact plant, were not detected in cell cultures. Several elicitors were supplied to cell cultures with the aim of increasing TS production, and elicitation was tested at different growth phases and by exposing cells for different periods. Methyl jasmonate and fungal mycelia from Trichoderma viridae and Fusarium moniliforme were able to significantly increase TS production if supplied at the early-exponential growth phase for 24 h. Based on the proposed link between proline and the phenylpropanoid pathways, proline accumulation in cell cultures was followed throughout a 14-day culture period, showing that it strictly reflected that of TS production. Moreover, exogenously supplied proline, and its analogue hydroxyproline, turned out to be very effective in increasing teucrioside production.     

Epistatic,synthetic, and balancing interactions among tubulin missense mutations affecting neurite growth in Caenorhabditis elegans

Mutations in tubulins affect microtubule (MT) dynamics and functions during neuronal differentiation and their genetic interaction provides insights into the regulation of MT functions. We previously used Caenorhabditis elegans touch receptor neurons to analyze the cellular impact of tubulin mutations and reported the phenotypes of 67 tubulin missense mutations, categorized into three classes: loss-of-function (lf), antimorphic (anti), and neomorphic (neo) alleles. In this study, we isolated 54 additional tubulin alleles through suppressor screens in sensitized backgrounds that caused excessive neurite growth. These alleles included 32 missense mutations not analyzed before, bringing the total number of mutations in our collection to 99. Phenotypic characterization of these newly isolated mutations identified three new types of alleles: partial lf and weak neo alleles of mec-7/β-tubulin that had subtle effects and strong anti alleles of mec-12/α-tubulin. We also discovered complex genetic interactions among the tubulin mutations, including the suppression of neo mutations by intragenic lf and anti alleles, additive and synthetic effects between mec-7 neo alleles, and unexpected epistasis, in which weaker neo alleles masked the effects of stronger neo alleles in inducing ectopic neurite growth. We also observed balancing between neo and anti alleles, whose respective MT-hyperstablizing and -destabilizing effects neutralized each other.     

neo-Clerodane diterpenoids from the leaf exudate of Dodonaea angustifolia

Phytochemical investigation of the leaf surface exudate of Dodonaea angustifolia L.f. yielded two new neo-clerodane diterpenes, neo-clerodan-3,13-dien-16,15:18,19-diolide (mkapwanin) and 15-methoxy-neo-clerodan-3,13-dien-16,15:18,19-diolide (15-methoxymkapwanin). In addition, ten known compounds were identified. The structures were determined on the basis of spectroscopic evidence. This additional chemical information could contribute towards solving the taxonomical controversy that exists between Dodonaea angustifolia and Dodonaea viscosa Jacq., which are morphologically similar.     

The effect of ethylene inhibitors （AgNO3, AVG）, an ethyleneliberating compound （CEPA） and aeration on the formation of protocorm-like bodies of hybrid Cymbidium （Orchidaceae）

Protocorm-like bodies （PLBs） or thin cell layers （TCLs） derived from PLBs of hybrid Cymbidium Twilight Moon ＇Day Light＇ can induce new or neo-PLBs on Teixeira Cymbidium （TC） medium, which contains 0.1 mg/L a- naphthaleneacetic acid, 0.1 mg/L kinetin, 2 g/L tryptone and 20 g/L sucrose, and is solidified with 8 g/L Bacto agar. This study aimed to assess the response ofneo-PLB formation to an ethylene-liberating compound （2-chloroethylphosphonic acid （CEPA））, to two ethylene inhibitors （silver nitrate （AgNO3） and aminoethoxyvinylglycine （AVG））, and to aeration （made possible by using Milliseal~ or autoclaved filter paper）. AgNO3 at 1 or 2 mg/L in TC medium significantly increased the fresh weight of PLBs while 1 mg/L of AgNO3 also showed a significant increase in the number ofneo-PLB from both half-PLBs and from tTCLs. In contrast, AVG and CEPA inhibited neo-PLB formation. Neo-PLB formation from half-PLB or TCL explants in the presence of aeration resulted in significantly lower neo-PLB weight. The use of AgNO3 and aeration are alternative means to mass produce neo-PLBs for micropropagation purposes.     

Successful storage of protocorm-like bodies of hybrid Cymbidium (Orchidaceae) under low temperature conditions

Low temperatures result in lower metabolic cellular activity, thus slowing down cell division and growth. This is advantageous where a plant scientist might seek to store important germplasm without the risks associated with low temperature storage. In this study, two cold temperatures above freezing, namely 4 and 10 °C, were tested to assess for how long PLBs could be preserved without a significant loss in regeneration ability (i.e., the ability to form neo-PLBs). Control treatments were cultured at 25 °C on Teixeira Cymbidium (TC) medium at a 16-h photoperiod at a photosynthetic photon flux density (PPFD) of 45 μmol m⁻² s⁻¹. For the cold treatments, each was replicated in the dark and at low light intensity (12-h photoperiod and a PPFD of 10 μmol m⁻² s⁻¹). All cultures were sub-cultured six times onto fresh medium every 60 days, for approximately 1 year. On the 7th subculture, all neo-PLBs were prepared uniformly and replated onto standard TC medium under light conditions described above for the control. 45 days after the 7th subculture and just before subcultures 1–6, the number of neo-PLBs per half-PLB was measured. The number of neo-PLBs that formed under different treatments depended strongly on the temperature and light conditions with most neo-PLBs forming under control conditions, although that number dropped significantly as the temperature was dropped to 10 °C and then even more to 4 °C, the same trend being observed when explants were cultured and subcultured under dim light, with organogenesis being more strongly negatively influenced in darkness. For all low-temperature treatments, as well as the dimmed light and darkness treatments, the number of neo-PLBs increased significantly when recultured, on the 7th subculture, onto control TC medium under control environmental conditions, almost as high as the control values. In contrast, the control values decreased, with significantly fewer neo-PLBs by the 7th subculture relative to the control, indicating that new PLBs should be induced from shoot cultures at least once a year to maintain their vitality.     

Teugin,a neo-clerodane diterpenoid from Teucrium fragile

From the aerial part of Teucrium fragile a new neo-clerodane diterpenoid, teugin, has been isolated. Its structure, 15,16-epoxy- 2β,6β-dihydroxy-neo-cleroda-3,13(16),14-triene-18,19:20,12S-diolide, was established mainly by spectroscopic means.     

Triterpene acids from Salvia and Teucrium species

The pentacyclic triterpene acids ursolic acid, oleanolic acid, micromeric acid, maslinic acid and 3-epi-maslinic acid have been isolated from several Salvia and Teucrium species.     

A comparative study of two clerodane diterpenes from Baccharis trimera (Less.) DC. on the influx and mobilization of intracellular calcium in rat cardiomyocytes

Baccharis trimera (Less.) D.C. (Asteraceae) is a medicinal species native to South America and used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. The aqueous extract (AE) of the aerial parts of this species presented two mainly constituents: the ent-clerodane diterpene (Fig. 1) and the neo-clerodane diterpene (Fig. 2). The objective of this work was to study their activities on the blockade of Ca²⁺-induced contractions in KCL-depolarized rat portal vein preparations, and on the influx and mobilization of cytosolic calcium in rat cardiomyocytes by fluorescence measurements. The results showed that both the neo- and the ent-clerodane diterpenes reduced the maximal contractions induced by CaCl₂, in KCl depolarized rat portal vein preparations, without modifying the EC₅₀. The data on the concentration of cytosolic calcium ([Ca²⁺]_c) showed that, while the neo-clerodane diterpene stimulates the mobilization of [Ca²⁺]_c in rat cardiomyocytes, this effect was not observed with the ent-clerodane diterpene. On the other hand, the influx of calcium was not altered by the neo-clerodane diterpene, but was reduced in the presence of the ent-clerodane diterpene, indicating that this compound induces a blockade of the voltage-dependent calcium channels.     

Ent-clerodane diterpenoids from six further species of Teucrium

Fifteen further species of the genus Teucrium have been investigated. Six of them contain known ent-clerodane diterpenoids; the others did not yield any diterpenoid. The chemotaxonomic significance of these results is discussed.     

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3′-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated M_r of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).     

Instability of Plastid DNA in the Nuclear Genome

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.     

neo-Clerodane diterpenes from Ajuga ciliata and their inhibitory activities on LPS-induced NO production

Two new neo-clerodane diterpenes, (12S)-6α-acetoxy-4α,18-epoxy-12-hydroxy-19-tigloyloxy-neo-clerod-13-en-15,16-olide (1) and 6α,18-diacetoxy-4α-hydroxy-19-tigloyloxy-neo-clerod-13-en-15,16-olide (2), along with three known analogs (3–5) have been isolated from the whole plants of Ajuga ciliata Bunge. Their structures were elucidated on the basis of spectroscopic data analyses (IR, ESI-MS, HR-ESI-MS, HMQC, HMBC, COSY, and NOESY). The inhibitory activities on LPS-induced NO production of these diterpenes were evaluated and compounds 1 and 5 showed inhibitory effects.     

Biodiversity characteristics of Teucrium polium species in Saudi Arabia

Teucrium (Lamiaceae) is a large and polymorphic genus distributed mainly in Europe, North Africa and in the temperate parts of Asia. In this study, the anatomical features of the leaf and stem of Teucrium polium are investigated. Teucrium has 19 taxa in Iran, which mainly grow in the Irano-Turanian region between 700 and 2000 m above sea level. T. polium belonging to sect. Polium, is a perennial herb growing on Lorestan province. The leaves clearly exhibit xeromorphy due to features such as thick cuticle layer, thick outer epidermal cell wall, high density of trichomes and thick palisade layer of the mesophyll. Anatomical studies on T. polium revealed that the stem shares the general characteristics of the Labiatae family. The aim of our approach was to study the morphological and taxonomical parameters for T. polium in Saudi Arabia. The results of this study showed that there was no influence of environment on the structure of stomata and trichomes as studying species with T.polium. In conclusion our study shows we have studied the geographical distribution of the species in Saudi Arabia and in the world.     

Neomycin- and spectinomycin-resistance replacement vectors for Bacillus subtilis

A plasmid is described for Bacillus subtilis that facilitates replacement of the widely used neomycin resistance gene (neo) with a spectinomycin resistance (spc_E) gene. A second plasmid is described that facilitates replacement of spc_S, associated with mini-Tn10 mutagenesis in B. subtilis, with neo. These plasmids can also function as integrative vectors for B. subtilis. They expand the scope of strain construction and gene analysis in B. subtilis.     

Chemical analysis and biological activities of the essential oil of Teucrium leucocladum Boiss. (Lamiaceae)

The chemical composition of the water-distilled essential oil and n-hexane–ether extract of Teucrium leucocladum Boiss. were determined by GLC and GLC–MS. Altogether 72 compounds were identified. The sesquiterpene alcohols, patchouli alcohol (31.24% and 29.66%) and α-cadinol (9.29% and 21.54%) were the main components in the oil and the extract, respectively. α-Cadinol was isolated and characterized using MS, ¹H- and ¹³C-NMR spectral analyses. Furthermore, the oil and the extract were tested for their bacterostatic and antifungal activities; they showed broad and potent activity against Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans. A marked larvicidal activity of the essential oil, n-hexane–ether extract and crude ethanolic extract was also observed against Culex pipiens, Musca domestica and Ceratitis capitata larvae.     

Identification of Tf1 integration events in S. pombe under nonselective conditions

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning the integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the Schizosaccharomyces pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418^S/neo⁺ Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418^S/neo⁺ clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1's ability to insert within silent regions of S. pombe's genome.     

Hastifolins A–G,antifeedant neo-clerodane diterpenoids from Scutellaria hastifolia

From the aerial parts of Scutellaria hastifolia, family Lamiaceae (Labiatae), seven neo-clerodane diterpenoids (hastifolins A–G) were isolated. The products are similar to the known scuteparvin and are characterized by being trans-cinnamoyl derivatives. Structures and stereochemistry were determined by intensive NMR investigation. Six of the products form three pairs of epimers at C-13. Hastifolins A–C showed significant antifeedant activity.