Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.     

A novel host shift and invaded range of a seed predator, Acanthoscelides macrophthalmus (Coleoptera: Chrysomelidae: Bruchinae), of an invasive weed, Leucaena leucocephala

An endophagous seed predator, Acanthoscelides macrophthalmus (Coleoptera: Chrysomelidae: Bruchinae), utilizes Neotropical Leucaena (Fabaceae: Mimosoideae). One of its hosts, Leucaena leucocephala , is a fast-growing nitrogen-fixing tree that serves as a multipurpose beneficial plant but eventually becomes an aggressive invader where it was introduced. Herein, we report A. macrophthalmus invasion of the Far East, South Asian tropics and subtropics (Japanese Pacific Islands, Taiwan, Southern China, Northern Thailand and Southern India). Of other field-collected mimosoid legumes, an introduced tree, Falcataria moluccana , in Taiwan was found to be used by the seed predator. Conversely, our published work review revealed that the seed predator had retained high host specificity to Leucaena species in its native and introduced regions. Acanthoscelides macrophthalmus was able to utilize aphagously postharvest mature seeds for oviposition and larval development, which is a trait of post-dispersal seed predators. We confirmed that A. macrophthalmus that was reared on L. leucocephala was able to utilize F. moluccana as well. Although the relatively high host specificity of the oligophagous beetle is suitable for controlling the weedy L. leucocephala , the potential host range expansion confirmed by this study must be cautioned.     

Investigation of the Listeria monocytogenes Scott A acid tolerance response and associated physiological and phenotypic features via whole proteome analysis

The global proteomic responses of the foodborne pathogen Listeria monocytogenes strain Scott A, during active growth and transition to the stationary growth phase under progressively more acidic conditions, created by addition of lactic acid and HCl, were investigated using label-free liquid chromatography/tandem mass spectrometry. Approximately 56% of the Scott A proteome was quantitatively assessable, and the data provides insight into its acquired acid tolerance response (ATR) as well as the relation of the ATR to the growth phase transition. Alterations in protein abundance due to acid stress were focused in proteins belonging to the L. monocytogenes common genome, with few strain-dependent proteins involved. However, one of the two complete prophage genomes appeared to enter lysogeny. During progressive acidification, the growth rate and yield were reduced 55% and 98%, respectively, in comparison to nonacidified control cultures. The maintenance of the growth rate was determined to be connected to activation of cytoplasmic pH homeostatic mechanisms while cellular reproductive-related and cell component turnover proteins were markedly more abundant in acid stressed cultures. Cell biomass accumulation was impeded predominantly due to repression of phosphodonor-linked enzymes involved with sugar phosphotransfer, glycolysis, and cell wall polymer biosynthesis. Acidification caused a shift from heterofermentation to an oxidatively stressed state in which ATP appears to be generated mainly through the pyruvate dehydrogenase/pyruvate oxidase/phosphotransacetylase/acetate kinase and branched chain acid dehydrogenase pathways. Analysis of regulons indicated energy conservation occurs due to repression by the GTP/isoleucine sensor CodY and also the RelA mediated stringent response. Whole proteome analysis proved to be an effective way to highlight proteins involved with the acquisition of the ATR.