Telomere Dynamics in Human Cells Reprogrammed to Pluripotency

Background

Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell “rejuvenation.”

Methodology/Principal Findings

We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres.

Conclusions/Significance

While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.     

Structure, distribution of species and inundation in a riparian forest of Rio Paraguai, Pantanal, Brazil

A phytosociological study was conducted in a riparian forest of the Rio Paraguai near Corumbá and Ladário in the state of Mato Grosso do Sul, Brazil. The topographic distribution of species was correlated with durations of the river's seasonal floodings and the cumulative time of flooding between 1974 and 1995. One hundred and eight contiguous 10×10 m plots were systematically established. All individuals with more than 15 cm of girth at breast height (gbh=1.3 m) were sampled. A total of 695 individuals distributed among 37 species, 35 genera and 23 families were found. The Shannon index (H′) for species diversity was 2.7. The highest importance value (IV) was found for Inga vera ssp. affinis, followed by Triplaris gardneriana, Ocotea diospyrifolia, Crataeva tapia and Vochysia divergens. The plots were classified into two groups according to their distance from the margin, applying Ward's method of classification and principal coordinate analysis (PCO) on the same Bray Curtis distance matrix. The topical environments were divided into four bands by Two-Way Indicator Species Analysis (TWINSPAN), and the species were also grouped into four groups. Flooding in these groups ranged from regular inundation in all 23 years of the historical series of the Ladário gauge to sporadic flooding for a maximum of 2 consecutive years.     

The neural correlates of desire

In an event-related fMRI study, we scanned eighteen normal human subjects while they viewed three categories of pictures (events, objects and persons) which they classified according to desirability (desirable, indifferent or undesirable). Each category produced activity in a distinct part of the visual brain, thus reflecting its functional specialization. We used conjunction analysis to learn whether there is a brain area which is always active when a desirable picture is viewed, regardless of the category to which it belongs. The conjunction analysis of the contrast desirable > undesirable revealed activity in the superior orbito-frontal cortex. This activity bore a positive linear relationship to the declared level of desirability. The conjunction analysis of desirable > indifferent revealed activity in the mid-cingulate cortex and in the anterior cingulate cortex. In the former, activity was greater for desirable and undesirable stimuli than for stimuli classed as indifferent. Other conjunction analyses produced no significant effects. These results show that categorizing any stimulus according to its desirability activates three different brain areas: the superior orbito-frontal, the mid-cingulate, and the anterior cingulate cortices.     

Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos

Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.     

Self-incompatibility, Inbreeding Depression and Crossing Potential in Five BrazilianPleurothallis (Orchidaceae) Species

Intra- and interspecific experimental pollinations were madeto determine the mating systems and the interspecific crossingpotential in individuals from 24 populations of five Pleurothallisspecies. Pleurothallis johannensis, P. ochreata and P. teresshow weak or partial self-incompatibility while P. adamantinensisand P.fabiobarrosii are more strictly self-incompatible. Wefound no differences in fruit set between intra- and interpopulationintraspecific crosses, and there was no correlation betweenfruit set and genetic variability or inbreeding in these species.All species are interfertile and showed no correlation betweengenetic similarity and crossing potential. We scored fruitsfor seed viability and observed a strong inbreeding depressionin all populations; however, there was no difference in seedviability among intrapopulation, interpopulation intraspecificand interspecific cross-pollinations. These species are pollinatedby flies with a behavioural pattern that facilitates self-pollination.Self-incompatibility and inbreeding depression are apparentlyimportant in the maintenance of the unusually high levels ofgenetic variability found in these species. As generally observedin other orchid species, barriers to hybridization between thesePleurothallis species are at the level of pollination. Copyright2001 Annals of Botany Company Pleurothallis, Orchidaceae, mating system, self-incompatibility, inbreeding depression, fruit set, seed set, polyembryony, interspecific compatibility     

Methacrylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of human serum albumin

Different biologands carrying synthetic adsorbents have been reported in the literature for protein separation. We have developed a novel and new approach to obtain high protein adsorption capacity utilizing 2-methacrylamidohistidine (MAH) as a bioligand. MAH was synthesized by reacting methacrylochloride and histidine. Spherical beads with an average size of 150–200 μm were obtained by the radical suspension polymerization of MAH and 2-hydroxyethyl-methacrylate (HEMA) conducted in an aqueous dispersion medium. p(HEMA-co-MAH) beads had a specific surface area of 17.6 m²/g. Synthesized MAH monomer was characterized by NMR. p(HEMA-co-MAH) beads were characterized by swelling test, FTIR and elemental analysis. Then, Cu(II) ions were incorporated onto the beads and Cu(II) loading was found to be 0.96 mmol/g. These affinity beads with a swelling ratio of 65%, and containing 1.6 mmol. MAH/g were used in the adsorption/desorption of human serum albumin (HSA) from both aqueous solutions and human serum. The adsorption of HSA onto p(HEMA-co-MAH) was low (8.8 mg/g). Cu(II) chelation onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA adsorption was observed at pH 3.0 Higher HSA adsorption was observed from human plasma (94.6 mg HSA/g). Adsorption of other serum proteins were obtained as 3.7 mg/g for fibrinogen and 8.5 mg/g for γ-globulin. The total protein adsorption was determined as 107.1 mg/g. Desorption of HSA was obtained using 0.1 M Tris/HCl buffer containing 0.5M NaSCN. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cu(II) chelated-p(HEMA-co-MAH) beads without significant decreases in the adsorption capacities.     

Variation of diastereoisomeric pyrrolizidine alkaloids in Pleurothallis (Orchidaceae)

The alkaloids of leaves and flowers of 18 populations of four Pleurothallis (Orchidaceae) species were characterized. Both leaves and flowers of individuals from all populations have two diasteroisomers of 1-hydroxymethylpyrrolizidine (RI=1290 and 1311). The 1290 isomer was the most abundant in all populations of P. johannensis (74-93%) and P. fabiobarrosii (63-93%), two morphologically closed species. On the other hand, in almost all populations of P. teres the 1311 isomer is the more abundant (61-95%), except in one population occurring disjunct from the core area distribution of the species. These results support the recent taxonomic revision for these three species. Almost all P. ochreata populations also have the 1290 as the most abundant isomer, except in one morphologically differentiated population situated at the southern extreme of the distribution of this species.     

Histological study of post-pollination events in Spathodea campanulata beauv. (Bignoniaceae), a species with late-acting self-incompatibility

The reproductive biology of Spathodea campanulata was investigated by means of hand-pollination experiments, observations of pollen tube growth using fluorescence microscopy, and serial sections of ovules in selfed and crossed pistils. Only cross-pollinated flowers developed fruits, and all selfed flowers abscised within 3-4 d. However, self pollen tubes grew successfully to the ovary, penetrating and fertilizing the majority of ovules by 48 h, indicating that S. campanulata is a species with late-acting self-incompatibility. The incidences of ovule penetration, fertilization and endosperm initiation were all significantly slower in selfed vs. crossed pistils, although no other signs of malfunctioning were detected. The possible role of such slow self pollen tube effectiveness as a recognition event is discussed within the context of the slow but not entirely suppressed self pollen tube growth reported for some species with conventional homomorphic self-incompatibility.     

Interspecies nuclear transfer: implications for embryonic stem cell biology

Accessibility of human oocytes for research poses a serious ethical challenge to society. This fact categorically holds true when pursuing some of the most promising areas of research, such as somatic cell nuclear transfer and embryonic stem cell studies. One approach to overcoming this limitation is to use an oocyte from one species and a somatic cell from another. Recently, several attempts to capture the promises of this approach have met with varying success, ranging from establishing human embryonic stem cells to obtaining live offspring in animals. This review focuses on the challenges and opportunities presented by the formidable task of overcoming biological differences among species.     

Oxidative Stress and Apoptosis in Relation to Exposure to Magnetic Field

We investigated the effect of extremely low-frequency electromagnetic field (ELF-EMF) with pulse trains exposure on lipid peroxidation, and, hence, oxidative stress in the rat liver tissue. The parameters that we measured were the levels of plasma alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase as well as plasma albumin, bilirubin, and total protein levels in 30 adult male Wistar rats exposed to ELF. We also determined the percentage of apoptotic and necrotic cells of the kidney extracts from the animals by flow cytometry method. Apoptotic cell death was further characterized by monitoring DNA degradation using gel electrophoresis. The results showed an increase in the levels of oxidative stress indicators, and the flow cytometric data suggested a possible relationship between the exposure to magnetic field and the cell death. We showed significantly lower necrotic cell percentages in experimental animals compared to either unexposed or sham control groups. However, DNA ladder analyses did not differentiate between the groups. Our results were discussed in relation to the response of biological systems to EMF.