Unding factors, suboptimal in vitro culture condition contributes to the poor embryonic development of reconstructed embryos following SCNT. The present study represents an attempt to optimize the culture conditions for the development of human SCNT embryos. Although no blastocyst was obtained following fibroblast nuclear transfer, there was a trend to an augmented development of reconstructed embryos cultured with media containing autocrine/paracrine growth factors. Results from the present study provide the basis for future use of autocrine/paracrine factors to facilitate the derivation of patient-specific embryonic stem cells. In conclusion, the present study demonstrated the utility of growth factor supplementation for optimal human early embryo development and blastocyst outgrowth. The findings may allow the design of better conditions for Solvent Yellow 14 site individual human embryo cultures, for estimating their developmental potentials using secretory products, and for the inclusion of growth factors in embryo transfer media to promote implantation. Although the present experimental design is based on the supplementation of endogenous growth factors diluted during assisted reproductive procedures, future studies on the potential side effects of these paracrine/autocrine factors on chromosomal numbers, genomic integrity, proteomic changes, and epigenetic modifications are essential before clinical use.Supporting InformationFigure SMorphology of reconstructed oocytes.(TIF)Table S1 Conventional RT-PCR primers for growthfactors. (TIF)Table S2 Quantitative RT-PCR primers for growth factors and receptors. (TIF)Author ContributionsConceived and 
designed the experiments: AJH RAR BB JQ. Performed the experiments: KK YC YS YC. Analyzed the data: KK YC YS YC. Wrote the paper: KK 18055761 AJW.
Methanogenesis is the pathway by which ion (H , Na ) gradients across the plasma membrane are generated to drive ATP synthesis, with the concomitant production of methane as an end product. Methanogens are strict anaerobes belonging to the Archaea domain, which can be found in a broad variety of environments such as anaerobic digesters of sewage treatment plants, landfills, rice paddies, lakes and in the sea sediments, among others [1]. Indeed, these organisms have an essential role in the global carbon cycle by transforming small carbon molecules such 
as methanol, methylamines, CO2+H2, formate, CO and acetate into methane. Because heavy metal pollution may develop in some of these habitats, methanogens may be exposed to this environmental stress with the consequent perturbation of the local ecology. Heavy metal pollution of water resources is now a widespread environmental and public health problem, as a result of their elevated toxicity, which may be exacerbated by their potential bio-magnification effect and accumulation throughout the ecological food webs. Pollution of coastal zones by heavy metals such as Cd, Pb, Hg and Ni, is a major environmental problem in some regions of the world [2]. Once in the marine environment, these contaminants accumulate in sediments [3]. Cadmium ocean pollution and BMS5 site mobilization has increased exponentially up to 300 thousands per decade, where 40 of the total current pollution derives from anthropogenic activities [4]. In some coastal zones in the Gulf of Mexico, up to 2,550 mg L21 (22.6 mM) of cadmium has been found, a value far higher than permissible [5]. In other seas and+ +oceans around the world, cadmium concentrations up to 20.5 mg L21 and.Unding factors, suboptimal in vitro culture condition contributes to the poor embryonic development of reconstructed embryos following SCNT. The present study represents an attempt to optimize the culture conditions for the development of human SCNT embryos. Although no blastocyst was obtained following fibroblast nuclear transfer, there was a trend to an augmented development of reconstructed embryos cultured with media containing autocrine/paracrine growth factors. Results from the present study provide the basis for future use of autocrine/paracrine factors to facilitate the derivation of patient-specific embryonic stem cells. In conclusion, the present study demonstrated the utility of growth factor supplementation for optimal human early embryo development and blastocyst outgrowth. The findings may allow the design of better conditions for individual human embryo cultures, for estimating their developmental potentials using secretory products, and for the inclusion of growth factors in embryo transfer media to promote implantation. Although the present experimental design is based on the supplementation of endogenous growth factors diluted during assisted reproductive procedures, future studies on the potential side effects of these paracrine/autocrine factors on chromosomal numbers, genomic integrity, proteomic changes, and epigenetic modifications are essential before clinical use.Supporting InformationFigure SMorphology of reconstructed oocytes.(TIF)Table S1 Conventional RT-PCR primers for growthfactors. (TIF)Table S2 Quantitative RT-PCR primers for growth factors and receptors. (TIF)Author ContributionsConceived and designed the experiments: AJH RAR BB JQ. Performed the experiments: KK YC YS YC. Analyzed the data: KK YC YS YC. Wrote the paper: KK 18055761 AJW.
Methanogenesis is the pathway by which ion (H , Na ) gradients across the plasma membrane are generated to drive ATP synthesis, with the concomitant production of methane as an end product. Methanogens are strict anaerobes belonging to the Archaea domain, which can be found in a broad variety of environments such as anaerobic digesters of sewage treatment plants, landfills, rice paddies, lakes and in the sea sediments, among others [1]. Indeed, these organisms have an essential role in the global carbon cycle by transforming small carbon molecules such as methanol, methylamines, CO2+H2, formate, CO and acetate into methane. Because heavy metal pollution may develop in some of these habitats, methanogens may be exposed to this environmental stress with the consequent perturbation of the local ecology. Heavy metal pollution of water resources is now a widespread environmental and public health problem, as a result of their elevated toxicity, which may be exacerbated by their potential bio-magnification effect and accumulation throughout the ecological food webs. Pollution of coastal zones by heavy metals such as Cd, Pb, Hg and Ni, is a major environmental problem in some regions of the world [2]. Once in the marine environment, these contaminants accumulate in sediments [3]. Cadmium ocean pollution and mobilization has increased exponentially up to 300 thousands per decade, where 40 of the total current pollution derives from anthropogenic activities [4]. In some coastal zones in the Gulf of Mexico, up to 2,550 mg L21 (22.6 mM) of cadmium has been found, a value far higher than permissible [5]. In other seas and+ +oceans around the world, cadmium concentrations up to 20.5 mg L21 and.
