The soil and plant biogeochemistry sampling design for The National Ecological Observatory Network

Human impacts on biogeochemical cycles are evident around the world, from changes to forest structure and function due to atmospheric deposition, to eutrophication of surface waters from agricultural effluent, and increasing concentrations of carbon dioxide (CO2) in the atmosphere. The National Ecological Observatory Network (NEON) will contribute to understanding human effects on biogeochemical cycles from local to continental scales. The broad NEON biogeochemistry measurement design focuses on measuring atmospheric deposition of reactive mineral compounds and CO2 fluxes, ecosystem carbon (C) and nutrient stocks, and surface water chemistry across 20 eco‐climatic domains within the United States for 30 yr. Herein, we present the rationale and plan for the ground‐based measurements of C and nutrients in soils and plants based on overarching or “high‐level” requirements agreed upon by the National Science Foundation and NEON. The resulting design incorporates early recommendations by expert review teams, as well as recent input from the larger natural sciences community that went into the formation and interpretation of the requirements, respectively. NEON\u27s efforts will focus on a suite of data streams that will enable end‐users to study and predict changes to biogeochemical cycling and transfers within and across air, land, and water systems at regional to continental scales. At each NEON site, there will be an initial, one‐time effort to survey soil properties to 1 m (including soil texture, bulk density, pH, baseline chemistry) and vegetation community structure and diversity. A sampling program will follow, focused on capturing long‐term trends in soil C, nitrogen (N), and sulfur stocks, isotopic composition (of C and N), soil N transformation rates, phosphorus pools, and plant tissue chemistry and isotopic composition (of C and N). To this end, NEON will conduct extensive measurements of soils and plants within stratified random plots distributed across each site. The resulting data will be a new resource for members of the scientific community interested in addressing questions about long‐term changes in continental‐scale biogeochemical cycles, and is predicted to inspire further process‐based research

Formation of free amino acids in rhizosphere and nonrhizosphere soil

Includes bibliographical references (page 362).Untreated samples of nonrhizosphere and soybean rhizosphere soils each contained about 15 identified free amino acids totaling 2 to 4 µg. per g. of soil; lysine was the most prevalent amino acid in each preparation. Numerous additional unidentified compounds occurred at concentrations estimated as 0.1 to 0.5 µg. per g. Treatment with glucose and potassium nitrate increased the amount of free amino acids to about 100 µg. per g. after 3 days. Concentrations declined after 3 days but still were 4 to 5 times that of the untreated control after 2 weeks' incubation. Glutamic acid was the dominant amino acid in all treated soils. Rhizosphere soil did not differ quantitatively from nonrhizosphere in samples treated with glucose, although a greater variety of ninhydrin reacting compounds was encountered in rhizosphere soil. Treated soils incubated at 20% field moisture capacity differed little in free amino acids from those held at 30%. The features of the free amino acid fraction are discussed

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al

Continuous flow isotope ratio mass spectrometry of carbon dioxide trapped as strontium carbonate

Includes bibliographical references (pages 756-757).The isotopic signal provided by differential discrimination against atmospheric carbon dioxide (13CO2) by C3 and C4 plant photosynthetic pathways is being widely used to study the processes of carbon (C) fixation, soil organic matter formation, and mineralization in nature. These studies have been facilitated by the availability of automated C and nitrogen (N) combustion analyzers (ANCA) combined with continuous flow isotope ratio mass spectrometers (CFIRMS). Analysis of 13CO2 in these instruments requires consistent sample mass for best precision, a requirement that is easily satisfied for soil and tissue samples by adjusting sample weight. Consistent CO2 sample size is much more difficult to achieve using gas handling systems for samples of headspace gases when CO2 concentrations vary widely. Long storage of gaseous samples also is difficult. Extended respiration studies are most easily conducted by trapping CO2 in alkali and conversion to an insoluble carbonate. Thermal decomposition of the carbonate in an on-line ANCA allows consistent and optimal CO2 sample mass to be obtained. The use of precipitated carbonates also facilitates storage of samples and enables full automation of sample analysis using an ANCA interfaced to a CFIRMS. Calcium (Ca), strontium (Sr), and barium (Ba) carbonates were tested. Strontium carbonate (SrCO3) with the addition of vanadium pentoxide (V2O5) as a combustion catalyst was found most suitable

Epigenetic Characterization of the FMR1 Gene and Aberrant Neurodevelopment in Human Induced Pluripotent Stem Cell Models of Fragile X Syndrome

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5′ untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology.FRAXA Research FoundationHarvard Stem Cell Institute (seed grant)Stanley Medical Research InstituteNational Institute of Mental Health (U.S.) (grant #R33MH087896

ROCK Inhibitor Is Not Required for Embryoid Body Formation from Singularized Human Embryonic Stem Cells

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications

Oral Insulin

Oral insulin is an exciting area of research and development in the field of diabetology. This brief review covers the various approaches used in the development of oral insulin, and highlights some of the recent data related to novel oral insulin preparation

Physical Passaging of Embryoid Bodies Generated from Human Pluripotent Stem Cells

Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1∶4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells

Identification of Five Developmental Processes during Chondrogenic Differentiation of Embryonic Stem Cells

Chondrogenesis is the complex process that leads to the establishment of cartilage and bone formation. Due to their ability to differentiate in vitro and mimic development, embryonic stem cells (ESCs) show great potential for investigating developmental processes. In this study, we used chondrogenic differentiation of ESCs as a model to analyze morphogenetic events during chondrogenesis.ESCs were differentiated into the chondrocyte lineage, forming small cartilaginous aggregates in suspension. Differentiated ESCs showed that chondrogenesis was typically characterized by five overlapping stages. During the first stage, cell condensation and aggregate formation was observed. The second stage was characterized by differentiation into chondrocytes and fibril scaffold formation within spherical aggregates. Deposition of cartilaginous extracellular matrix and cartilage formation were hallmarks of the third stage. Apoptosis of chondrocytes, hypertrophy and/or degradation of cartilage occurred during the fourth stage. Finally, during the fifth stage, bone replacement with membranous calcified tissues took place.We demonstrate that ESCs show the chondrogenic differentiation pathway from the pluripotent stem cell to terminal skeletogenesis through these five stages in vitro. During each stage, morphological changes acquired in preceding stages played an important role in further development as a scaffold or template in subsequent stages. The study of chondrogenesis via ESC differentiation may be informative to our further understanding of skeletal growth and regeneration