Bifunctional oligo(ethylene glycol) decorated surfaces which permit covalent protein immobilization and resist protein adsorption

In this article, surface coatings derived from homo-bifunctional tri(ethylene glycol) (EG₃) and hexa(ethylene glycol) (EG₆) molecules which have two terminal aldehyde groups are reported. These homo-bifunctional molecules can be used to functionalize amine-terminated surfaces through crosslinking one aldehyde group to surface amine groups, while leaving the other aldehyde group available for covalent immobilization of proteins. Best of all, after reducing remaining aldehyde groups on the surface with a reducing agent, sodium borohydride, the surface becomes oligo(ethylene glycol) (OEG)-terminated. The OEG-terminated surface can resist nonspecific protein adsorption, a feature that is often required for many biosensors and biomedical devices. Although some mixed self-assembled monolayers formed from two different organothiols also permit covalent protein immobilization and resist nonspecific protein adsorption, the procedure reported herein requires only one type of homo-bifunctional molecule and can be applied to both silicon and gold surfaces.     

Isolation of Paenibacillus sp. and Assessment of its Potential for Enhancing Mineral Weathering

One mineral-solubilizing bacterial strain designated KT was isolated from a soil in Henan Province, China. The full-length 16S rRNA gene sequence showed 95.2 to 96.7% similarity to the bacteria of the genus Paenibacillus, indicating that the strain KT belonged to the genus Paenibacillus. The potential of this strain to release potassium from silicate minerals was investigated using a potassium-bearing rock as the sole source of potassium to support its growth. After inoculation for 7 days, the concentrations of water-soluble Al, Ca and Fe released from the potassium-bearing rock in active bacterial culture were higher than those from the control with autoclaved inoculum, but the concentration of water-soluble K in active bacterial culture was similar to that in the control. The concentrations of HNO₃-extractable Al, Ca, Fe and K from the bacterial culture were also higher than those in the control. These results showed strain KT was able to release potassium from potassium-bearing rock. These results have important implications for extraction of potassium from rocks to support plant growth.     

Microbial Diversity and Community Structure on Corroding Concretes

The objective of this study was to analyze bacterial diversity in two different concrete samples to understand the dominant types of bacteria that may contribute to concrete corrosion. Two concrete samples, HN-1 from the sunny side and HN-2 from dark and damp side, were collected from Zijin Mountain in Nanjing and genomic DNA was extracted. The partial bacterial 16S rRNA gene fragment was PCR amplified and two clone libraries were constructed. Amplified ribosomal DNA restriction analysis (ARDRA) was performed by digestion of the 16S rRNA gene and each unique restriction fragment polymorphism pattern was designated as an operational taxonomic unit (OTU). Phylogenetic trees of bacterial 16S rDNA nucleotide sequences were constructed. Sample HN-1 and HN-2 contained 21 OTUs and 26 OTUs, respectively. Proteobacteria and Planctomycetes were the predominant bacteria in both samples, and they are distributed among Herbaspirillum, Archangium, Phyllobacteriaceae and Planctomycetaceae. Cyanobacteria and Rubrobacter sp. are dominant in HN-1; while Acidobacteriaceae, Adhaeribacter sp. and Nitrospira sp. are predominant in HN-2. This distribution pattern was consistent with local environmental conditions of these two samples. The inferred physiological characteristics of these bacteria, based on relatedness of the DNA clone sequences to cultivated species, revealed different mechanisms of concrete corrosion depending on the local environmental conditions.     

TRPC6 channel‐mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK,PI3K,and CAMKIV signaling

The non‐selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin‐induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient‐mediated changes in synaptic plasticity, ranging from calmodulin‐mediated Ras‐induced signaling cascades comprising the mitogen‐activated protein kinase, PI3K signal transduction pathways as well as Ca²⁺/calmodulin‐dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6‐mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen‐activated protein kinase/extracellular signal‐regulated kinases, phosphatidylinositide 3‐kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP‐response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract‐mediated antidepressant activity.

     

Multispecies coexistence of trees in tropical forests: spatial signals of topographic niche differentiation increase with environmental heterogeneity

Neutral and niche theories give contrasting explanations for the maintenance of tropical tree species diversity. Both have some empirical support, but methods to disentangle their effects have not yet been developed. We applied a statistical measure of spatial structure to data from 14 large tropical forest plots to test a prediction of niche theory that is incompatible with neutral theory: that species in heterogeneous environments should separate out in space according to their niche preferences. We chose plots across a range of topographic heterogeneity, and tested whether pairwise spatial associations among species were more variable in more heterogeneous sites. We found strong support for this prediction, based on a strong positive relationship between variance in the spatial structure of species pairs and topographic heterogeneity across sites. We interpret this pattern as evidence of pervasive niche differentiation, which increases in importance with increasing environmental heterogeneity.     

The Years of the Monkey

The fact that mammals are diploid sets a barrier to rapidly understand the function of non-coding and coding genes in the genome. Recently, Yang et al. reported successful derivation of monkey haploid embryonic stem cells from parthenotes, which provide an effective platform for studying mammalian gene function and enable reverse genetic screening of genes for recessive phenotypes in monkeys.According to the Zodiac in the Chinese Calendar, the next year of the monkey is not slated until February 2016, but a recent paper in this month''s Cell Research suggests that it may have arrived early for the field of stem cell biology. In a stunning technical “Tour de Force”, Jinsong Li and his colleagues report for the first time the generation of several independent haploid monkey embryonic stem (ES) cell lines¹, building on the previous work from their lab and others that described the generation of murine haploid ES cell lines^2,3,4,5 (Figure 1). They first activated metaphase II monkey oocytes with ionomycin followed by cycloheximide treatment. These activated oocytes could develop into blastocysts in vitro and haploid ES cells (haESCs) can be derived by culturing the inner cell mass in a standard monkey ES cell culture system and using Hoechst FACS technique. Remarkably, one of the cell lines remained stable during long term passage, obviating the need for FACS sorting for the haploid cell lines during subsequent propagation. The cell lines can be genetically manipulated by insertional mutagenesis or by PiggyBac transposon technology, suggesting the possibility of genome-wide screening strategies. In this regard, a series of parallel scientific advances suggest that this technology platform may be particularly timely as the field of stem cell biology moves towards regenerative medicine and therapeutics.Open in a separate windowFigure 1The scheme of parthenogenetic (PG) and androgenetic (AG) haploid embryonic stem cells (haESCs) derivation. (A) For the generation of PG-haESCs, metaphase II oocytes were activated with either strontium chloride (SrCl₂) for mice or ionomycin/cycloheximide (CHX) for monkeys and further cultivated to the blastocyst stage. With the help of Hoechst FACS technique, PG-haESCs can be derived. (B) For the generation of AG-haESCs, metaphase II oocytes were enucleated followed by sperm injection. In addition, the reconstructed oocytes were activated with SrCl₂ for mice and further developed to the blastocyst stage in vitro. AG-haESCs can be derived by several rounds of Hoechst FACS based on DNA contents. The derivation of non-human primate AG-haESCs has not been reported yet.For many years, it has proven quite difficult to engineer site-specific mutations, knock-ins, and knock-outs in human ES or induced pluripotent stem (iPS) cells, and only a handful of genetically engineered lines have been created by conventional homologous recombination strategies⁶. However, recent advances in RNA-guided nuclease technology has led to a marked improvement in the efficiency of the knockout of genes in human pluripotent stem cells⁷, suggesting that it may be possible to create knock-out haploid non-human primate (NHP) ES cell lines that harbor specific disease genes and surrogate reporter readouts, and then to look for genetic complementation that could identify critical genes that could be potential drug targets. A library of individual NHP haploid ES cell lines that harbor a loss-of-function mutation across the entire NHP genome could find multiple uses in quickly identifying signaling pathways in differentiated cell types. Given recent advances in screening in human ES and iPS cell lines⁸, direct drug screening on the haploid monkey ES cell lines should also be possible. In addition, it will likely be possible to set up genome-wide screening to systematically identify entire network of genes that drive specific differentiation events, and early steps of primate organogenesis. If androgenetic NHP haploid cell lines can be developed (see Figure 1), a leap in the efficiency of the generation of monkey KO animal models could be envisioned over the long term. In this regard, the recent generation of chimeric monkeys⁹, as well as future technical advances related to this achievement, could become of significant interest.At the same time, the study indirectly raises the query as to the need for monkey model systems when the technology for genetic manipulation in the mouse is without peer, and human ES and iPS cell lines can now be easily generated and genetically manipulated. The recent pronouncement of the termination of NIH support for primate research (http://news.sciencemag.org/people-events/2013/06/nih-will-retire-most-research-chimps-end-many-projects), along with the growing awareness of the need to re-examine the need for NHP models, suggests that there must be very solid scientific grounds for pursuing NHP model systems in the future.In this regard, a growing body of evidence is now pointing to the lack of fidelity of mouse models of human disease to the in vivo human setting, a problem that has plagued cancer therapeutics for decades. Recently, the lack of predictability of human responses from models of murine sepsis has been cogently made¹⁰, and the divergence in the physiology of mice and humans, particularly in terms of metabolism and cardiovascular, are enormous. The complexity and scalability of primate versus murine organogenesis also may be an issue. For example, the human heart is 10 000 times larger than the murine, has a much larger diversity of cell types, and a level of tertiary morphology that is not found in the murine heart (for review see¹¹). Murine cardiogenesis is largely completed with 48 h, while human cardiogenesis occurs over months, and recent studies that suggest a much larger diversity and markedly extended period of proliferation of the family of heart progenitors in the human fetal versus murine heart¹². To date, there are no approved drugs that have come from genetically engineered murine models of cardiovascular (CV) disease, and the biggest CV drugs have actually been discovered based on human genetics (statins, PCSK9, etc.). The increased importance of CV side effects for new drugs in the diabetes space, as well as for other chronic diseases, points to the importance of their study in more sophisticated primate systems, as all these drugs (Avandia, Vioxx, etc.) had cleared conventional screening in rodent model systems. Given the above, we may have to put the Chinese Calendar on auto-repeat mode, as we enter the “Years of the Monkey” in this decade and the next.