Dynamics of Autotrophic Marine Planktonic Thaumarchaeota in the East China Sea

The ubiquitous and abundant distribution of ammonia-oxidizing Thaumarchaeota in marine environments is now well documented, and their crucial role in the global nitrogen cycle has been highlighted. However, the potential contribution of Thaumarchaeota in the carbon cycle remains poorly understood. Here we present for the first time a seasonal investigation on the shelf region (bathymetry≤200 m) of the East China Sea (ECS) involving analysis of both thaumarchaeal 16S rRNA and autotrophy-related genes (acetyl-CoA carboxylase gene, accA). Quantitative PCR results clearly showed a higher abundance of thaumarchaeal 16S and accA genes in late-autumn (November) than summer (August), whereas the diversity and community structure of autotrophic Thaumarchaeota showed no statistically significant difference between different seasons as revealed by thaumarchaeal accA gene clone libraries. Phylogenetic analysis indicated that shallow ecotypes dominated the autotrophic Thaumarchaeota in the ECS shelf (86.3% of total sequences), while a novel non-marine thaumarchaeal accA lineage was identified in the Changjiang estuary in summer (when freshwater plumes become larger) but not in autumn, implying that Changjiang freshwater discharge played a certain role in transporting terrestrial microorganisms to the ECS. Multivariate statistical analysis indicated that the biogeography of the autotrophic Thaumarchaeota in the shelf water of the ECS was influenced by complex hydrographic conditions. However, an in silico comparative analysis suggested that the diversity and abundance of the autotrophic Thaumarchaeota might be biased by the ‘universal’ thaumarchaeal accA gene primers Cren529F/Cren981R since this primer set is likely to miss some members within particular phylogenetic groups. Collectively, this study improved our understanding of the biogeographic patterns of the autotrophic Thaumarchaeota in temperate coastal waters, and suggested that new accA primers with improved coverage and sensitivity across phylogenetic groups are needed to gain a more thorough understanding of the role of the autotrophic Thaumarchaeota in the global carbon cycle.     

?-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca²⁺] <1 µM, ß-adrenergic stimulation increased luminal Ca²⁺ activation of single RyR channels, decreased luminal Mg²⁺ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg²⁺. At cytoplasmic [Ca²⁺] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg²⁺ and Ca²⁺ inhibition of RyRs. The K_a and maximum levels of cytoplasmic Ca²⁺ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low-affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.     

Diet Shift and Its Impact on Foraging Behavior of Siberian Crane (Grus Leucogeranus) in Poyang Lake

The study of habitat selection and diet has a long history in ecology. This is often used to assess the functional roles of wetland in biodiversity conservation. Shifting habitat and diet may be one of the survival strategies during extremely adverse conditions. Therefore, sudden changes in habitat selection may indicate the deterioration of the habitat quality, and management interventions are necessary. Siberian crane (Grus leucogeranus) became critically endangered due to loss of habitat, and is currently a global conservation focus. Every winter, more than 95% of the species'' global population congregates at Poyang Lake, and feeds on tubers of Vallisneria spiralis in shallow water and mudflat habitat. In this study, we reported the first sighting of large numbers of Siberian cranes foraging at wet meadows, where they fed on a different plant, Potentilla limprichtii due to extreme scarcity of their preferred tuber. To understand how well the cranes adapted to such unusual habitat, field surveys to assess the distribution of cranes across different habitats, and food availability in each habitat were carried out in the winter of 2011. Field observations on crane behaviors at different habitats were also conducted. Results show that cranes displayed significantly different behavior patterns when using the wet meadow, compared to the crane''s optimal habitat - shallow water and mudflat. Both juveniles and adults spent significantly less time foraging, and more time alerting in meadows than in shallow waters and mudflats. These results indicated that the meadow might be a suboptimal wintering ground for Siberian crane, which helped the cranes survive from extreme unfavorable conditions. To some degree, this finding alleviates the general concern over the fluctuating of its food resources which was caused by hydrological disturbances. However, more studies are needed to assess the consequences of such diet and habitat shift for crane survival.     

End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

Background

Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening.

Results

Herein, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform’s capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, and proteogenic and metabolic output analysis.

Conclusions

Taken together, we demonstrate the microfluidic platform’s potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis.

     

Evaluation of Liver Ischemia-Reperfusion Injury in Rabbits Using a Nanoscale Ultrasound Contrast Agent Targeting ICAM-1

ObjectiveTo assess the feasibility of ultrasound molecular imaging in the early diagnosis of liver ischemia-reperfusion injury (IRI) using a nanoscale contrast agent targeting anti-intracellular adhesion molecule-1 (anti-ICAM-1).MethodsThe targeted nanobubbles containing anti-ICAM-1 antibody were prepared using the avidin-biotin binding method. Human hepatic sinusoidal endothelial cells (HHSECs) were cultured at the circumstances of hypoxia/reoxygenation (H/R) and low temperature. The rabbit liver IRI model (I/R group) was established using the Pringle’s maneuver. The time-intensity curve of the liver contrast ultrasonographic images was plotted and the peak intensity, time to peak, and time of duration were calculated.ResultsThe size of the targeted nanobubbles were 148.15 ± 39.75 nm and the concentration was 3.6–7.4 × 10⁹/ml, and bound well with the H/R HHSECs. Animal contrast enhanced ultrasound images showed that the peak intensity and time of duration of the targeted nanobubbles were significantly higher than that of common nanobubbles in the I/R group, and the peak intensity and time of duration of the targeted nanobubbles in the I/R group were also significantly higher than that in the SO group.ConclusionThe targeted nanobubbles have small particle size, stable characteristic, and good targeting ability, which can assess hepatic ischemia-reperfusion injury specifically, noninvasively, and quantitatively at the molecular level.     

Genome-Wide Identification,Characterization, and Stress-Responsive Expression Profiling of Genes Encoding LEA (Late Embryogenesis Abundant) Proteins in Moso Bamboo (Phyllostachys edulis)

Late embryogenesis abundant (LEA) proteins have been identified in a wide range of organisms and are believed to play a role in the adaptation of plants to stress conditions. In this study, we performed genome-wide identification of LEA proteins and their coding genes in Moso bamboo (Phyllostachys edulis) of Poaceae. A total of 23 genes encoding LEA proteins (PeLEAs) were found in P. edulis that could be classified to six groups based on Pfam protein family and homologous analysis. Further in silico analyses of the structures, gene amount, and biochemical characteristics were conducted and compared with those of O. sativa (OsLEAs), B. distachyon (BdLEAs), Z. mays (ZmLEAs), S. bicolor (SbLEAs), Arabidopsis, and Populus trichocarpa. The less number of PeLEAs was found. Evolutionary analysis revealed orthologous relationship and colinearity between P. edulis, O. sativa, B. distachyon, Z. mays, and S. bicolor. Analyses of the non-synonymous (Ka) and synonymous (Ks)substitution rates and their ratios indicated that the duplication of PeLEAs may have occurred around 18.8 million years ago (MYA), and divergence time of LEA family among the P. edulis-O. sativa and P. edulis–B. distachyon, P. edulis-S. bicolor, and P. edulis-Z. mays was approximately 30 MYA, 36 MYA, 48 MYA, and 53 MYA, respectively. Almost all PeLEAs contain ABA- and (or) stress-responsive regulatory elements. Further RNA-seq analysis revealed approximately 78% of PeLEAs could be up-regulated by dehydration and cold stresses. The present study makes insights into the LEA family in P. edulis and provides inventory of stress-responsive genes for further functional validation and transgenic research aiming to plant genetic improvement of abiotic stress tolerance.     

Hindcasting Historical Breeding Conditions for an Endangered Salamander in Ephemeral Wetlands of the Southeastern USA: Implications of Climate Change

The hydroperiod of ephemeral wetlands is often the most important characteristic determining amphibian breeding success, especially for species with long development times. In mesic and wet pine flatwoods of the southeastern United States, ephemeral wetlands were a common landscape feature. Reticulated flatwoods salamanders (Ambystoma bishopi), a federally endangered species, depend exclusively on ephemeral wetlands and require at least 11 weeks to successfully metamorphose into terrestrial adults. We empirically modeled hydroperiod of 17 A. bishopi breeding wetlands by combining downscaled historical climate-model data with a recent 9-year record (2006–2014) of observed water levels. Empirical models were subsequently used to reconstruct wetland hydrologic conditions from 1896–2014 using the downscaled historical climate datasets. Reconstructed hydroperiods for the 17 wetlands were highly variable through time but were frequently unfavorable for A. bishopi reproduction (e.g., only 61% of years, using a conservative estimate of development time [12 weeks], were conducive to larval development and metamorphosis). Using change-point analysis, we identified significant shifts in average hydroperiod over the last century in all 17 wetlands. Mean hydroperiods were shorter in recent years than at any other point since 1896, and thus less suitable for A. bishopi reproduction. We suggest that climate change will continue to impact the reproductive success of flatwoods salamanders and other ephemeral wetland breeders by reducing the number of years these wetlands have suitable hydroperiods. Consequently, we emphasize the importance of conservation and management for mitigating other forms of habitat degradation, especially maintenance of high quality breeding sites where reproduction can occur during appropriate environmental conditions.