Folate receptor-mediated boron-10 containing carbon nanoparticles as potential delivery vehicles for boron neutron capture therapy of nonfunctional pituitary adenomas

Invasive nonfunctional pituitary adenomas (NFPAs) are difficult to completely resect and often develop tumor recurrence after initial surgery. Currently, no medications are clinically effective in the control of NFPA. Although radiation therapy and radiosurgery are useful to prevent tumor regrowth, they are frequently withheld because of severe complications. Boron neutron capture therapy (BNCT) is a binary radiotherapy that selectively and maximally damages tumor cells without harming the surrounding normal tissue. Folate receptor (FR)-targeted boron-10 containing carbon nanoparticles is a novel boron delivery agent that can be selectively taken up by FR-expressing cells via FR-mediated endocytosis. In this study, FR-targeted boron-10 containing carbon nanoparticles were selectively taken up by NFPAs cells expressing FR but not other types of non-FR expressing pituitary adenomas. After incubation with boron-10 containing carbon nanoparticles and following irradiation with thermal neutrons, the cell viability of NFPAs was significantly decreased, while apoptotic cells were simultaneously increased. However, cells administered the same dose of FR-targeted boron-10 containing carbon nanoparticles without neutron irradiation or received the same neutron irradiation alone did not show significant decrease in cell viability or increase in apoptotic cells. The expression of Bcl-2 was down-regulated and the expression of Bax was up-regulated in NFPAs after treatment with FR-mediated BNCT. In conclusion, FR-targeted boron-10 containing carbon nanoparticles may be an ideal delivery system of boron to NFPAs cells for BNCT. Furthermore, our study also provides a novel insight into therapeutic strategies for invasive NFPA refractory to conventional therapy, while exploring these new applications of BNCT for tumors, especially benign tumors.     

Simultaneous Analysis of Anthocyanin and Non-Anthocyanin Flavonoid in Various Tissues of Different Lotus (Nelumbo) Cultivars by HPLC-DAD-ESI-MSn

A validated HPLC-DAD-ESI-MSⁿ method for the analysis of non-anthocyanin flavonoids was applied to nine different tissues of twelve lotus genotypes of Nelumbo nucifera and N. lutea, together with an optimized anthocyanin extraction and separation protocol for lotus petals. A total of five anthocyanins and twenty non-anthocyanin flavonoids was identified and quantified. Flavonoid contents and compositions varied with cultivar and tissue and were used as a basis to divide tissues into three groups characterized by kaempferol and quercetin derivatives. Influences on flower petal coloration were investigated by principal components analyses. High contents of kaempferol glycosides were detected in the petals of N. nucifera while high quercetin glycoside concentrations occurred in N. lutea. Based on these results, biosynthetic pathways leading to specific compounds in lotus tissues are deduced through metabolomic analysis of different genotypes and tissues and correlations among flavonoid compounds.     

LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin Whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression

There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF_1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF_1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.     

c-Abl Kinase Is a Regulator of αvβ3 Integrin Mediated Melanoma A375 Cell Migration

Integrins are heterodimeric transmembrane receptors that physically link the extracellular matrix (ECM) to the intracellular actin cytoskeleton, and are also signaling molecules that transduce signals bi-directionally across the plasma membrane. Integrin regulation is essential for tumor cell migration in response to growth factors. c-Abl kinase is a nonreceptor tyrosine kinase and is critical for signaling transduction from various receptors. Here we show that c-Abl kinase is involved in A375 cell migration mediated by α_vβ₃ integrin in response to PDGF stimulation. c-Abl kinase colocalizes with α_vβ₃ integrin dynamically and affects α_vβ₃ integrin affinity by regulating its cluster. The interaction between c-Abl kinase and α_vβ₃ integrin was dependent on the activity of c-Abl kinase induced by PDGF stimulation, but was not dependent on the binding of α_vβ₃ integrin with its ligands, suggesting that c-Abl kinase is not involved in the outside-in signaling of α_vβ₃ integrin. Talin head domain was required for the interaction between c-Abl kinase and α_vβ₃ integrin, and the SH3 domain of c-Abl kinase was involved in its interaction with talin and α_vβ₃ integrin. Taken together, we have uncovered a novel and critical role of c-Abl kinase in α_vβ₃ integrin mediated melanoma cell migration.     

Developmental Potential of Prepubertal Mouse Oocytes Is Compromised Due Mainly to Their Impaired Synthesis of Glutathione

Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca²⁺ reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS) levels increased, Ca²⁺ storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the γ-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca²⁺ store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.     

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.