Serial extraction of endosperm drillings (SEED)—A method for detecting transgenes and proteins in single viable maize kernels

We have developed a method for detecting a transgene and its protein product in maize endosperm that allows the kernel to be germinated after analysis. This technique could be highly useful for several monocots and dicots. Our method involves first sampling the endosperm with a hand-held rotary grinder so that the embryo is preserved and capable of germination. This tissue is then serially extracted, first with SDS-PAGE sample buffer to extract proteins, then with an aqueous buffer to extract DNA. The product of the transgene can be detected in the first extract by SDS-PAGE with visualization by total protein staining or immuno-blot detection. The second extract can be purified and used as template DNA in PCR reactions to detect the transgene. This method is particularly useful for screening transgenic kernels in breeding experiments and testing for gene silencing in kernels

The Association of Two Invasive Shrubs, Common Buckthorn (Rhamnus Cathartica) and Tartarian Honeysuckle (Lonicera Tatarica), with Oak Communities in the Midwestern United States

Oak forests throughout North America are declining due to changes in disturbance regimes that have led to increased competition from other tree and shrub species. We evaluated associations between oak regeneration, the occurrence of two common invasive shrubs (common buckthorn (Rhamnus cathartica L.) and Tartarian honeysuckle (Lonicera tatarica L.)), and forest edges in oak forests in a portion of the midwestern United States where bur (Quercus macrocarpa Michx.), red (Quercus rubra L.), and white oak (Quercus alba L.) were historically dominant. We found poor recruitment of oaks in comparison to other, more shade-tolerant tree species. Results further revealed a strong stand-scale association between bur oak, open canopy conditions, high soil nutrient levels, and the presence of common buckthorn and Tartarian honeysuckle; these same site characteristics were disassociated with red and white oak. Within red and white oak stands, however, the presence of the invasive shrubs was more pronounced near forest edges. While oak recruitment is hampered throughout stands, our research suggests that predominant constraints may vary based on soil and light gradients found along forest edges.This article is from Canadian Journal of Forest Research 41 (2011): 1981, doi:10.1139/X11-112.</p

Serial extraction of endosperm drillings (SEED)—A method for detecting transgenes and proteins in single viable maize kernels

We have developed a method for detecting a transgene and its protein product in maize endosperm that allows the kernel to be germinated after analysis. This technique could be highly useful for several monocots and dicots. Our method involves first sampling the endosperm with a hand-held rotary grinder so that the embryo is preserved and capable of germination. This tissue is then serially extracted, first with SDS-PAGE sample buffer to extract proteins, then with an aqueous buffer to extract DNA. The product of the transgene can be detected in the first extract by SDS-PAGE with visualization by total protein staining or immuno-blot detection. The second extract can be purified and used as template DNA in PCR reactions to detect the transgene. This method is particularly useful for screening transgenic kernels in breeding experiments and testing for gene silencing in kernels.This article is published as Sangtong, Varaporn, Erik C. Mottl, Mary Jane Long, Michael Lee, and M. Paul Scott. "Serial extraction of endosperm drillings (seed)—A method for detecting transgenes and proteins in single viable maize kernels." Plant Molecular Biology Reporter 19, no. 2 (2001): 151-158.</p

Clinical Efficacy of ONC201 in H3K27M-Mutant Diffuse Midline Gliomas Is Driven by Disruption of Integrated Metabolic and Epigenetic Pathways.

UNLABELLED: Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction. SIGNIFICANCE: The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction. This article is featured in Selected Articles from This Issue, p. 2293