By about 2.0 billion years ago (Ga), there is evidence for a period best known for its extended, apparent geochemical stability expressed famously in the carbonate–carbon isotope data. Despite the first appearance and early innovation among eukaryotic organisms, this period is also known for a rarity of eukaryotic fossils and an absence of organic biomarker fingerprints for those organisms, suggesting low diversity and relatively small populations compared to the Neoproterozoic era. Nevertheless, the search for diagnostic biomarkers has not been performed with guidance from paleoenvironmental redox constrains from inorganic geochemistry that should reveal the facies that were most likely hospitable to these organisms. Siltstones and shales obtained from drill core of the ca. 1.3–1.4 Ga Roper Group from the McArthur Basin of northern Australia provide one of our best windows into the mid‐Proterozoic redox landscape. The group is well dated and minimally metamorphosed (of oil window maturity), and previous geochemical data suggest a relatively strong connection to the open ocean compared to other mid‐Proterozoic records. Here, we present one of the first integrated investigations of Mesoproterozoic biomarker records performed in parallel with established inorganic redox proxy indicators. Results reveal a temporally variable paleoredox structure through the Velkerri Formation as gauged from iron mineral speciation and trace‐metal geochemistry, vacillating between oxic and anoxic. Our combined lipid biomarker and inorganic geochemical records indicate at least episodic euxinic conditions sustained predominantly below the photic zone during the deposition of organic‐rich shales found in the middle Velkerri Formation. The most striking result is an absence of eukaryotic steranes (4‐desmethylsteranes) and only traces of gammacerane in some samples—despite our search across oxic, as well as anoxic, facies that should favor eukaryotic habitability and in low maturity rocks that allow the preservation of biomarker alkanes. The dearth of Mesoproterozoic eukaryotic sterane biomarkers, even within the more oxic facies, is somewhat surprising but suggests that controls such as the long‐term nutrient balance and other environmental factors may have throttled the abundances and diversity of early eukaryotic life relative to bacteria within marine microbial communities. Given that molecular clocks predict that sterol synthesis evolved early in eukaryotic history, and (bacterial) fossil steroids have been found previously in 1.64 Ga rocks, then a very low environmental abundance of eukaryotes relative to bacteria is our preferred explanation for the lack of regular steranes and only traces of gammacerane in a few samples. It is also possible that early eukaryotes adapted to Mesoproterozoic marine environments did not make abundant steroid lipids or tetrahymanol in their cell membranes.

Mercapto-silica spheres with controllable size from ∼150 nm to ∼3.5 μm and narrow size distribution have been prepared in water using a one-pot synthesis, in which 3-mercaptopropyltrimethoxysilane (MPS) was used as the sole silica source and ammonia as the base catalyst. The hydrolysis of MPS at the early stage of the reaction produces amphiphilic silicate species which initiate the self-emulsification of the system and lead to the formation of oil-in-water emulsion droplets. Further hydrolysis and condensation promote the nucleation and growth of the mercapto-silica spheres inside the emulsion droplets. These mercapto-silica spheres are both structurally and functionally different from typical silica particles prepared from silicon alkoxides. Understanding the formation mechanism allows systematic tuning of the size of mercapto-silica spheres in a wide range by changing the amount of precursor, the concentration of ammonia, the amount of additional surfactants, and the reaction time. We find that Ostwald ripening may occur quickly if the spheres are kept in the reaction solution, resulting in significant broadening of the particle size distribution. In order to obtain uniform and stable samples, it is important to quench the growth of the mercapto-silica spheres by separating them from the original reaction mixture and then storing them in solvents that can prevent further ripening.

Alterations in multiple neurochemical systems have been reported in animal and human studies of posthypoxic myoclonus. It is impossible, however, to establish causative relationships between the observed changes and the myoclonic movements from these studies. Therefore, to establish causative links between neurochemical changes and myoclonus, ligands that target neurotransmitter systems that are altered in posthypoxic myoclonus were microinjected into the lateral ventricles of normal rats to identify the changes that can produce myoclonus. Of the ligands that were tested, only the GABAA antagonists produced myoclonus after intracerebroventricular administration, suggesting the importance of disinhibition of GABAergic systems in myoclonus. To further examine the role of GABA in myoclonus, GABAergic antagonists were microinjected into the nucleus reticularis of the thalamus (NRT), an area of the brain in which extensive pathologic changes are seen in posthypoxic animals. GABAA, but not GABAB, antagonists produced myoclonus after microinjection into the NRT. Earlier investigators have further reported the ability of GABAA antagonists to produce myoclonus after microinjection into the caudate. The data therefore suggest that disruption of activity at GABAA receptors at any one of a number of levels in the neural axis can produce myoclonus.

Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study, we investigated the activation of Src family of cytoplasmic tyrosine kinases (SFKs) and two SFK substratesdCUB-domain containing protein 1 (CDCP1) and protein kinase C d (PKCd)din 56 formalin-fixed, paraffin-embedded (FFPE) TNBCs. Expression of SFK phosphorylated at Y416 (SFK_pY416 þ ) in tumor cells was strongly associated with phosphorylation of CDCP1 and PKCd (CDCP1_ pY743 þ and PKCd_pY311 þ ), as assessed by immunohistochemistry, indicating increased SFK activity in situ. To enable biochemical analysis, protein extraction from FFPE tissue was optimized. Cleaved CDCP1 isoform (70 kDa) was expressed to a varying degree in all samples but only phosphorylated in TNBC tumor cells that expressed SFK_pY416. Interestingly, active SFK was found to be biphosphorylated (SFK_pY416 þ /pY527 þ ). Biphosphorylated active SFK was observed more frequently in forkhead box protein A1 (FOXA1) À TNBCs. In addition, in SFK_pY416 À samples, FOXA1 þ TNBC tended to be SFK_pY527 þ (classic inactive SFK), and FOXA1 À TNBC tended to be SFK_pY527 À (SFK poised for activation). Strong SFK_pY416 staining was also observed in tumor-infiltrating lymphocytes in a subset of TNBCs with high tumor-infiltrating lymphocyte content. This report will facilitate protein biochemical analysis of FFPE tumor samples and justifies the development of therapies targeting the SFK/ CDCP1/PKCd pathway for TNBC treatment.

Breast cancer stem cells (BCSCs) have been characterized as a fraction of cells in primary tumors that are drug resistant and have metastatic potential. Ganglioside GD2 has been shown by us and others as a marker for BCSCs. Furthermore, nutrient deprivation associated metabolic stress seen during tumor progression is reportedly associated with the cancer stem cell phenotype. We hypothesized that metabolic stress could induce spontaneous generation of GD2+ BCSCs during tumor progression. To test our hypothesis, we cultured breast cancer cell lines MDA-MB-231 and SUM159 at low seeding density and measured percentage and absolute number of GD2+ cells daily. Flow cytometry analysis revealed that the percentage of GD2+ cells increased from 4.5 ± 2.5 on day 2 to 15 ± 3.8% on day 5 in MDA MB-231 cells and from 8.5 ± 2.8% on day2 to 28 ± 6.2% on day 5 in SUM159 cells (both designated as triple-negative breast cancer, TNBC). To investigate this phenomenon in-vivo, we injected GFP+ MDA-MB-231 cells in NSG mice mammary fat pads and examined GD2 expression in the implanted tumors weekly. Interestingly, we noticed that the percentage of GD2+ also increased from 12 ± 1.5% on week 1 to 30 ± 2.5% on week 6. Next, SUM159 cells were cultured in either nutrient rich (NR, i.e., 10% serum) or nutrient deprived (ND, 1% serum) for 4 days. We found that the percentage of GD2+ cells in NR medium at the end of 4 day culture was ~20% of the total cell population, whereas in ND medium was almost 50%. We then tested the effects of nutrient rich environment on GD2 expression by refreshing the media daily. Interestingly, cells that received fresh media had lower number of GD2+ cells (15 ± 1.5%) compared to cells cultured in the same medium for 4 days (33 ± 2.5%). Our data suggests that nutrient deprivation induces a stem cell phenotype in TNBC cells. Next, we performed global metabolic profiling (i.e., for a total of 300 biochemical metabolites) using a mass spectroscopy-based approach. We profiled SUM159 cells cultured with NR vs. ND medium (set-1); GD2+ vs GD2- SUM159 cells (set -2); GD2+ vs GD2- MDA-MB-231 cell (set-3). Metabolites associated with amino acid metabolism, in particular glutathione metabolism, including glutamyl-alanine, 5-oxy-proline, proline, glutamine, and glutathione itself were found to be most highly up-regulated in GD2+ compared to GD2- cells and also in cells cultured in serum starved compared to serum rich conditions. Further analysis of these metabolites and their association with GD2+ cell signature raveled that gamma-glutamyl transferase (GGT5), was one of the most highly up-regulated (>150-fold) gene across all the groups. GGT is expressed on cell surface and transfers glutamyl group to amino acids, which then get transported across the membrane. In cancer, cells expressing GGT has been shown to be resistant to chemotherapeutic agents including cisplatin. Targeting glutathione metabolism could be future therapeutic strategy to inhibit BCSC growth in TNBC. Citation Format: Battula VL, Piyaranthna B, Nguyen K, Sun JC, Jin F, Coarfa C, Nagireddy P, Andreeff M. Metabolic stress induces GD2 expression and cancer stem cell phenotype in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-01.