The growth of plant embryos in vitro. Preliminary experiments on the role of accessory substances

The aseptic culture of plant embryos isolated from the seed dates back to the work of Brown and Morris,(1) Hannig(2) and Dietrich.(3) More recent contributions to our knowledge concerning the culture in vitro of excised embryos have been made by Tukey,(4) Brunner(5) and LaRue,(6) among others. It has been recognized by, for example, Ray(7) that the embryo culture technique offers a useful tool for biochemical investigations, and it has also been recognized(4,8) that it may be used as a practical measure to circumvent the abortion of embryos. It has, however, been found that in general the growth of the excised embryo, even upon a medium containing essential inorganic materials and sugar, is far less than that of normal intact seedlings. This has led to the suggestion(5) that "accessory growth factors" which are needed in minute amounts, are required by the developing plant as they are by the developing animal organism. The present work, as well as that of Kogl and Haagen-Smit,(9) furnish final proof that this is the case; that these accessory substances, although normally furnished by the seed, may be replaced to some extent by pure compounds added in small amounts to the embryo culture medium. These investigations, taken up early in 1936, are concerned particularly with orienting experiments undertaken with an ultimate view toward the elucidation of the nature and mode of action of these accessory growth factors. The embryo culture technique is here to be used as a tool in the "hormonal" analysis of plant development

Design, Synthesis, and Evaluation of Site-Specific Hsp90 Inhibitors

The heat shock protein 90 (Hsp90) family of molecular chaperones is responsible for the conformational maturation of nascent polypeptides and refolding of denatured proteins. Hsp90 is known to play an important role in the regulation of cell signaling, survival and proliferation, transforming it into a promising target for the treatment of several diseases, including cancer. Proteins associated with all six hallmarks of cancer, as described by Weinberg, are Hsp90 clients. Since these proteins depend upon Hsp90, its inhibition has the potential to simultaneously disrupt all six and halt the malignant progression using a single small molecule. Hsp90 is abundantly expressed in the cell and accounts for 1-2% of the total cellular protein, making itone of the most prevalent proteins in eukaryotic cells. The Hsp90 isoforms are responsible for the conformational maintenance of greater than 150 proteins. There are four distinct isoforms, including Hsp90á, Hsp90â, GRP94 and TRAP1. Hsp90á and â are located in the cytoplasm; Hsp90á is the major inducible form, while Hsp90â is the constitutively active form. In contrast, GRP94 is found in the endoplasmic reticulum and TRAP1 is located in the mitochondrial matrix. Hsp90 exists as a homodimer consisting of an N- and C-terminal domain, connected by a middle domain. The Hsp90 N-terminus contains an ATP binding site, responsible for the ATPase activity of the chaperone associated with folding of clients. Small molecules that preferentially bind to this nucleotide binding site inhibit the ability of Hsp90 to properly fold polypeptides, ultimately tagging them for degradation through the ubiquitin-proteasome pathway. Natural product inhibitors of the N-terminal ATP binding site include the ansamycin antibiotic geldanamycin (GDA) and the macrocyclic lactone radicicol (RDC). Blagg and co-workers have previously reported the chimeric N-terminal inhibitor radamide, which combines the resorcinol portion of RDC and the quinone portion of GDA through a flexible linker. Radamide manifests a slightly greater binding affinity for GRP94 (Kd = 0.52 ìM) versus cytosolic Hsp90 (Kd = 0.87 ìM). This phenomenon is elucidated by examining the unique binding conformations adopted by radamide when crystal structures of were obtained using each isoform. When bound to Hsp90, radamide exhibits a linear conformation, while when bound to GRP94 the quinone portion is bent towards a pocket that is not accessible in Hsp90á or â. By employing conformational constraint, it was proposed that the quinone portion of radamide could exhibit the bent conformation seen in the natural products and result in increased affinity for Hsp90 as well as selective binding to the cytosolic isoforms. In contrast to radamide, a recent paper by Gasiewicz and co-workers examined the effects of flavones and (-)-Epigallocatechin-3-Gallate (EGCG), the major polyphenolic catechin found in green tea, on the aryl hydrocarbon receptor (AHR). Due to its diverse medicinal properties, EGCG has been proposed as a potential treatment for several diseases. The study by Gasiewicz revealed that EGCG functions through a different mechanism of action than the known flavone antagonists, interacting with the Hsp90 C-terminus rather than the AHR. It was confirmed that EGCG binds specifically to the C-terminal nucleotide binding site, either within or proximal to the site where the natural product novobiocin is thought to bind. Previous studies have determined that EGCG manifests an IC50 of ~150 ìM against MCF-7 cells. Upon examination of an overlay of EGCG and novobiocin, the structural similarities of the coumarin and the catechin cores become apparent. It was proposed that design of molecules based on the structure-activity relationships established for novobiocin may result in Hsp90-specific inhibitors based upon the EGCG scaffold

Supermarket Loss Estimates for Fresh Fruit, Vegetables, Meat, Poultry, and Seafood and Their Use in the ERS Loss-Adjusted Food Availability Data

A certain amount of food in supermarkets is deemed unusable (“food loss”) because of moisture loss, spoilage, and other causes. This study analyzed updated food loss estimates for fresh fruit, vegetables, meat, poultry, and seafood obtained through a competitive grant with the Perishables Group, Inc. This independent consulting firm compared supplier shipment data with point-of-sale data from six large national and regional supermarket retailers to identify loss in 2005 and 2006. The new estimates, when incorporated into the ERS Loss-Adjusted Food Availability data, had little impact on aggregate per capita food loss estimates in 2006 because the new estimates were, on average, close to the previous loss assumptions. The new estimates increased annual per capita estimates of fresh fruit available at the retail level by 0.7 pounds (0.6 percent), 4.2 pounds (2.7 percent) for fresh vegetables, and 4.8 pounds (2.7 percent) for fresh meat, poultry, and seafood. The commodity-specific food loss estimates are more accurate than in previous years.Conversion factor, food loss, fruit, meat, poultry, seafood, supermarket, vegetables, Agribusiness, Agricultural Finance, Financial Economics,

A Review of Aviation Manufacturing and Supply Chain Processes

This paper provides an overview of modern aviation manufacturing and supply chain practices, including machining processes, 3D printing processes, and automation processes. Continuous improvement efforts in both manufacturing and supply chain practices are necessary for the industry to meet the increase in production demand of 35,000 aircraft over the next two decades

Editorial: The next generation of tools and technologies for studying human neurons in a dish

Despite their prevalence, disorders of the brain, including neurodevelopmental, neurodegenerative, and other mental illnesses, have historically been some of the most challenging to treat. The enormous economic burden coupled with a paucity of curative therapies highlights an urgent need for new approaches and targets to tackle these diseases. One biologically relevant and transformative method that has been employed is the use of induced pluripotent stem cells (iPSCs) to generate neurons that closely mimic those found within the human brain (Mohamed et al., 2019). Advances in this area have enabled investigators to readily grow many of the cell types found within the human brain on a dish, increasing our understanding of mechanisms and targets that are implicated in these diseases and helping to facilitate translational efforts (Fermini et al., 2018)

CK2 Chemical Probes: Past, Present, and Future

Protein kinase casein kinase 2 (CK2/CSNK2) is a pleiotropic kinase involved in many cellular processes and, accordingly, has been identified as a potential target for therapeutic intervention for multiple indications. Significant research effort has been invested into identifying CK2 inhibitors as potential drug candidates and potent and selective CK2 chemical probes to interrogate CK2 function. Here, we review the small molecule inhibitors reported for CK2 and discuss various orthosteric, allosteric, and bivalent inhibitors of CK2. We focus on the pyrazolo[1,5-a]pyrimidines and naphthyridines, two chemotypes that have been extensively explored for chemical probe development. We highlight the uptake and demonstrated utility of the pyrazolo[1,5-a]pyrimidine chemical probe SGC-CK2-1 by the scientific community in cellular studies. Finally, we propose criteria for an ideal in vivo chemical probe for investigating CK2 function in a living organism. While no compound currently meets these metrics, we discuss ongoing and future directions in the development of in vivo chemical probes for CK2

Evaluation of a Selective Chemical Probe Validates That CK2 Mediates Neuroinflammation in a Human Induced Pluripotent Stem Cell-Derived Mircroglial Model

Novel treatments for neurodegenerative disorders are in high demand. It is imperative that new protein targets be identified to address this need. Characterization and validation of nascent targets can be accomplished very effectively using highly specific and potent chemical probes. Human induced pluripotent stem cells (hiPSCs) provide a relevant platform for testing new compounds in disease relevant cell types. However, many recent studies utilizing this platform have focused on neuronal cells. In this study, we used hiPSC-derived microglia-like cells (MGLs) to perform side-by-side testing of a selective chemical probe, SGC-CK2-1, compared with an advanced clinical candidate, CX-4945, both targeting casein kinase 2 (CK2), one of the first kinases shown to be dysregulated in Alzheimer’s disease (AD). CK2 can mediate neuroinflammation in AD, however, its role in microglia, the innate immune cells of the central nervous system (CNS), has not been defined. We analyzed available RNA-seq data to determine the microglial expression of kinases inhibited by SGC-CK2-1 and CX-4945 with a reported role in mediating inflammation in glial cells. As proof-of-concept for using hiPSC-MGLs as a potential screening platform, we used both wild-type (WT) MGLs and MGLs harboring a mutation in presenilin-1 (PSEN1), which is causative for early-onset, familial AD (FAD). We stimulated these MGLs with pro-inflammatory lipopolysaccharides (LPS) derived from E. coli and observed strong inhibition of the expression and secretion of proinflammatory cytokines by simultaneous treatment with SGC-CK2-1. A direct comparison shows that SGC-CK2-1 was more effective at suppression of proinflammatory cytokines than CX-4945. Together, these results validate a selective chemical probe, SGC-CK2-1, in human microglia as a tool to reduce neuroinflammation

Protein proximity networks and functional evaluation of the casein kinase 1 gamma family reveal unique roles for CK1γ3 in WNT signaling

Aberrant activation or suppression of WNT/β-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and dark. Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the β-catenin-dependent and β-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated β-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and β-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway