Peroxisome inspired hybrid enzyme nanogels for chemodynamic and photodynamic therapy

Peroxisome, a special cytoplasmic organelle, possesses one or more kinds of oxidases for hydrogen peroxide (H2O2) production and catalase for H2O2 degradation, which serves as an intracellular H2O2 regulator to degrade toxic peroxides to water. Inspired by this biochemical pathway, we demonstrate the reactive oxygen species (ROS) induced tumor therapy by integrating lactate oxidase (LOx) and catalase (CAT) into Fe3O4 nanoparticle/indocyanine green (ICG) co-loaded hybrid nanogels (designated as FIGs-LC). Based on the O2 redistribution and H2O2 activation by cascading LOx and CAT catalytic metabolic regulation, hydroxyl radical (·OH) and singlet oxygen (1O2) production can be modulated for glutathione (GSH)-activated chemodynamic therapy (CDT) and NIR-triggered photodynamic therapy (PDT), by manipulating the ratio of LOx and CAT to catalyze endogenous lactate to produce H2O2 and further cascade decomposing H2O2 into O2. The regulation reactions of FIGs-LC significantly elevate the intracellular ROS level and cause fatal damage to cancer cells inducing the effective inhibition of tumor growth. Such enzyme complex loaded hybrid nanogel present potential for biomedical ROS regulation, especially for the tumors with different redox state, size, and subcutaneous depth.

Ferroptosis induces membrane blebbing in placental trophoblasts

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp)- forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins, and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis, caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp- phosphatidylethanolamine in the cell membrane promoted its stretchability. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.

OR02-03 Role of Phosphorylated Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in the Regulation of Germ Cell Specific MRNAs in Chromatoid Bodies During Spermiogenesis

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX 25) is a member of the DEAD-box family of RNA helicases which play an essential role in spermatogenesis. There are two species of GRTH, the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in the Japanese azoospermic men which resulted in the lack of phospho-GRTH (pGRTH) in in vitro studies. GRTH knock-in (KI) mice with insertion of the human mutant GRTH gene show loss of the cytoplasmic 61 KDa phospho-species with preservation of the non-phospho nuclear form. KI mice are sterile, lack elongated spermatids and spermatozoa with arrest at step 8 of round spermatids (RS) which contain chromatoid bodies (CB) markedly reduced in size. CB is a non-membranous, cytoplasmic organelle present adjacent to the nucleus of RS, where mRNAs bound to GRTH transported from nucleus to cytoplasmic sites are temporarily stored, translationally repressed for later transport to polyribosomes for translation at specific stages of spermiogenesis. Owing to the specific function of CBs and importance of pGRTH in spermatid elongation, CBs isolated from germ cells of WT and GRTH KI mice were used for subsequent experiments. CBs isolated from GRTH KI mice are smaller, highly condensed and lack the nuage texture of CBs in WT mice. We observed the absence of pGRTH in CB of round spermatids of GRTH KI mice. Also, MVH protein (recognized CB marker protein) was decreased in the CB of GRTH KI mice. Expression of genes related to spermatid regulation, chromatin compaction, remodeling (TP1 and 2, PRM1 and 2, GRTH, TSSK6, HMG2, GCNF, RNF8, TDRD 1, 6, 7 and 9) analyzed by qPCR were markedly reduced in the CB of GRTH KI mice compared to WT. No change was observed in the expression of bromodomain mRNAs and protein, indicating that pGRTH does not participate in the translational regulation of this protein class at the level of this organelle. Notably, mRNAs of TP2, PRM2 and GRTH which associated with GRTH protein were co-localized with MVH protein in the CB. This indicated the relevance of GRTH as a binder/transport protein of key chromatin remodelers for ensuring their mRNA repression/stability within the CB. In addition, GRTH binding to genes essential for spermatid development and regulation (TP1 and 2, PRM1 and 2, GRTH, TSSK6, RNF8 and GCNF) were also found to be markedly decreased in the CB KI mice. These results demonstrate the importance of pGRTH in the maintenance of biochemical composition/structure of the CB and role in spermatid regulation, chromatin compaction, spermatid development and completion of spermatogenesis.

Complete chloroplast genomes of all six Hosta species occurring in Korea: molecular structures, comparative, and phylogenetic analyses

Background The genus Hosta is a group of economically appreciated perennial herbs consisting of approximately 25 species that is endemic to eastern Asia. Due to considerable morphological variability, the genus has been well recognized as a group with taxonomic problems. Chloroplast is a cytoplasmic organelle with its own genome, which is the most commonly used for phylogenetic and genetic diversity analyses for land plants. To understand the genomic architecture of Hosta chloroplasts and examine the level of nucleotide and size variation, we newly sequenced four (H. clausa, H. jonesii, H. minor, and H. venusta) and analyzed six Hosta species (including the four, H. capitata and H. yingeri) distributed throughout South Korea. Results The average size of complete chloroplast genomes for the Hosta taxa was 156,642 bp with a maximum size difference of ~ 300 bp. The overall gene content and organization across the six Hosta were nearly identical with a few exceptions. There was a single tRNA gene deletion in H. jonesii and four genes were pseudogenized in three taxa (H. capitata, H. minor, and H. jonesii). We did not find major structural variation, but there were a minor expansion and contractions in IR region for three species (H. capitata, H. minor, and H. venusta). Sequence variations were higher in non-coding regions than in coding regions. Four genic and intergenic regions including two coding genes (psbA and ndhD) exhibited the largest sequence divergence showing potential as phylogenetic markers. We found compositional codon usage bias toward A/T at the third position. The Hosta plastomes had a comparable number of dispersed and tandem repeats (simple sequence repeats) to the ones identified in other angiosperm taxa. The phylogeny of 20 Agavoideae (Asparagaceae) taxa including the six Hosta species inferred from complete plastome data showed well resolved monophyletic clades for closely related taxa with high node supports. Conclusions Our study provides detailed information on the chloroplast genome of the Hosta taxa. We identified nucleotide diversity hotspots and characterized types of repeats, which can be used for developing molecular markers applicable in various research area.

Duo in a Mystical Realm—Nuclear Lipid Droplets and the Inner Nuclear Membrane

The lipid droplet (LD) is a cytoplasmic organelle, but it also exists in the nucleus under some conditions or in some cell types. New studies have revealed that nuclear LDs do not occur by haphazard entry of cytoplasmic LDs. Instead, they are generated by specific mechanisms that are increasingly understood. The inner nuclear membrane (INM) plays a critical role in nuclear LD formation in both mammalian hepatocytes and budding yeast, although in significantly different ways. Hepatocyte nuclear LDs derive from precursors of very low-density lipoprotein lacking apolipoprotein B-100, which form in the endoplasmic reticulum lumen and accumulate in intranuclear extensions of the perinuclear space called type I nucleoplasmic reticulum. In contrast, nuclear LDs in yeast are generated by triglyceride synthesized in the INM. Nuclear LDs in hepatocytes and budding yeast are both instrumental in the regulation of phospholipid synthesis; however, again they function in different ways. As the full functional importance is as yet unknown, the close relationship of nuclear LDs and the INM is an attractive target of research from both physiological and pathological perspectives.

Metformin Suppresses Expression of Pre-Fibrotic Enzyme Lysyl-Oxidase Levels through Inhibition of UPR in MPN Cells

The endoplasmic reticulum (ER) is a cytoplasmic organelle required for proper protein folding and sorting. When protein load exceeds the capacity of the ER, the accumulation of the misfolded proteins triggers a quality control response known as the unfolded protein response (UPR). Recent studies have revealed that increased activation of the UPR is one of the hallmarks of hematological malignancies. In addition, excess UPR is associated with pro-fibrotic conditions, i.e., pulmonary fibrosis, renal fibrosis and kidney fibrosis.Lysyl-oxidase (LOX) is an enzyme that regulates the crosslinking of extracellular matrix proteins, such as collagen, and enhances a fibrotic phenotype. Recent studies revealed that the genes of LOX family members are upregulated in myeloproliferative neoplasms (MPN). Furthermore, megakaryocytes and platelets derived from MPN patients had increased LOX protein concentrations in comparison with healthy subjects. It is also reported that the UPR is one of the important regulators of LOX. Based on this knowledge, we speculated that the enhanced UPR might be involved in the elevation of LOX in JAK2V617F harboring MPN and may contribute to the development of myelofibrosis. To study this hypothesis, we initially confirmed that JAK2V617F-positive cell lines(HEL and SET-2) show enhanced activation of UPR, including phosphorylation of eIF2-alpha, nuclear localization of ATF6 and XBP1s and upregulation of glucose response protein 78 (GRP78). We also confirmed the presence of elevated levels of LOX in these cells. Treatment of the cells with a chemical chaperone, namely, tauroursodeoxycholic acid (TUDCA), suppressed UPR and decreased expression of LOX, suggesting that UPR is responsible for elevation of LOX in MPN cells. To analyze whether JAK2V617F is responsible for the enhanced UPR and expression of LOX in MPN cells, we treated the cells with JAK1/JAK2 inhibitor ruxolitinib. Unexpectedly, ruxolitinib did not block UPR nor expression of LOX. Previously, we reported that metformin, which is a member of the biguanide family, inhibited growth of MPN cells through activation of AMPK and PP2A (Kawashima et al. Exp.Hematol, 2016). Furthermore, a number of studies demonstrated that metformin suppressed UPR in several cancer cells through activation of AMPK. Therefore, we investigated whether metformin could block UPR and LOX levels in these cells. As expected, metformin reduced the phosphorylation levels of eIF2alpha and nuclear localization of XBP1 and ATF6. Expression of GRP78 was also blocked by metformin. These results clearly indicated that metformin suppressed UPR in MPN cells. Metformin also blocked the activation of the molecules required for proteins synthesis including p70S6kinase, mTOR and 4E-PB1. These results indicated that suggested that metformin blocked aberrant UPR through inhibition of enhanced protein synthesis. Importantly, we also confirmed that LOX expression was also suppressed by metformin in MPN cells.Our observations indicated that the UPR is enhanced in MPN cells irrespective of JAK2V617F, and metformin could block this process leading to suppression of LOX levels. Combination of ruxolitinib with metformin might be a new and attractive method to suppress myelofibrosis in MPN. Disclosures Kirito: Novartis Pharma KK: Honoraria.