Genetic Insights into the Extremely Dwarf Hibiscus syriacus var. micranthus: Complete Chloroplast Genome Analysis and Development of a Novel dCAPS Marker.

This study explored the chloroplast (cp) genomes of three Hibiscus syriacus (HS) specimens endemic to Korea possessing unique ornamental and conservation values: the dwarf H. syriacus var. micranthus (HSVM), renowned for its small stature and breeding potential; HS 'Tamra', a cultivar from Korea's southernmost islands, noteworthy for its distinctive beauty; and HS Natural Monument no. 521 (N.M.521), a specimen of significant lifespan and height. Given the scarcity of evolutionary studies on these specimens, we assembled and analyzed their cp genomes. We successfully assembled genomes spanning 160,000 to 160,100 bp and identified intraspecific variants. Among these, a unique ATA 3-mer insertion in the trnL-UAA region was identified in HSVM, highlighting its value as a genetic resource. Leveraging this finding, we developed a novel InDel dCAPS marker, which was validated across 43 cultivars, enhancing our ability to distinguish HSVM and its derivatives from other HS cultivars. Phylogenetic analysis involving 23 Malvaceae species revealed that HSVM forms a clade with woody Hibiscus species, closely associating with N.M.520, which may suggest a shared ancestry or parallel evolutionary paths. This investigation advances our understanding of the genetic diversity in Korean HS and offers robust tools for accurate cultivar identification, aiding conservation and breeding efforts.

Expansion of human hematopoietic stem cells by inhibiting translation.

Hematopoietic stem cell (HSC) transplantation using umbilical cord blood (UCB) is a potentially life-saving treatment for leukemia and bone marrow failure but is limited by the low number of HSCs in UCB. The loss of HSCs after ex vivo manipulation is also a major obstacle to gene editing for inherited blood disorders. HSCs require a low rate of translation to maintain their capacity for self-renewal, but hematopoietic cytokines used to expand HSCs stimulate protein synthesis and impair long-term self-renewal. We previously described cytokine-free conditions that maintain but do not expand human and mouse HSCs ex vivo. Here we performed a high throughput screen and identified translation inhibitors that allow ex vivo expansion of human HSCs while minimizing cytokine exposure. Transplantation assays show a ~5-fold expansion of long-term HSCs from UCB after one week of culture in low cytokine conditions. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the importance of regulated proteostasis in HSC maintenance and expansion. This expansion method maintains and expands human HSCs after CRISPR/Cas9 editing of the BCL11A+58 enhancer, overcoming a major obstacle to ex vivo gene correction for human hemoglobinopathies.

Effects of Drip Irrigation-Fertilization on Growth, Flowering, Photosynthesis and Nutrient Absorption of Containerized Seedlings of Hibiscus syriacus L. (Haeoreum).

The amount of irrigation and fertilization should be considered first for the production and standardization of high-quality H. syriacus L. seedlings using container seedlings. This study was conducted to investigate the optimal conditions suitable for container cultivation of hibiscus by analyzing growth and physiological responses according to the control of irrigation and fertilization. Therefore, in this study, H. syriacus L. for. Haeoreum (3-year-old hardwood cutting propagation), a fast-growing, was transplanted into a 40 L container. The irrigation amount per container was adjusted (0.2, 0.3 and 0.4 ton/yr/tree), and the amount of fertilizer applied (0, 69.0, 138.0 and 207.0 g/yr/tree). The growth rate according to the irrigation-fertilization treatment was higher in the 0.3 ton-138.0 g/yr/tree irrigation-fertilization treatment (p < 0.001). Total biomass yield and seedling quality index (SQI) were highest in the 0.3 ton-138.0 g/yr/tree irrigation-fertilization treatment (p < 0.001). The higher the fertilization concentration, the faster the flowering and the longer the flowering. The photosynthetic capacity of H. syriacus L. was reduced in bare root seedling cultivation and container-non-fertilized treatment. The chlorophyll fluorescence response was also affected by bare root cultivation and containerized seedling cultivation fertilization. Nutrient vector diagnosis showed "nutritional suitability" in the 0.3 ton-138.0 g/yr/tree treatment. Overall, containerized seedling cultivation was superior in growth, photosynthetic performance, photochemical efficiency, and nutrient storage capacity compared to bare root cultivation. These results be expected to contribute not only to the industrial production of excellent container seedlings of H. syriacus L. but also to the production of other woody plants.

Developmental fidelity is imposed by genetically separable RalGEF activities that mediate opposing signals.

The six C. elegans vulval precursor cells (VPCs) are induced to form the 3°-3°-2°-1°-2°-3° pattern of cell fates with high fidelity. In response to EGF signal, the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade is necessary to induce 1° fate and synthesis of DSL ligands for the lateral Notch signal. In turn, LIN-12/Notch receptor is necessary to induce neighboring cells to become 2°. We previously showed that, in response to graded EGF signal, the modulatory LET-60/Ras-RGL-1/RalGEF-RAL-1/Ral signal promotes 2° fate in support of LIN-12. In this study, we identify two key differences between RGL-1 and RAL-1. First, deletion of RGL-1 confers no overt developmental defects, while previous studies showed RAL-1 to be essential for viability and fertility. From this observation, we hypothesize that the essential functions of RAL-1 are independent of upstream activation. Second, RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2° fate via canonical GEF-dependent activation of RAL-1. Conversely, RGL-1 promotes 1° fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1°-promoting AGE-1/PI3-Kinase-PDK-1-AKT-1 cascade. Additionally, animals lacking RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions to orchestrate opposing 1°- and 2°-promoting modulatory cascades to decrease developmental stochasticity. We speculate that such switches are broadly conserved but mostly masked by paralog redundancy or essential functions.

The Signaling Network Controlling C. elegans Vulval Cell Fate Patterning.

EGF, emitted by the Anchor Cell, patterns six equipotent C. elegans vulval precursor cells to assume a precise array of three cell fates with high fidelity. A group of core and modulatory signaling cascades forms a signaling network that demonstrates plasticity during the transition from naïve to terminally differentiated cells. In this review, we summarize the history of classical developmental manipulations and molecular genetics experiments that led to our understanding of the signals governing this process, and discuss principles of signal transduction and developmental biology that have emerged from these studies.

Ral Signals through a MAP4 Kinase-p38 MAP Kinase Cascade in C. elegans Cell Fate Patterning.

C. elegans vulval precursor cell (VPC) fates are patterned by an epidermal growth factor (EGF) gradient. High-dose EGF induces 1° VPC fate, and lower dose EGF contributes to 2° fate in support of LIN-12/Notch. We previously showed that the EGF 2°-promoting signal is mediated by LET-60/Ras switching effectors, from the canonical Raf-MEK-ERK mitogen-activated protein (MAP) kinase cascade that promotes 1° fate to the non-canonical RalGEF-Ral that promotes 2° fate. Of oncogenic Ras effectors, RalGEF-Ral is by far the least well understood. We use genetic analysis to identify an effector cascade downstream of C. elegans RAL-1/Ral, starting with an established Ral binding partner, Exo84 of the exocyst complex. Additionally, RAL-1 signals through GCK-2, a citron-N-terminal-homology-domain-containing MAP4 kinase, and PMK-1/p38 MAP kinase cascade to promote 2° fate. Our study delineates a Ral-dependent developmental signaling cascade in vivo, thus providing the mechanism by which lower EGF dose is transduced.

Similarities and differences between alpha-tocopherol and gamma-tocopherol in amelioration of inflammation, oxidative stress and pre-fibrosis in hyperglycemia induced acute kidney inflammation.

BACKGROUND/OBJECTIVES: Diabetes mellitus (DM) is a major chronic disease which increases global health problems. Diabetes-induced renal damage is associated with inflammation and fibrosis. Alpha (AT) and gamma-tocopherols (GT) have shown antioxidant and anti-inflammatory effects in inflammation-mediated injuries. The primary aim of this study was to investigate effects of AT and GT supplementations on hyperglycemia induced acute kidney inflammation in alloxan induced diabetic mice with different levels of fasting blood glucose (FBG). MATERIALS/METHODS: Diabetes was induced by injection of alloxan monohydrate (150 mg/kg, i.p) in ICR mice (5.5-week-old, male) and mice were subdivided according to their FBG levels and treated with different diets for 2 weeks; CON: non-diabetic mice, m-DMC: diabetic control mice with mild FBG levels (250 mg/dl ≤ FBG ≤ 450 mg/dl), m-AT: m-DM mice fed AT supplementation (35 mg/kg diet), m-GT: m-DM mice with GT supplementation (35 mg/kg diet), s-DMC: diabetic control mice with severe FBG levels (450 mg/dl < FBG), s-AT: s-DM mice with AT supplementation, s-GT: s-DM mice with GT supplementation. RESULTS: Both AT and GT supplementations showed similar beneficial effects on NFκB associated inflammatory response (phosphorylated inhibitory kappa B-α, interleukin-1ß, C-reactive protein, monocyte chemotactic protein-1) and pre-fibrosis (tumor growth factor ß-1 and protein kinase C-II) as well as an antioxidant emzyme, heme oxygenase-1 (HO-1) in diabetic mice. On the other hands, AT and GT showed different beneficial effects on kidney weight, FBG, and oxidative stress associated makers (malondialdehyde, glutathione peroxidase, and catalase) except HO-1. In particular, GT significantly preserved kidney weight in m-DM and improved FBG levels in s-DM and malondialdehyde and catalase in m- and s-DM, while AT significantly attenuated FBG levels in m-DM and improved glutathione peroxidase in m- and s-DM. CONCLUSIONS: The results suggest that AT and GT with similarities and differences would be considered as beneficial nutrients to modulate hyperglycemia induced acute renal inflammation. Further research with careful approach is needed to confirm beneficial effects of tocopherols in diabetes with different FBG levels for clinical applications.

Stromal cell-derived factor-1α and macrophage migration-inhibitory factor induce metastatic behavior in CXCR4-expressing colon cancer cells.

Metastasis of cancer cells is a major cause of death in cancer patients. The process of cancer metastasis includes the proliferation of primary cancer cells, local invasion, intravasation and cancer cell survival in blood flow, extravasation and attachment to secondary organs and metastatic growth in a new environment. In these mechanisms of cancer metastasis, CXC chemokine receptor 4 (CXCR4) and its ligand play an important role. Stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is well known as a ligand of CXCR4, and macrophage migration-inhibitory factor (MIF) has recently become known as a ligand of CXCR4. In many types of cancers including breast, pancreatic and colorectal cancer (CRC), CXCR4/SDF-1α has been investigated in metastasis-related cancer behavior, which include cell proliferation, adhesion, migration and invasion. However, CXCR4/MIF has rarely been investigated in the metastatic behavior of colon cancer cells. In this report, the effect of SDF-1α or MIF was studied on cell cycle, cell proliferation, adhesion and migration of the CXCR4-expressing colon cancer cell line SW480. SDF-1α or MIF caused a decrease in the number of cells in G0/G1 phase and an increase in the numbers of cells in S and G2/M phases. In addition, SDF-1α or MIF caused an increase in cell proliferation, cell adhesion to fibronectin and migration. AMD3100, a CXCR4 antagonist, attenuated these effects, which included increased cell proliferation, adhesion and migration due to treatment of CXCR4-expressing colon cancer cells with SDF-1α or MIF. In conclusion, SDF-1α or MIF affects the metastasis-related behaviors of CXCR4-expressing colon cancer cells.

Identification of genes upregulated by pinewood nematode inoculation in Japanese red pine.

Pine wilt disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, has destroyed huge areas of pine forest in East Asia, including Japan, China and Korea. No protection against PWN has been developed, and the responses of pine trees at the molecular level are unrecorded. We isolated and analyzed upregulated or newly induced genes from PWN-inoculated Japanese red pine (Pinus densiflora Sieb. et Zucc.) by using an annealing control primer system and suppression subtractive hybridization. Significant changes occurred in the transcript abundance of genes with functions related to defense, secondary metabolism and transcription, as the disease progressed. Other gene transcripts encoding pathogenesis-related proteins, pinosylvin synthases and metallothioneins were also more abundant in PWN-inoculated trees than in non-inoculated trees. Our report provides fundamental information on the molecular mechanisms controlling the biochemical and physiological responses of Japanese red pine trees to PWN invasion.