Unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating Ethylene and Abscisic acid pathway

Submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates Galactosyltransferase (GALT9) and their expression is inversely affected at 24 hours of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild type and MIM775 Arabidopsis shoot. A similar recovery phenotype of galt9 mutant indicates the role of miR775-GALT9 module in post-submergence recovery. We predicted Golgi-localized GALT9 to be potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29, and ORE1), ethylene signalling (EIN2 and EIN3) and ABA biosynthesis (NCED3) pathway genes in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role of miR775-GALT9 module in post-submergence recovery through a crosstalk with ethylene and ABA pathway.

C-kit+VEGFR-2+ Mesenchymal Stem Cells Differentiate into Cardiovascular Cells and Repair Infarcted Myocardium after Transplantation

Background: Resent preclinical studies and clinical trails prove that transplantation of mesenchymal stem cells (MSCs) is a promised therapy for ischemic diseases. However, the properties of c-kit+ cells in MSCs remain unclear. We investigated the differential potential of c-kit+VEGFR-2+ MSCs and evaluated their effects on repairing the infarcted myocardium after transplantation. Methods: c-kit+VEGFR-2+ MSCs were isolated from rat bone marrow. Gene expression profile of the cells was examined with RNA-sequencing. Differential potential of the cells was determined after induction with VEGF, TGF-β and BMP-2 for 2 weeks. Improvement of cardiac function and repair of the infarcted myocardium were assessed at 4 weeks after transplantation of the cells preconditioned with hypoxia and serum deprivation. Results: Gene expression profile revealed that the upregulated genes are enrichment of genes related to immune process and cell differentiation. The cells represented a potential of differentiation towards endothelial cell, smooth muscle cells and cardiamyocytes. In hypoxic condition, secretion of VEGF, SCF and SDF-1α from the cells was increased. VEGF and SCF promoted proliferation and migration of the cells. VEGF could induce the cells to incorporate to the microvessels. After transplantation of the preconditioned cells into the infarcted myocardium, cardiac function was improved, scar size of the infarcted myocardium was decreased, and angiogensis and myocardium repair were enhanced significantly. With preconditioning and delivery by fibrin gel, survival of the cells in the ischemic tissue was augmented. Conclusion: These results suggest that c-kit+VEGFR-2+ MSCs have a potential of differentiation towards cardiovascular cells. SCF/c-kit and VEGF/VEGFR-2 singnalling pathways regulate proliferation, migration and differentiation of the cells. Transplantation of c-kit+VEGFR-2+ MSCs may enhance repair of the infarcted myocardium effectively.

Molecular Characterization and Response of Prolyl Hydroxylase Domain (PHD) Genes to Hypoxia Stress in Hypophthalmichthys molitrix

As an economically and ecologically important freshwater fish, silver carp (Hypophthalmichthys molitrix) is sensitive to low oxygen tension. Prolyl hydroxylase domain (PHD) proteins are critical regulators of adaptive responses to hypoxia for their function of regulating the hypoxia inducible factor-1 alpha subunit (HIF-1α) stability via hydroxylation reaction. In the present study, three PHD genes were cloned from H. molitrix by rapid amplification of cDNA ends (RACE). The total length of HmPHD1, HmPHD2, and HmPHD3 were 2981, 1954, and 1847 base pair (bp), and contained 1449, 1080, and 738 bp open reading frames (ORFs) that encoded 482, 359, and 245 amino acids (aa), respectively. Amino acid sequence analysis showed that HmPHD1, HmPHD2, and HmPHD3 had the conserved prolyl 4-hydroxylase alpha subunit homolog domains at their C-termini. Meanwhile, the evaluation of phylogeny revealed PHD2 and PHD3 of H. molitrix were more closely related as they belonged to sister clades, whereas the clade of PHD1 was relatively distant from these two. The transcripts of PHD genes are ubiquitously distributed in H. molitrix tissues, with the highest expressional level of HmPHD1 and HmPHD3 in liver, and HmPHD2 in muscle. After acute hypoxic treatment for 0.5 h, PHD genes of H. molitrix were induced mainly in liver and brain, and different from HmPHD1 and HmPHD2, the expression of HmPHD3 showed no overt tissue specificity. Furthermore, under continued hypoxic condition, PHD genes exhibited an obviously rapid but gradually attenuated response from 3 h to 24 h, and upon reoxygenation, the transcriptional expression of PHD genes showed a decreasing trend in most of the tissues. These results indicate that the PHD genes of H. molitrix are involved in the early response to hypoxic stress, and they show tissue-specific transcript expression when performing physiological regulation functions. This study is of great relevance for advancing our understanding of how PHD genes are regulated when addressing the hypoxic challenge and provides a reference for the subsequent research of the molecular mechanisms underlying hypoxia adaptation in silver carp.

The impacts of oxygen and pentoxifylline in hypoxic condition

Introduction:Among major trauma patients in the emergency department, the leading cause of morbidity and mortality is a hemorrhagic shock. The low oxygen flow with hypovolemia in trauma patients is believed to play a significant role. Hence, oxygen supply is essential in severe trauma patients with massive hemorrhage. This study aimed to investigate the effect of oxygen supply in hypoxic condition and variable treatments such as pentoxifylline (PTX), glycerol, hypertonic saline (HTS), protease inhibitor, and dexamethasone (DEXA) in macrophage and T cells. Method:Nitric oxide synthase (iNOS) and macrophage migration inhibitory factor (MIF) were measured for macrophage. MIF, interleukin (IL)-2, and IL-8 were measured for T cells. T cell viability was measued by MTT assay. Results: Pentoxifylline decreased iNOS expression mostly followed by glycerol under hypoxia. Under the hyperoxia, PTX and other treatments decreased iNOS expressions in macrophage. MIF expression was lowered with PTX under hypoxia. PTX, glycerol, HTS, and protease inhibitor were effective under hyperoxia in macrophage. PTX increased T cell survival under hypoxia. Under the hyperoxia, IL-2 expressions were upregulated with PTX, glycerol, and HTS. PTX and other treatments were effective for IL-8. Our results indicate that the PTX and the other agents tested reversed the effects of stimulation of lipopolysaccharide, PGE2 in hypoxia or hypoxia. Conclusion:Our study demonstrated potential usefulness in improving immune systems during severe inflammatory conditions similar to septic shock possibly caused by massive hemorrhage.

Terapi Oksigen Hiperbarik Meningkatkan Kualitas Hidup Pasien Glioblastoma Multiforme Stadium Akhir Dinilai Berdasarkan Neurologic Assessment Neuro Oncology Score

Patients with end-stage glioblastoma multiforme (GBM) that cannot be operated or treated with chemotheraphy and radiotheraphy have increased clinical complaints, thus affecting the patients’ quality of lifes (QoL). Hyperbaric oxygen therapy (HBOT) could alters hypoxic condition in tumor tissue with a different cascade from wound healing process. This study was aimed to assess whether the QoL of end-stage GBM patients could improve with the administration of HBOT assessed by decreased clinical complaints based on NANO score. The study was carried out at the Surgery Department of Prof. Dr. R. D. Kandou Hospital Manado, using pre and posttrial design. Each sample was assessed for the NANO score before HBOT, after one time, three times, five times, and 10 times of HBOT. The results showed that there was a change in the NANO score before and after 10 times of HBOT. The decrease in the NANO score occurred more quickly after the therapy. In the last two measurements, the NANO score according to the RVC model decreased from one to two units. The decreased NANO score occurred quite regularly with variations in the score that changed from time to time. In conclusion, HBOT can improve the QoL of patients with end-stage GBM and reduce the NANO score which is an assessment of clinical complaints of the patients.Keywords: glioblastoma multiforme; hyperbaric oxygen therapy; NANO score Abstrak: Penderita glioblastoma multiforme (GBM) stadium akhir yang tidak dapat dilakukan operasi, kemoterapi dan radioterapi memiliki keluhan klinis yang semakin meningkat sehingga memengaruhi kualitas hidup. Terapi oksigen hiperbarik (TOHB) dapat mengubah kondisi hipoksia pada jaringan tumor dengan kaskade yang berbeda dari proses penyembuhan luka. Penelitian ini bertujuan untuk menilai apakah kualitas hidup pasien GBM stadium akhir dapat membaik dengan pemberian TOHB, dinilai dengan menurunnya keluhan klinis berdasarkan NANO score. Penelitian dilakukan di Bagian Bedah RSUP Prof. Dr. R. D. Kandou Manado, dengan menggunakan pre and posttrial design. Pada setiap sampel dilakukan penilaian NANO score sebelum TOHB, setelah satu kali, tiga kali, lima kali, dan 10 kali TOHB. Hasil penelitian mendapatkan adanya perubahan NANO score sebelum TOHB dan setelah 10 kali TOHB. Penurunan NANO score berlangsung lebih cepat pasca terapi tersebut. Dalam dua pengukuran terakhir, NANO score menurut model RVC turun dari satu hingga dua satuan. Pada grafik, hasil penurunan NANO score terjadi cukup beraturan dengan variasi score yang berubah dari waktu ke waktu. Simpulan penelitian ini ialah TOHB dapat meningkatkan kualitas hidup penderita GBM stadium akhir dan menurunkan NANO score yang menjadi penilainan keluhan klinis penderita.Kata kunci: glioblastoma multiforme; terapi oksigen hiperbarik; NANO score

Implantation of bovine hydroxyapatite and secretome with different oxygen concentration may improve massive bone defect regeneration: An experimental study on animal model

The most widely used biomaterials in the treatment of massive bone defects are allograft bone or metal implants. The current problem is that the availability of allographs is limited and metal implants are very expensive. Mass production of secretome can make bone reconstruction of massive bone defects using a scaffold more effective and efficient. This study aims to prove bone regeneration in massive bone defects using bovine hydroxyapatite reconstruction with normoxic and hypoxic secretome conditions using collagen type 1 (COL1), alkaline phosphate (ALP), osteonectin (ON), and osteopontin (OPN) parameters. This is an in vivo study using male New Zealand white rabbits aged 6–9 months. The research was carried out at the Biomaterials Center—Tissue Bank, Dr. Soetomo Hospital for the manufacturer of bovine hydroxyapatite (BHA) and secretome BM-MSC culture under normoxic and hypoxic conditions, and UNAIR Tropical Disease Institute for implantation in experimental animals. Data analysis was carried out with the one-way ANOVA statistical test and continued with the Post Hoc test LSD statistical test to determine whether or not there were significant differences between groups. There were significant differences between hypoxic to normoxic group and hypoxic to BHA group at day-30 observation using ALP, COL 1, ON, and OPN parameters. Meanwhile, there is only osteonectin parameter has significant difference at day-30 observation. At day-60 observation, only OPN parameter has significant differences between hypoxic to normoxic and hypoxic to BHA group. Between day-30 and day-60 observation, BHA and normoxic groups have a significant difference at all parameters, but in hypoxic group, there are only difference at ALP, COL 1, and ON parameters. Hypoxic condition BM-MSC secretome with BHA composite is superior and could be an option for treating bone defect.

UDCA Inhibits Hypoxic Hepatocellular Carcinoma Cell–Induced Angiogenesis Through Suppressing HIF-1α/VEGF/IL-8 Intercellular Signaling

Background: A hypoxic microenvironment may induce angiogenesis and promote the development of hepatocellular carcinoma (HCC). The aim of this study was to evaluate whether ursodeoxycholic acid (UDCA) may inhibit hypoxic HCC cell–induced angiogenesis and the possible mechanisms.Methods: Tube formation and matrigel plug angiogenesis assays were used to evaluate angiogenesis in vitro and in vivo, respectively. Real-time PCR, enzyme-linked immunosorbent assay, and Western blot were used to evaluate the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and IL-8, respectively. Dual-luciferase reporter assay was applied to assess the reporter gene expression of hypoxia-response element (HRE).Results: UDCA antagonized hypoxic Huh 7 cell-induced tube formation of EA.hy 926 cells. In HCC cells, UDCA inhibited hypoxia-induced upregulation of VEGF and IL-8 both in mRNA and protein levels. UDCA also inhibited IL-8–induced angiogenesis in vitro and in vivo through suppressing IL-8–induced phosphorylation of ERK. The levels of HIF-1α mRNA and protein and HRE-driven luciferase activity in HCC cells were upregulated by hypoxia and were all inhibited by UDCA. The proteasome inhibitor MG132 antagonized the effect of UDCA on HIF-1α degradation. In hypoxic condition, the phosphorylation of ERK and AKT was obviously increased in HCC cells, which was suppressed by UDCA. Transfection of the HIF-1α overexpression plasmid reversed the effects of UDCA on hypoxic HCC cell–induced angiogenesis, HRE activity, and expressions of IL-8 and VEGF.Conclusions: Our results demonstrated that UDCA could inhibit hypoxic HCC cell–induced angiogenesis through suppressing HIF-1α/VEGF/IL-8–mediated intercellular signaling between HCC cells and endothelial cells.

Hypoxic in vitro culture reduces histone lactylation and impairs pre-implantation embryonic development in mice

Background Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated. Results We examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate. Conclusions We provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.