A chromosome-scale genome sequence of pitaya (Hylocereus undatus) provides novel insights into the genome evolution and regulation of betalain biosynthesis.

Pitaya (Hylocereus) is the most economically important fleshy-fruited tree of the Cactaceae family that is grown worldwide, and it has attracted significant attention because of its betalain-abundant fruits. Nonetheless, the lack of a pitaya reference genome significantly hinders studies focused on its evolution, as well as the potential for genetic improvement of this crop. Herein, we employed various sequencing approaches, namely, PacBio-SMRT, Illumina HiSeq paired-end, 10× Genomics, and Hi-C (high-throughput chromosome conformation capture) to provide a chromosome-level genomic assembly of 'GHB' pitaya (H. undatus, 2n = 2x = 22 chromosomes). The size of the assembled pitaya genome was 1.41 Gb, with a scaffold N50 of ~127.15 Mb. In total, 27,753 protein-coding genes and 896.31 Mb of repetitive sequences in the H. undatus genome were annotated. Pitaya has undergone a WGT (whole-genome triplication), and a recent WGD (whole-genome duplication) occurred after the gamma event, which is common to the other species in Cactaceae. A total of 29,328 intact LTR-RTs (~696.45 Mb) were obtained in H. undatus, of which two significantly expanded lineages, Ty1/copia and Ty3/gypsy, were the main drivers of the expanded genome. A high-density genetic map of F1 hybrid populations of 'GHB' × 'Dahong' pitayas (H. monacanthus) and their parents were constructed, and a total of 20,872 bin markers were identified (56,380 SNPs) for 11 linkage groups. More importantly, through transcriptomic and WGCNA (weighted gene coexpression network analysis), a global view of the gene regulatory network, including structural genes and the transcription factors involved in pitaya fruit betalain biosynthesis, was presented. Our data present a valuable resource for facilitating molecular breeding programs of pitaya and shed novel light on its genomic evolution, as well as the modulation of betalain biosynthesis in edible fruits.

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

Inhibition of extracellular signal-regulated kinase/calpain-2 pathway reduces neuroinflammation and necroptosis after cerebral ischemia-reperfusion injury in a rat model of cardiac arrest.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is the leading cause of poor neurological prognosis after cardiopulmonary resuscitation (CPR). We previously reported that the extracellular signal-regulated kinase (ERK) activation mediates CIRI. Here, we explored the potential ERK/calpain-2 pathway role in CIRI using a rat model of cardiac arrest (CA). METHODS: Adult male Sprague-Dawley rats suffered from CA/CPR-induced CIRI, received saline, DMSO, PD98059 (ERK1/2 inhibitor, 0.3 mg/kg), or MDL28170 (calpain inhibitor, 3.0 mg/kg) after spontaneous circulation recovery. The survival rate and the neurological deficit score (NDS) were utilized to assess the brain function. Hematoxylin stain, Nissl staining, and transmission electron microscopy were used to evaluate the neuron injury. The expression levels of p-ERK, ERK, calpain-2, neuroinflammation-related markers (GFAP, Iba1, IL-1ß, TNF-α), and necroptosis proteins (TNFR1, RIPK1, RIPK3, p-MLKL, and MLKL) in the brain tissues were determined by western blotting and immunohistochemistry. Fluorescent multiplex immunohistochemistry was used to analyze the p-ERK, calpain-2, and RIPK3 co-expression in neurons, and RIPK3 expression levels in microglia or astrocytes. RESULTS: At 24 h after CA/CPR, the rats in the saline-treated and DMSO groups presented with injury tissue morphology, low NDS, ERK/calpain-2 pathway activation, and inflammatory cytokine and necroptosis protein over-expression in the brain tissue. After PD98059 and MDL28170 treatment, the brain function was improved, while inflammatory response and necroptosis were suppressed by ERK/calpain-2 pathway inhibition. CONCLUSION: Inflammation activation and necroptosis involved in CA/CPR-induced CIRI were regulated by the ERK/calpain-2 signaling pathway. Inhibition of that pathway can reduce neuroinflammation and necroptosis after CIRI in the CA model rats.

Edaravone Ameliorates Renal Warm Ischemia-Reperfusion Injury by Downregulating Endoplasmic Reticulum Stress in a Rat Resuscitation Model.

BACKGROUND: This study was conducted to explore whether the effect of edaravone (5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol3-one, EDR) can ameliorate renal warm ischemia-reperfusion injury (IRI) by modulating endoplasmic reticulum stress (ERS) and its downstream effector after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in a rat model. METHODS: The rats (n=10) experienced anaesthesia and intubation followed by no CA inducement were defined as the Sham group. Transoesophageal alternating current stimulation was employed to establish 8 min of CA followed by conventional CPR for a resuscitation model. The rats with successful restoration of spontaneous circulation (ROSC) randomly received EDR (3 mg/kg, EDR group, n=10) or equal volume normal saline solution (the NS group, n=10). At 24 hr after ROSC, serum creatinine (SCR), blood urea nitrogen (BUN) levels, and cystatin-C (Cys-C) levels were determined and the protein level of glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), extracellular signal-regulated kinase (ERK), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), Bax/Bcl-2, and caspase-3 were detected by Western blot method. RESULTS: At 24 hrs after ROSC, SCR, BUN and Cys-C were obviously increased and the proteins expression, including GRP78, CHOP and p-ERK1/2, cleaved-caspase 3 Bax/Bcl-2 ratio, were significantly upregulated in the NS group compared with the Sham group (p<0.05). The remarkable improvement of these adverse outcomes was observed in the EDR group (p<0.05). CONCLUSION: In conclusion, we found that EDR ameliorates renal warm IRI by downregulating ERS and its downstream effectors in a rat AKI model evoked by CA/CPR. These data may provide evidence for future therapeutic benefits of EDR against AKI induced by CA/CPR.

The incidence of acute kidney injury following cardiac arrest and cardiopulmonary resuscitation in a rat model.

OBJECTIVE: In the current study, we investigated the incidence of acute kidney injury (AKI) induced by cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) and whether such an AKI can recover spontaneously in rats. METHODS: We used transesophageal alternating current stimulation to establish 7 min of CA rat model followed by conventional CPR. The experimental rats were randomly divided into three groups (n = 20 per group) according to the different time points after restoration spontaneous circulation (ROSC): the ROSC 24 h, ROSC 48 h, and ROSC 72 h group. The diagnosis of rat AKI refers to the 2012 KDIGO adult AKI diagnostic criteria. The severity of AKI quantified by the serum creatinine (SCR), blood urea nitrogen (BUN) levels and histological features of renal tissue. RESULTS: The incidence rates of AKI in ROSC 24 h, ROSC 48 h, and ROSC 72 h group were 65%, 45%, and 42.9%. Moreover, the values of SCR and BUN were highest at ROSC 24 h, and then gradually decreased with the time of ROSC. The histological changes of the renal tissues such as glomerular collapse, renal tubular cell swelling, and inflammatory cell infiltration had also observed. CONCLUSION: The incidence of AKI in rats was high after suffering from CA and CPR, but renal function improved with the prolongation of ROSC time, indicating the ability of the kidney to self-repair.

[Effect of Astragalus membranaceus var. mongholicus seed extracts on seed germination and seedling growth of different Codonopsis pilosula caltiver].

OBJECTIVE: To reveal the allelopathy effect of Astragalus membranaceus var. mongholicus seeds and provide information for the intercrop production. METHOD: The A. membranaceus. var. mongholicus seeds were soaked in distilled water for different time (12, 24, 36, 48, 60 h) , and then the seed extracts were used to study their effects on the seed germination, seedling growth and development of two Codonopsis pilosula. RESULT: The A. membranaceus var. mongholicus seeds contained some allelopathy compounds. Their soaked liquid had significantly influence on the seed germination and seedling growth of C. pilosula. The seed germination rate, germination power, germination index and vigor index of two C. pilosula calrivar were improved and then inhabited with soaking time elongation. The extract soaking for 24 h significantly improved the germination traits but the extract for 60 h appeared different degrees of inhibiting vigor. The seed extracts soaking ranging between 12 and 60 h all significantly improved the above plant growth of C. pilosula but significant inhibited their radicle growth in length. And with the soaking time elongation the facilitation effect weakened and the inhibiting effect enhanced, especially more significant in the C. pilosula caltivar (Baitiaodangshen). CONCLUSION: The A. membranaceus var. mongholicus seeds have allelopathic compounds and the endogenous inhibitor can be extracted when soaked for more than 24 h in water with intact seeds, resulting in improvement of seed germination rate. The C. pilosula could be intercropped in A. membranaceus var. mongholicus field, however, when intercroped it should notice that the intercrop proportion should vary with the caltivar.

Morphology study of freeze-drying mononuclear cells of human cord blood.

The research on haematopoietic stem cells of human cord blood has become more important recently. At present, cord blood is mainly preserved at ultra-low temperatures. In the former study, we compared the effects of preserving mononuclear cells (MNC) and whole human cord blood by freeze-drying. This time, a further study was conducted on freeze-drying mononuclear cells. Samples in the presence of PVP, sucrose, mannitol and FBS were firstly frozen to -38 degrees C. Afterwards, they were vacuum-dried at a selected shelf temperature of -30 degrees C for the main drying stage, and then vacuum-dried at 15 degrees C for the second drying stage. The entire time of freeze-drying process was 41 hours. Samples were stored at room temperature for 7 days prior to evaluation. Subsequently, the dried samples were resuspended in an isotonic phosphate-buffered saline solution. The residual moisture content was 6.5 +/- 0.87%. The recovery of the cells was tested by a haemacytometer, and the numerical cell count recovery of rehydrated MNC increased by 8%. Morphology of the fresh and rehydrated MNC was analyzed respectively using standard light microscopy, scanning electron microscope and transmission electron microscope. The results showed that karyons changed and cytoplasm decreased after rehydration, but it is still unknown that whether these changes will influence the proliferative ability of the stem cells.

Freeze-drying of mononuclear cells and whole blood of human cord blood.

The research on haematopoietic stem cells of human cord blood has become more important recently. People have concentrated on the preservation of cord blood stem cells. At present, cord blood can be preserved at ultra-low temperatures. In this study, we try to preserve cord blood and its constituents by freeze-drying. The experiments on both the mononuclear cell content and the whole blood of human cord blood were carried out respectively. The samples were frozen firstly by different cooling protocols in the presence of PVP, sucrose, and mannitol. Afterwards, they were vacuum-dried at a selected shelf temperature of -30 degree C for the main drying stage, and then vacuum-dried at 15 degree C for the second drying stage. The entire time of the freeze drying was 52 hours. Samples were stored at room temperature for 2 days prior to evaluation. Subsequently, the dried samples were suspended in an isotonic phosphate-buffered saline solution. The recovery of the cells were tested by a haemacytometer, and the highest cell numerical count recovery of MNC was 75.0 percent (SD = 4.1 percent) (P = 0.01), obtained in the protocol of 40 percent PVP + 20 percent sucrose + 10 percent Mannitol. The viability of the nucleated cells measured by PI staining and the ratio of the number of CD34+ to the number of lymphocytes (by the FITC anti-human CD34+ conjugated antibody method) were measured using a flow cytometer (FCM). The protocol of 40 percent PVP + 20 percent sucrose + 10 percent fetal bovine serum had the highest viability of 98.6 percent (SD = 0.7 percent) (P = 0.01). The highest ratio of CD34+ to lymphocytes was 1.2%, and the highest recovery of CD34+ was 68.4 percent (SD = 39.5 percent) (P = 0.05). Comparing the results of the lyophilized MNC subfraction with that of the whole blood, the lyophilization of the isolated MNC was more successful than that of whole blood.

[Photochemical Effect of the Photosensitizer Hemoporphyrin Derivative on MCF-7 Cell Line and Umbilical Cord Blood Hematopoietic Cells]

To study the effect of hemoporphyrin derivative(HPD) combined with laser irradiation on human breast tumor cell line MCF-7 and normal human umbilical cord blood-derived hematopoietic cells by using MTT assay. The results showed that HPD plus laser irradiation was more efficient for killing MCF-7 cells than normal human umbilical cord blood-derived hematopoietic cells. The photochemical effect with laser irradiation were higher than with light irradiation and it's effect on MCF-7 cells was higher gradually with the increase of HPD concentration.

Application of Free-flow Electrophoresis to the Separation of Murine Lymphocytes.

Free-flow electrophoresis offers not only the all-in-solution separation process but also very gentle separation conditions, and it can be used for continuous separation and preparation, so it has been widely applied in the fields of biochemistry and cell biology. Using this technique to separate insoluble particles such as cells, both high separation efficiency and high activity and viability could be obtained. Murine lymphocytes were separated in low ionic strength triethanolamine buffer by free-flow electrophoresis, the cell fractions were detected and characterized by means of UV spectrometry, immune fluorescence labeling and flow cytometry. Results indicated that T and B lymphocytes were well separated with high viability.