Auxin apical dominance governed by the OsAsp1-OsTIF1 complex determines distinctive rice caryopses development on different branches.

In rice (Oryza sativa), caryopses located on proximal secondary branches (CSBs) have smaller grain size and poorer grain filling than those located on apical primary branches (CPBs), greatly limiting grain yield. However, the molecular mechanism responsible for developmental differences between CPBs and CSBs remains elusive. In this transcriptome-wide expression study, we identified the gene Aspartic Protease 1 (OsAsp1), which reaches an earlier and higher transcriptional peak in CPBs than in CSBs after pollination. Disruption of OsAsp1 expression in the heterozygous T-DNA line asp1-1+/-eliminated developmental differences between CPBs and CSBs. OsAsp1 negatively regulated the transcriptional inhibitor of auxin biosynthesis, OsTAA1 transcriptional inhibition factor 1 (OsTIF1), to preserve indole-3-acetic acid (IAA) apical dominance in CPBs and CSBs. IAA also facilitated OsTIF1 translocation from the endoplasmic reticulum (ER) to the nucleus by releasing the interaction of OsTIF1 with OsAsp1 to regulate caryopses IAA levels via a feedback loop. IAA promoted transcription of OsAsp1 through MADS29 to maintain an OsAsp1 differential between CPBs and CSBs during pollination. Together, these findings provide a mechanistic explanation for the distributed auxin differential between CPBs and CSBs to regulate distinct caryopses development in different rice branches and potential targets for engineering yield improvement in crops.

Clinical grade lentiviral vector purification and quality control requirements.

Lentiviral vectors have been proven to be a powerful tool in gene therapies that includes the ability to perform long-term gene editing in both dividing and non-dividing cells. In order to meet the rising demand for clinical-grade lentiviral vectors for future clinical trials and requirements by regulatory agencies, new methods and technologies were developed, including the rapid optimization of production and purification processes. However, gaps still exist in achieving ideal yields and recovery rates in large-scale manufacturing process steps. The downstream purification process is a critical step required to obtain a sufficient quantity and high-quality lentiviral vectors products, which is challenged by the low stability of the lentiviral vector particles and large production volumes associated with the manufacturing process. This review summarizes the most recent and promising technologies and enhancements used in the large-scale purification process step of lentiviral vector manufacturing and aims to provide a significant contribution towards the achievement of providing sufficient quantity and quality of lentiviral vectors in scalable processes.

Transcriptomic identification of long noncoding RNAs and their hormone-associated nearby coding genes involved in the differential development of caryopses localized on different branches in rice.

Long noncoding RNAs (lncRNAs) play important regulatory roles in caryopsis development and grain size in rice. However, whether there exist differences in lncRNA expression between caryopses located on primary branches (CPB) and caryopses located on secondary branches (CSB) that contribute to their differential development remains elusive. Here, we performed transcriptome-wide analysis to identify 2,273 lncRNAs expressed in CPB and CSB at 0, 5, 12, and 20 days after flowering (DAF). Although these lncRNAs were widely distributed, the majority were located in intergenic regions of the 12 rice chromosomes. Based on gene expression cluster analysis, lncRNAs expressed in CPB and CSB were clustered into two subtypes in a position-independent manner: one includes 0- and 5-DAF CPB and CSB, and 12-DAF CSB; the second includes 12-DAF CPB and 20-DAF CPB and CSB. Furthermore, according to the expression value of each lncRNA, K-means cluster analysis revealed 135 early-stage, 116 middle-stage, and 114 late-stage expression-delayed lncRNAs in CSB. Then, we analyzed the expression values of the expression-delayed lncRNAs and nearby coding genes (100 kb upstream and downstream of the lncRNAs), and found 631 lncRNA-mRNA pairs, including 258 lncRNAs and 571 nearby coding genes, some of which are related to hormone-regulated grain development. These results suggested that expression-delayed lncRNAs in CSB may regulate the development of CPB and CSB, providing insight into the mechanism underlying the developmental differences between CPB and CSB, and the differences in grain yield.

OsHSD2 interaction with and phosphorylation by OsCPK21 is essential for lipid metabolism during rice caryopsis development.

Rice calcium-dependent protein kinase 21 (OsCPK21) is specifically and highly expressed throughout reproductive development and plays a critical role in rice pollen development by indirectly regulating the MIKC*-type MADS box transcription factor. However, little is known about the function of OsCPK21 in rice caryopsis development. In this study, we performed an in vitro pull-down experiment followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and identified hydroxysteroid dehydrogenase 2 (HSD2) as a candidate OsCPK21-interacting protein in 25 DAF (days after flowering) rice caryopses. Then, we verified the interaction between OsCPK21 and OsHSD2 using yeast two-hybrid and bimolecular fluorescence assays and revealed the in vitro phosphorylation of OsHSD2 by OsCPK21. Furthermore, oscpk21 and oshsd2 mutants were generated by the CRISPR/Cas9 technique, and we found that the lipid profiles were drastically changed in both oscpk21 and oshsd2, implying that OsHSD2 phosphorylated by OsCPK21 regulates lipid abundance in caryopsis development, thereby providing a potential target for the genetic improvement of rice grain quality in future lipid-related breeding and biotechnology applications.

Prognostic nomograms for patients undergoing radical operation for stage I-III appendiceal adenocarcinoma: A surveillance, epidemiology, and end results database analysis.

Objective: To develop nomograms for effective prediction of cause-specific survival (CSS) and overall survival (OS) of patients who underwent radical operation for stage I-III appendiceal adenocarcinoma. Methods: Clinical information from the surveillance, epidemiology, and end results database was retrieved from 2004 to 2015 and subjected to multivariate analysis to explore variables that affect the OS and CSS. Results were used to construct nomograms to assess the 1-, 3-, and 5-year OS and CSS rates, then their calibration accuracy and discriminative power were examined using Kaplan-Meier curves, calibration plots, and C statistics. Results: Overall, 1,241 patients were included in the analysis. We found 7 and 5 factors that could independently alter the prognosis, then used for creating nomograms for evaluating the OS and CSS, respectively, with respective C-index values of 0.741 (95% confidence interval [CI]: 0.729-0.754) and 0.747 (95% CI: 0.733-0.762). Calibration and receiver operating characteristic curves further revealed excellent predictive performance. Conclusions: We successfully built highly accurate nomograms for evaluating the 1-, 3-, and 5-year CSS and OS rates in subjects who underwent radical operation for stage I-III appendiceal adenocarcinoma. Further studies, involving prospective validations, are required to validate these nomograms.

Transcriptomic analysis reveals the contribution of auxin on the differentially developed caryopses on primary and secondary branches in rice.

Generally, the caryopses located on proximal secondary branches (CSB) have smaller grain size and slower and poorer filling rate than those on apical primary branches (CPB) in rice, which greatly limits the grain yield potential fulfillment. However, the key regulators determining the developmental differences between CPB and CSB remain elusive. Here, we have performed transcriptomic analysis in CPB and CSB at four developmental stages [0, 5, 12 and 20 days after fertilization (DAF)] using high-throughput RNA-sequencing technique. Based on gene expression cluster analysis, the genes expressed in CPB and CSB were clustered into two subtypes in a positional-independent manner: one includes 0- and 5-DAF CPB and CSB, and 12-DAF CSB; another includes 12-DAF CPB, 20-DAF CPB and CSB. Moreover, according to the expression value of each gene, K-mean cluster analysis showed that the K4 to K6 classifiers contain the genes highly expressed in 5-DAF CPB and 12-DAF CSB, which were enriched in DNA synthesis, protein synthesis and cell proliferation mainly responsible for grain size decision. Then, functional enrichment analysis in Gene Ontology database showed that auxin-related genes were relatively enriched, indicating that auxin might be the key determinant for gene expression in K4 to K6 classifiers. Finally, the application of exogenous IAA in CSB before fertilization promoted gene expression, caryopsis development and grain weight closer to that in CPB, providing a molecular framework to optimize CSB development and potential targets for increasing grain yield.

[Protective effects of curcumin on neonatal rats with necrotizing enterocolitis].

OBJECTIVE: This study examined the effects of curcumin on intestinal histopathological changes, cyclooxygenase-2 (COX-2) expression, and tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) concentrations in neonatal rats with necrotizing enterocolitis (NEC), in order to investigate the effects of curcumin against NEC. METHODS: Forty neonatal rats were randomly divided into four groups (n=10 each): normal control, solvent control, NEC model, and curcumin intervention. The general situations of rats were observed for 3 consecutive days, and the rats were then sacrificed on the 4th day. Intestinal tissues were obtained for examining the histopathological changes, COX-2 expression, and TNF-alpha and IL-10 concentrations. RESULTS: Curcumin treatment ameliorated the general situations and histopathological signs in rats with NEC. TNF-alpha and IL-10 concentrations in the NEC model and the curcumin intervention groups increased significantly compared with those in the normal and solvent control groups (p<0.05). The concentration of TNF-alpha decreased (p<0.05), while the concentration of IL-10 increased significantly in the curcumin intervention group in comparison with the NEC model group (p<0.05). Immunohistochemistry results indicated that the positive expression of COX-2 in the curcumin intervention group was significantly lower than that in the NEC model group. CONCLUSIONS: Curcumin has protective effects against NEC in neonatal rats, possibly through inhibiting COX-2 expression, reducing TNF-alpha content, and increasing IL-10 content.

Responses of Antioxidant Defense and Immune Gene Expression in Early Life Stages of Large Yellow Croaker (Pseudosciaena crocea) Under Methyl Mercury Exposure.

Early life stages of marine organisms are the most sensitive stages to environment stressors including pollutants. In order to understand the toxicological effects induced by MeHg exposure on juveniles of large yellow croaker (Pseudosciaena crocea), a toxicity test was performed wherein fish were exposed to sub-lethal concentrations of MeHg under laboratory conditions (18 ± 1°C; 26 ± 1 in salinity). After 30 days of 0-4.0 µg L-1 MeHg exposure, SOD activity was significantly decreased in the 0.25, 1.0, and 4.0 µg L-1 treatments; while CAT activity was significantly increased in the 4.0 µg L-1 treatments; GSH level, GPx activity were significantly elevated in the 4.0 µg L-1 treatments, respectively. Meanwhile, malondialdehyde content was also significantly increased in the 1.0 and 4.0 µg L-1 treatments with respect to the control. Acetylcholinesterase activity was significantly decreased by 18.3, 25.2, and 21.7% in the 0.25, 1.0, and 4.0 µg L-1 treatments, respectively. The expression of TCTP, GST3, Hsp70, Hsp27 mRNA were all up-regulated in juveniles with a dose-dependent manner exposed to MeHg. These results suggest that large yellow croaker juveniles have the potential to regulate the levels of antioxidant enzymes and initiate immune response in order to protect fish to some extent from oxidative stress induced by MeHg.

Conformational Characteristics of Rice Hexokinase OsHXK7 as a Moonlighting Protein involved in Sugar Signalling and Metabolism.

Hexokinase (HXK) as a moonlighting protein involves in glucose metabolism and signalling to regulate growth and development in plants. Therefore, the clarification for the structural properties of OsHXK7 (Oryza sativa Hexokinase 7) is essential to understand its role mechanism associated with the Glc signalling and metabolism. In this study, the structural characteristics of OsHXK7 (Oryza sativa Hexokinase 7) were identified. In the fluorescence spectrum, the Trp peak representing OsHXK7 binding to D-glucose (D-Glc) and 2-deoxyglucose (2-dG) showed an obvious blue shift. The distinct change in the secondary structure of OsHXK7 after binding to Glc was also detected in circular dichroism spectra. Using superimposed modelling, OsHXK7 showed a Glc-induced structural change, in which the 76th glycine, 148th serine and 256th tryptophan were contained within the pocket region. It was further shown by site-directed mutagenesis that the 76th glycine and the 256th tryptophan, but not the 148th serine, are the pivotal sites of OsHXK7 that maintain its catalytic activity and intrinsic blue shift fluorescence. These results suggest that OsHXK7 binding to Glc leads to a conformational change, that is likely essential for the function of OsHXK7 in Glc signalling and metabolism during plant growth and development.

FRET-based glucose imaging identifies glucose signalling in response to biotic and abiotic stresses in rice roots.

Glucose is the primary energy provider and the most important sugar-signalling molecule, regulating metabolites and modulating gene expression from unicellular yeast to multicellular plants and animals. Therefore, monitoring intracellular glucose levels temporally and spatially in living cells is an essential step for decoding the glucose signalling in response to biotic and abiotic stresses. In this study, the genetically encoded FRET (Förster resonance energy transfer) nanosensors, FLIPglu-2µ∆13 and FLIPglu-600µΔ13, were used to measure cytosolic glucose dynamics in rice plants. First, we found that the FRET signal decreased in response to external glucose in a concentration-dependent manner. The glucose concentration at which the cytosolic level corresponded to the K0.5 value for FLIPglu-2µΔ13 was approximately 10.05µM, and that for FLIPglu-600µΔ13 was 0.9mM, respectively. The substrate selectivity of nanosensors for glucose and its analogues is D-Glucose>2-deoxyglucose>3-O-methylglucose>L-Glucose. We further showed that the biotic elicitors (flg22 and chitin) and the abiotic elicitors (osmotic stress, salinity and extreme temperature) induce the intracellular glucose increases in the detached root segments of transgenic rice containing FLIPglu-2µΔ13 in a stimulus-specific manner, but not in FLIPglu-600µΔ13 transgenic lines. These results demonstrated that FRET nanosensors can be used to detect increases in intracellular glucose within the physiological range of 0.2-20µM in response to various stimuli in transgenic rice root cells, which indicated that intracellular glucose may act as a potential secondary messenger to connect extracellular stimuli with cellular physiological responses in plants.

GpDSR7, a Novel E3 Ubiquitin Ligase Gene in Grimmia pilifera Is Involved in Tolerance to Drought Stress in Arabidopsis.

The growth and development of plants under drought stress depends mainly on the expression levels of various genes and modification of proteins. To clarify the molecular mechanism of drought-tolerance of plants, suppression subtractive hybridisation cDNA libraries were screened to identify drought-stress-responsive unigenes in Grimmia pilifera, and a novel E3 ubiquitin ligase gene, GpDSR7, was identified among the 240 responsive unigenes. GpDSR7 expression was induced by various abiotic stresses, particularly by drought. GpDSR7 displayed E3 ubiquitin ligase activity in vitro and was exclusively localised on the ER membrane in Arabidopsis mesophyll protoplasts. GpDSR7-overexpressing transgenic Arabidopsis plants showed a high water content and survival ratio under drought stress. Moreover, the expression levels of some marker genes involved in drought stress were higher in the transgenic plants than in wild-type plants. These results suggest that GpDSR7, an E3 ubiquitin ligase, is involved in tolerance to drought stress at the protein modification level.

Four AUXIN RESPONSE FACTOR genes downregulated by microRNA167 are associated with growth and development in Oryza sativa.

MicroRNA167 (miR167), as a conserved miRNA, has been implicated in auxin signalling by regulating the expression of certain auxin response factor (ARF) genes to determine the plant developmental process. Among the 10 MIR167 genes of rice, the precursor structures derived from MIR167a, MIR167b and MIR167c produce miR167 with high efficiency. To explore the biological function of miR167 in rice, four of its predicted target genes, OsARF6, OsARF12, OsARF17 and OsARF25, were identified in vivo. Although the expression levels of miR167 and its target OsARFs did not show an obvious negative correlation, the enhanced miR167 level in transgenic rice overexpressing miR167 resulted in a substantial decrease in mRNA levels of the four OsARF genes. Moreover, the transgenic rice plants were small in stature with remarkably reduced tiller number. These results suggest that miR167 is important for the appropriate expression of at least four OsARFs, which mediate the auxin response, to contribute to the normal growth and development of rice.