Near-freezing temperature suppresses avocado (Persea americana Mill.) fruit softening and chilling injury by maintaining cell wall and reactive oxygen species metabolism during storage.

To enhance the postharvest quality of avocado (Persea americana Mill.) fruit, this study investigates alterations in cell wall metabolism and reactive oxygen species (ROS) metabolism during near-freezing temperature (NFT) storage, and explores their impact on fruit softening. The fruit was stored at 25 °C, 5 °C, 2 °C, and NFT, respectively. NFT storage retarded firmness loss and chilling injury in comparison with 25 °C, 5 °C, and 2 °C. NFT storage delayed the decrease of ionic-soluble pectin (ISP) and cellulose (CLL) contents by suppressing cell wall degradation enzyme activities. Correlation analysis showed that cell wall degradation enzyme activities were positively correlated to rates of ethylene release and respiration. Moreover, NFT storage maintained higher levels of DPPH and ABTS scavenging abilities, activities of superoxide dismutase, peroxidase, and catalase, as well as ascorbate-glutathione cycle (ascorbic acid, glutathione, glutathione disulfide, ascorbate peroxidase, cycle-related enzymes), thereby inhibited the increase of ROS content, malondialdehyde content, and cell membrane permeability. Fruit firmness and chilling injury were correlated with the contents of hydrogen (H2O2), superoxide anion (O2.-), ISP, and CLL. These results suggested that NFT could suppress fruit softening and chilling injury by inhibiting cell wall degradation through delaying respiration and ethylene production and suppressing ROS production via activation of antioxidant systems, thereby maintaining quality and prolonged storage life during avocado fruit storage.

Genome-wide exploration of the GDSL-type esterase/lipase gene family in rapeseed reveals several BnGELP proteins active during early seedling development.

The Gly-Asp-Ser-Leu (GDSL)-type esterase/lipase proteins (GELP) are one of the most important families of lipolytic enzymes and play prominent roles in seed germination and early seedling establishment through mobilizing the lipids stored in seeds. However, there are no comprehensive studies systematically investigating the GELP gene family in Brassica napus (BnGELP), and their biological significance to these physiological processes are far from understood. In the present study, a total of 240 BnGELP genes were identified in B. napus cultivar "Zhongshuang 11" (ZS11), which is nearly 2.3-fold more GELP genes than in Arabidopsis thaliana. The BnGELP genes clustered into 5 clades based on phylogenetic analysis. Ten BnGELPs were identified through zymogram analysis of esterase activity followed by mass spectrometry, among which five clustered into the clade 5. Gene and protein architecture, gene expression, and cis-element analyses of BnGELP genes in clade 5 suggested that they may play different roles in different tissues and in response to different abiotic stresses. BnGELP99 and BnGELP159 were slightly induced by cold, which may be attributed to two low-temperature responsive cis-acting regulatory elements present in their promoters. An increased activity of esterase isozymes by cold was also observed, which may reflect other cold inducible esterases/lipases in addition to the ten identified BnGELPs. This study provides a systemic view of the BnGELP gene family and offers a strategy for researchers to identify candidate esterase/lipase genes responsible for lipid mobilization during seed germination and early seedling establishment.

Genome-Wide Identification of the Cytochrome P450 Superfamily Genes and Targeted Editing of BnCYP704B1 Confers Male Sterility in Rapeseed.

The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar "Zhongshuang 11" (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes.

Comparative full-length transcriptome analysis by Oxford Nanopore Technologies reveals genes involved in anthocyanin accumulation in storage roots of sweet potatoes (Ipomoea batatas L.).

Background: Storage roots of sweet potatoes (Ipomoea batatas L.) with different colors vary in anthocyanin content, indicating different economically agronomic trait. As the newest DNA/RNA sequencing technology, Oxford Nanopore Technologies (ONT) have been applied in rapid transcriptome sequencing for investigation of genes related to nutrient metabolism. At present, few reports concern full-length transcriptome analysis based on ONT for study on the molecular mechanism of anthocyanin accumulation leading to color change of tuberous roots of sweet potato cultivars. Results: The storage roots of purple-fleshed sweet potato (PFSP) and white-fleshed sweet potato (WFSP) at different developmental stages were subjected to anthocyanin content comparison by UV-visible spectroscopy as well as transcriptome analysis at ONT MinION platform. UV-visible spectrophotometric measurements demonstrated the anthocyanin content of PFSP was much higher than that of WFSP. ONT RNA-Seq results showed each sample generated average 2.75 GB clean data with Full-Length Percentage (FL%) over 70% and the length of N50 ranged from 1,192 to 1,395 bp, indicating reliable data for transcriptome analysis. Subsequent analysis illustrated intron retention was the most prominent splicing event present in the resulting transcripts. As compared PFSP with WFSP at the relative developmental stages with the highest (PH vs. WH) and the lowest (PL vs. WL) anthocyanin content, 282 and 216 genes were up-regulated and two and 11 genes were down-regulated respectively. The differential expression genes involved in flavonoid biosynthesis pathway include CCoAOMT, PpLDOX, DFR, Cytochrome P450, CHI, and CHS. The genes encoding oxygenase superfamily were significantly up-regulated when compared PFSP with WFSP at the relative developmental stages. Conclusions: Comparative full-length transcriptome analysis based on ONT serves as an effective approach to detect the differences in anthocyanin accumulation in the storage roots of different sweet potato cultivars at transcript level, with noting that some key genes can now be closely related to flavonoids biosynthesis. This study helps to improve understanding of molecular mechanism for anthocyanin accumulation in sweet potatoes and also provides a theoretical basis for high-quality sweet potato breeding.

Genome-Wide Identification and Analysis of NAC Transcription Factor Family in Two Diploid Wild Relatives of Cultivated Sweet Potato Uncovers Potential NAC Genes Related to Drought Tolerance.

NAC (NAM, ATAF1/2, and CUC2) proteins play a pivotal role in modulating plant development and offer protection against biotic and abiotic stresses. Until now, no systematic knowledge of NAC family genes is available for the food security crop, sweet potato. Here, a comprehensive genome-wide survey of NAC domain-containing proteins identified 130 ItbNAC and 144 ItfNAC genes with full length sequences in the genomes of two diploid wild relatives of cultivated sweet potato, Ipomoea triloba and Ipomoea trifida, respectively. These genes were physically mapped onto 15 I. triloba and 16 I. trifida chromosomes, respectively. Phylogenetic analysis divided all 274 NAC proteins into 20 subgroups together with NAC transcription factors (TFs) from Arabidopsis. There were 9 and 15 tandem duplication events in the I. triloba and I. trifida genomes, respectively, indicating an important role of tandem duplication in sweet potato gene expansion and evolution. Moreover, synteny analysis suggested that most NAC genes in the two diploid sweet potato species had a similar origin and evolutionary process. Gene expression patterns based on RNA-Seq data in different tissues and in response to various hormone, biotic or abiotic treatments revealed their possible involvement in organ development and response to various biotic/abiotic stresses. The expression of 36 NAC TFs, which were upregulated in the five tissues and in response to mannitol treatment, was also determined by real-time quantitative polymerase chain reaction (RT-qPCR) in hexaploid cultivated sweet potato exposed to drought stress. Those results largely corroborated the expression profile of mannitol treatment uncovered by the RNA-Seq data. Some significantly up-regulated genes related to drought stress, such as ItbNAC110, ItbNAC114, ItfNAC15, ItfNAC28, and especially ItfNAC62, which had a conservative spatial conformation with a closely related paralogous gene, ANAC019, may be potential candidate genes for a sweet potato drought tolerance breeding program. This analysis provides comprehensive and systematic information about NAC family genes in two diploid wild relatives of cultivated sweet potato, and will provide a blueprint for their functional characterization and exploitation to improve the tolerance of sweet potato to abiotic stresses.

Prognostic value of baseline hemoglobin-to-red blood cell distribution width ratio in small cell lung cancer: A retrospective analysis.

BACKGROUND: This study aimed to investigate the prognostic value of baseline hemoglobin-to-red blood cell distribution width ratio (HRR) in patients with small cell lung cancer (SCLC). METHODS: We retrospectively analyzed the medical records of patients with newly diagnosed SCLC who had received first-line chemotherapy at the Department of Pulmonary Oncology of the PLA 307 Hospital between January 2008 and October 2018. The optimal cutoff value of the continuous variables was determined using the X-tile software. Univariate and multivariate analyses were conducted using Cox proportional hazard models. The Kaplan-Meier method was used for survival analysis, with differences tested using the log-rank test. RESULTS: A total of 146 patients were included. The cutoff value for HRR was determined as 0.985. Statistically significant differences were observed in sex, smoking history, stage, radiotherapy combination, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, hemoglobin, and red blood cell distribution width between the high and low HRR groups. The median overall survival (OS) was nine and 17.5 months in the low and high HRR groups, respectively (P < 0.001). The median progression-free survival (PFS) was five and 8.5 months, respectively (P < 0.001). Univariate and multivariate analyses showed low HRR to be an independent predictor of a poor prognosis for OS (hazard ratio = 3.782; 95% confidence interval, 2.151-6.652; P < 0.001) and PFS (hazard ratio = 2.112; 95% confidence interval, 1.195-3.733; P = 0.01) in SCLC. CONCLUSION: Low HRR was associated with poorer OS and PFS in patients with SCLC and can be a potentially valuable prognostic factor for these patients. KEY POINTS: The prognostic value of the baseline hemoglobin-to-red blood cell distribution width ratio was evaluated in patients with small cell lung cancer. In this population, this ratio was an independent predictor of overall survival and progression-free survival. This ratio, an inexpensive and routine parameter, can be used as a prognostic factor in small cell lung cancer.

Circulating plasma lncRNAs as novel markers of EGFR mutation status and monitors of epidermal growth factor receptor-tyrosine kinase inhibitor therapy.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations predict tumor response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). However, even patients with EGFR-sensitive mutations in NSCLC have limited efficacy with EGFR-TKI. Studies have shown that long noncoding RNA (lncRNA) is related to diagnosis and prognosis with NSCLC. This study aimed to explore the correlation between lncRNA in NSCLC patients with EGFR mutation status and EGFR-TKI efficacy. METHODS: The amplification-refractory mutation system method was used to test the EGFR mutation status in tumor tissues and pleural effusions of NSCLC patients. Three EGFR-mutant patients and three EGFR wild-type patients were selected. Differential lncRNA was performed on the pleural effusions of the two selected groups of patients using Clariom D Human chip technology. Five lncRNAs significantly associated with EGFR mutation status were screened by FC value and GO analysis, and then evaluated by real-time quantitative polymerase chain reaction in NSCLC patients' pleural effusions. Three were further analyzed in NSCLC patients' plasma. RESULTS: There were 61 significant differences in lncRNA between EGFR mutation-positive and wild-type patients. Among them, SCARNA7, MALAT1, NONHSAT017369, NONHSAT051892, and FTH1P2 were significantly associated with EGFR mutation status. SCARNA7, MALAT1, and NONHSAT017369 showed consistent results with plasma in pleural effusions compared to EGFR wild-type, all upregulated in the EGFR mutation group. CONCLUSION: This study shows that lncRNAs can be used not only as potential biomarkers for predicting the mutation status of EGFR and the efficacy of EGFR-TKI, but also for monitoring the efficacy of EGFR-TKI.

Single-nucleotide polymorphisms (rs342275, rs342293, rs7694379, rs11789898, and rs17824620) showed significant association with lobaplatin-induced thrombocytopenia.

This study aimed to investigate single-nucleotide polymorphisms (SNPs) associated with lobaplatin-induced thrombocytopenia in patients with advanced lung cancer in China. Thirty-nine patients who received lobaplatin-based chemotherapy in the 307 Hospitals of Chinese People's Liberation Army from April 2017 to March 2018 were enrolled as study subjects. Peripheral blood DNA was extracted, and 79 candidate SNP positions were selected. A Sanger sequencing platform was employed to measure genotypes for locating the SNP positions associated with lobaplatin-induced thrombocytopenia. Of the 79 candidate genes, SNPs rs342275 and rs7694379 were significantly associated with lobaplatin-induced decrease in platelet (PLT) count (P < 0.05). SNPs rs342275, rs342293, rs11789898, and rs17824620 showed significant association with lobaplatin-induced lowest PLT counts (P < 0.05). SNPs rs342275, rs342293, rs11789898, rs17824620, and rs7694379 can be used as predictors of thrombocytopenia induced by lobaplatin-based chemotherapy in patients with advanced lung cancer in China.

Doxycycline Induces Apoptosis and Inhibits Proliferation and Invasion of Human Cervical Carcinoma Stem Cells.

BACKGROUND: Cancer stem cells (CSCs) are proposed to be responsible for high recurrence rate in cervical carcinoma. Reagents that can suppress the proliferation and differentiation of CSCs would provide new opportunities to fight against tumor recurrence. Doxycycline has been reported as a potential anti-cancer compound. However, few studies investigated its inhibitory effect against cervical cancer stem cells. METHODS: HeLa cells were cultured in cancer stem cell conditional media in a poly-hema-treated dish. In this non-adhesive culture system, HeLa cells were treated with cisplatin until some cells survived and formed spheroids, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every three days for five times. The tumor xenografts with CSC enrichment were cultured in cancer stem cell specific medium again to form tumorsphere, which we called HeLa-CSCs. Expression of cancer stem cell markers in HeLa-CSCs was measured by flow cytometry and qPCR. HeLa-CSCs were then treated with doxycycline. Proliferation and differentiation rates were determined by the size of spheres formed in vitro and tumor formed in vivo. RESULTS: We developed a new strategy to selectively enrich CSCs from human cervical carcinoma cell line HeLa, and these HeLa-CSCs are CD133+/CD49f+ cell populations with significantly enhanced expression of stem cell markers. When these HeLa-CSCs were treated with doxycycline, the colony formation, proliferation, migration and invasion, and differentiation were all suppressed. Meanwhile, stem cell markers SOX-2, OCT-4, NANOG, NOTCH and BMI-1 decreased in doxycycline treated cells, so as the surface markers CD133 and CD49f. Furthermore, proliferation markers Ki67 and PCNA were also decreased by doxycycline treatment in the in vivo xenograft mouse model. CONCLUSIONS: Cancer stem cells are enriched from sphere-forming and chemoresistant HeLa-derived tumor xenografts in immunodeficient mice. Doxycycline inhibits proliferation, invasion, and differentiation, and also induces apoptosis of these HeLa-CSCs in vitro and in vivo.

Preparation and structural evolution of SiO(2)-TiO(2) pillared layered manganese oxide nanocomposite upon intercalating reaction.

SiO(2)-TiO(2) pillared layered manganese oxide nanohybrid was successfully synthesized by preliminarily expanding the interlayer of H-type layered manganese oxide using dodecylamine, followed by reacting it with a mixture solution of titanium isopropoxide and tetraethylorthosilicate. The basal spacing and the pillared agent content of the obtained materials connected with the length of intercalated n-alkylamine, incorporated Si/Ti molar ratios and the solvothermal treatment temperature. The structural evolution of SiO(2)-TiO(2) pillared layered manganese oxide nanohybrid was characterized by XRD, DSC-TGA, SEM, IR, N(2) adsorption-desorption and element analyses. TiO(2) particles exhibited a stronger affinity for the negatively charged manganese layers, and the TiO(2) particles incorporated were independently intercalated without any distinct chemical bonding with the co-intercalated SiO(2) particles. SiO(2)-TiO(2) pillared layered manganese oxide nanohybrid had a BET surface area of 98 m(2)/g with a gallery height of about 1.43 nm between layers. The obtained SiO(2)-TiO(2) co-pillared layered manganese oxide nanohybrid is expected as a selective catalyst, or an improved battery material.