Transcriptomic and biochemical analysis of upland cotton (Gossypium hirsutum) and a chromosome segment substitution line from G. hirsutum × G. barbadense in response to Verticillium dahliae infection.

BACKGROUND: Verticillium wilt (VW), also known as "cotton cancer," is one of the most destructive diseases in global cotton production that seriously impacts fiber yield and quality. Despite numerous attempts, little significant progress has been made in improving the VW resistance of upland cotton. The development of chromosome segment substitution lines (CSSLs) from Gossypium hirsutum × G. barbadense has emerged as a means of simultaneously developing new cotton varieties with high-yield, superior fiber, and resistance to VW. RESULTS: In this study, VW-resistant investigations were first conducted in an artificial greenhouse, a natural field, and diseased nursery conditions, resulting in the identification of one stably VW-resistant CSSL, MBI8255, and one VW-susceptible G. hirsutum, CCRI36, which were subsequently subjected to biochemical tests and transcriptome sequencing during V991 infection (0, 1, and 2 days after inoculation). Eighteen root samples with three replications were collected to perform multiple comparisons of enzyme activity and biochemical substance contents. The findings indicated that VW resistance was positively correlated with peroxidase and polyphenol oxidase activity, but negatively correlated with malondialdehyde content. Additionally, RNA sequencing was used for the same root samples, resulting in a total of 77,412 genes, of which 23,180 differentially expressed genes were identified from multiple comparisons between samples. After Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the expression profiles identified using Short Time-series Expression Miner, we found that the metabolic process in the biological process, as well as the pathways of phenylpropanoid biosynthesis and plant hormone signal transduction, participated significantly in the response to VW. Gene functional annotation and expression quantity analysis indicated the important roles of the phenylpropanoid metabolic pathway and oxidation-reduction process in response to VW, which also provided plenty of candidate genes related to plant resistance. CONCLUSIONS: This study concentrates on the preliminary response to V991 infection by comparing the VW-resistant CSSL and its VW-susceptible recurrent parent. Not only do our findings facilitate the culturing of new resistant varieties with high yield and superior performance, but they also broaden our understanding of the mechanisms of cotton resistance to VW.

QTL mapping and genetic effect of chromosome segment substitution lines with excellent fiber quality from Gossypium hirsutum × Gossypium barbadense.

Chromosome segment substitution lines (CSSLs) are ideal materials for identifying genetic effects. In this study, CSSL MBI7561 with excellent fiber quality that was selected from BC4F3:5 of CCRI45 (Gossypium hirsutum) × Hai1 (Gossypium barbadense) was used to construct 3 secondary segregating populations with 2 generations (BC5F2 and BC5F2:3). Eighty-one polymorphic markers related to 33 chromosome introgressive segments on 18 chromosomes were finally screened using 2292 SSR markers which covered the whole tetraploid cotton genome. A total of 129 quantitative trait loci (QTL) associated with fiber quality (103) and yield-related traits (26) were detected on 17 chromosomes, explaining 0.85-30.35% of the phenotypic variation; 39 were stable (30.2%), 53 were common (41.1%), 76 were new (58.9%), and 86 had favorable effects on the related traits. More QTL were distributed in the Dt subgenome than in the At subgenome. Twenty-five stable QTL clusters (with stable or common QTL) were detected on 22 chromosome introgressed segments. Finally, the 6 important chromosome introgressed segments (Seg-A02-1, Seg-A06-1, Seg-A07-2, Seg-A07-3, Seg-D07-3, and Seg-D06-2) were identified as candidate chromosome regions for fiber quality, which should be given more attention in future QTL fine mapping, gene cloning, and marker-assisted selection (MAS) breeding.

Chemokine (C-X-C motif) receptor 3-positive B cells link interleukin-17 inflammation to protumorigenic macrophage polarization in human hepatocellular carcinoma.

UNLABELLED: B cells consistently represent abundant cellular components in tumors; however, direct evidence supporting a role for B cells in the immunopathogenesis of human cancers is lacking, as is specific knowledge of their trafficking mechanisms. Here, we demonstrate that chemokine (C-X-C motif) receptor 3-positive (CXCR3(+)) B cells constitute approximately 45% of B-cell infiltrate in human hepatocellular carcinoma (HCC) and that their levels are positively correlated with early recurrence of HCC. These cells selectively accumulate at the invading edge of HCC and undergo further somatic hypermutation and immunoglobulin G-secreting plasma cell differentiation. Proinflammatory interleukin-17(+) cells are important for the induction of epithelial cell-derived CXCR3 ligands CXCL9, CXCL10, and CXCL11, which subsequently promote the sequential recruitment and further maturation of CXCR3(+) B cells. More importantly, we provide evidence that CXCR3(+) B cells, but not their CXCR3(-) counterparts, may operate in immunoglobulin G-dependent pathways to induce M2b macrophage polarization in human HCC. Depletion of B cells significantly suppresses M2b polarization and the protumorigenic activity of tumor-associated macrophages and restores the production of antitumorigenic interleukin-12 by those cells in vivo. CONCLUSION: Selective recruitment of CXCR3(+) B cells bridges proinflammatory interleukin-17 response and protumorigenic macrophage polarization in the tumor milieu, and blocking CXCR3(+) B-cell migration or function may help defeat HCC.

Altered B cell immunoglobulin signature exhibits potential diagnostic values in human colorectal cancer.

Antibody-secreting B cells have long been considered the central element of gut homeostasis; however, tumor-associated B cells in human colorectal cancer (CRC) have not been well characterized. Here, we show that the clonotype, phenotype, and immunoglobulin subclasses of tumor-infiltrating B cells have changed compared to adjacent normal tissue B cells. Remarkably, the tumor-associated B cell immunoglobulin signature alteration can also be detected in the plasma of patients with CRC, suggesting that a distinct B cell response was also evoked in CRC. We compared the altered plasma immunoglobulin signature with the existing method of CRC diagnosis. Our diagnostic model exhibits improved sensitivity compared to the traditional biomarkers, CEA and CA19-9. These findings disclose the altered B cell immunoglobulin signature in human CRC and highlight the potential of using the plasma immunoglobulin signature as a non-invasive method for the assessment of CRC.

Exosomal transfer of p-STAT3 promotes acquired 5-FU resistance in colorectal cancer cells.

BACKGROUND: Acquired resistance remains a limitation of the clinical use of 5-fluorouracil (5-FU). Because exosomes, are important vesicles participating in intercellular communication, their contribution to the development of acquired 5-FU resistance needs to be elucidated. In this study, we aimed to examine the underlying mechanisms of exosomes from 5-FU resistant cells (RKO/R) in sustaining acquired 5-FU resistance in sensitive cells (RKO/P). METHODS: Exosomes from a 5-FU-resistant cell line (RKO/R) and its parental cell line RKO/P were isolated and co-cultured with 5-FU-sensitive cells. Real-time cellular analysis (RTCA) and FACS analysis were used to examine cell viability and apoptosis. Exosomal protein profiling was performed using shotgun proteomics. Inhibitors and siRNAs were applied to study the involvement of selected proteins in 5-FU resistance. The effect of exosomal p-STAT3 (Tyr705) on the caspase cascade was examined by western blotting (WB) and high content analysis. Xenograft models were established to determine whether exosomal p-STAT3 can induce 5-FU resistance in vivo. RESULTS: Our results indicated that exosomes from RKO/R cells significantly promoted cell survival during 5-FU treatment. Proteomics and WB analysis results indicated that GSTP1 and p-STAT3 (Tyr705) were enriched in exosomes from RKO/R cells. Inhibition of p-STAT3 re-sensitized RKO/P cells to 5-FU via caspase cascade. Furthermore, p-STAT3 packaged by exosomes from RKO/R cells increased resistance of tumor cells to 5-FU in vivo. CONCLUSIONS: Our results reveal a novel mechanism by which p-STAT3-containing exosomes contribute to acquired 5-FU resistance in CRC. This study suggests a new option for potentiating the 5-FU response and finding biomarkers for chemotherapy resistance.

[Effects of p-hydroxybenzoic acid and phloroglucinol on mitochondria function and root growth in cotton (Gossypium hirsutum L.) seedling roots.] / å¯¹ç¾ŸåŸºè‹¯ç”²é ¸å’Œé—´è‹¯ä¸‰é šå¯¹æ£‰èŠ±å¹¼è‹—æ ¹ç³»çº¿ç²’ä½“åŠŸèƒ½å’Œæ ¹ç³»ç”Ÿé•¿çš„å½±å“.

With early-maturing cotton cultivar CCRI-50 widely grown in China as experimental material, water culture experiment was conducted to study the effects of p-hydroxybenzoic acid and phloroglucinol with different concentrations (0.8, 4.0, and 20.0 mmol·L-1) on generation rate of reactive oxygen, changes of antioxidant enzyme activities and mitochondria function of cotton roots. Results showed that p-hydroxybenzoic acid and phloroglucinol treatments inhibited the cotton root growth, reduced SOD, POD, CAT and H+-ATPase activities in root mitochondria, increased the generation rate of O2-· and H2O2 content. In addition, they also increased the opening of mitochondrial permeability transition pores (MPTP), decreased the membrane fluidity and cytochrome c/a (Cyt c/a). Difference of mitochondria function between p-hydroxybenzoic acid and phloroglucinol treatments was minor at concentration of 0.8 mmol·L-1, while the inhibition to root growth and mitochondria function under treatment of p-hydroxybenzoic acid at concentration of 4.0 and 20.0 mmol·L-1 was stronger than that of phloroglucinol. Above all, p-hydroxybenzoic acid and phloroglucinol inhibited antioxidant enzyme activity and mitochondrial function in cotton seedling roots, and the inhibition depended on dose of phenolic acids. The inhibition to root growth and mitochondria function between p-hydroxybenzoic acid and phloroglucinol treatment was different, and p-hydroxybenzoic acid had stronger inhibition than phloroglucinol at the concentration more than 4.0 mmol·L-1.

[Effects of nitrogen application rates on nitrogen uptake and utilization of direct-seeded cotton after wheat harvest].

An experiment was carried out to study the effects of different nitrogen application rates (0, 60, 120, 150, 180 and 240 kg N · hm⁻²) on the nitrogen uptake, utilization and distribution of short season cotton cultivar (CCRI-50) which was directly seeded after wheat harvest. Results showed that nitrogen application increased the nitrogen uptake of cotton at different growth stages, with the highest increment at the peak flowering-boll opening stage. Nitrogen application changed the percentages of nitrogen uptake among different growth stages. The percentages of nitrogen uptake decreased from seedling to peak flowering stage, but increased from peak flowering to boll maturing stage. In addition, nitrogen application reduced the decreasing speed of nitrogen concentration in middle and upper fruiting branches at later growth stages. Direct-seeded cotton had nitrogen and biomass accumulation in the lower and middle reproductive branches. With the nitrogen application of 150-180 kg · hm⁻², the lint yield, NARE (nitrogen apparent recovery efficiency) and the economic coefficient of biomass and nitrogen were relatively higher, and the eigenvalues of dynamic model of nitrogen content and nitrogen accumulation were relatively coordinate. Excessively high nitrogen application (over 180 kg N · hm⁻²) decreased biomass and nitrogen amount of reproductive organ in lower and middle branches, narrow rise of yield, and lower nitrogen use efficiency. However, excessively low nitrogen application (lower than 150 kg N · hm⁻²) also resulted in lower economic coefficient of biomass and nitrogen and yield. These results suggested the optimum nitrogen application rate 150-180 kg N · hm⁻² for direct-seeded cotton after wheat harvest in lower reaches of Yangtze River.

[Effects of nitrogen application rate on potassium uptake and utilization of direct-seeded cotton after wheat harvest]. / æ–½æ°®é‡å¯¹éº¦åŽç›´æ’­æ£‰é’¾ç´ å¸æ”¶åˆ©ç”¨çš„å½±å“.

By using cotton cultivar CCRI-50 as material, field experiments were conducted in the summer seasons of 2013 and 2014 at the experimental station of Jiangsu Academy of Agricultural Sciences (Nanjing, China) to study the effects of different nitrogen application rates (0, 60, 120, 150, 180 and 240 kg N·hm-2) on the potassium uptake and utilization of the cotton plant that was direct-seeded after wheat harvest. Data suggested that the elevated nitrogen application rates increased the cotton potassium uptake of all growth stages, and the largest increment was observed at the peak flowering-boll opening stage. Nitrogen application also changed the uptake percentage of potassium uptake of each stage, i.e., the percentage of potassium uptake decreased in the stage from seedling to peak flowering, while increased in the stage from peak flowering to boll maturing. In addition, the elevated nitrogen applications reduced the decreasing rate of nitrogen concentration in upper fruiting branches, but promoted the decreasing rate in middle and low fruiting branches at later growth stages. As the nitrogen application rate increased, the marginal effect of potassium uptake (promoted amount of potassium uptake due to 1 kg increase of N application) increased first and then decreased, and the lint production efficiency of potassium descended steadily. In cotton plants that were direct-seeded after wheat harvest, potassium and biomass were mainly accumulated in the lower and middle fruiting branches. At the 150 and 180 kg N·hm-2 application levels, much more potassium was allocated to the reproductive organs and the characters and the eigenvalues of simulated curves of potassium concentration and total potassium accumulation were more optimized than those at the higher or the lower N application levels. At the high nitrogen application (more than 180 kg N·hm-2) level, the marginal effect of potassium uptake and lint production efficiency decreased, and at the lower nitrogen application (less than 150 kg N·hm-2) level, lint yield was lower due to the decrease of economic coefficient of biomass and potassium in the middle and low fruiting branches.

Dendritic cell-elicited B-cell activation fosters immune privilege via IL-10 signals in hepatocellular carcinoma.

B cells are prominent components of human solid tumours, but activation status and functions of these cells in human cancers remain elusive. Here we establish that over 50% B cells in hepatocellular carcinoma (HCC) exhibit an FcγRIIlow/- activated phenotype, and high infiltration of these cells positively correlates with cancer progression. Environmental semimature dendritic cells, but not macrophages, can operate in a CD95L-dependent pathway to generate FcγRIIlow/- activated B cells. Early activation of monocytes in cancer environments is critical for the generation of semimature dendritic cells and subsequent FcγRIIlow/- activated B cells. More importantly, the activated FcγRIIlow/- B cells from HCC tumours, but not the resting FcγRIIhigh B cells, without external stimulation suppress autologous tumour-specific cytotoxic T-cell immunity via IL-10 signals. Collectively, generation of FcγRIIlow/- activated B cells may represent a mechanism by which the immune activation is linked to immune tolerance in the tumour milieu.

Polarization of Tissue-Resident TFH-Like Cells in Human Hepatoma Bridges Innate Monocyte Inflammation and M2b Macrophage Polarization.

The existence, regulation, and functions of IL21+ immune cells are poorly defined in human cancers. Here, we identified a subset of protumorigenic IL21+ TFH-like cells in human hepatocellular carcinoma. These cells were the major source of IL21 in tumors and represented about 10% of the CD4+ T-cell population at levels comparable with the TFH cells present in lymph nodes. However, these TFH-like cells displayed a unique CXCR5-PD-1lo/-BTLA-CD69hi tissue-resident phenotype with substantial IFNγ production, which differed from the phenotype of TFH cells. Toll-like receptor 4 (TLR4)-elicited innate monocyte inflammation was important for IL21+ TFH-like cell induction in tumors, and activation of STAT1 and STAT3 was critical for TFH-like cell polarization in this process. Importantly, the TFH-like cells operated in IL21-IFNγ-dependent pathways to induce plasma cell differentiation and thereby create conditions for protumorigenic M2b macrophage polarization and cancer progression. Thus, induction of TFH-like cells links innate inflammation to immune privilege in tumors. SIGNIFICANCE: We identified a novel protumorigenic IL21+ TFH-like cell subset with a CXCR5-PD-1- BTLA-CD69hi tissue-resident phenotype in hepatoma. TLR4-mediated monocyte inflammation and subsequent T-cell STAT1 and STAT3 activation are critical for TFH-like cell induction. TFH-like cells operate via IL21-IFNγ pathways to induce plasma cells and create conditions for M2b macrophage polarization. Cancer Discov; 6(10); 1182-95. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.

PD-1hi Identifies a Novel Regulatory B-cell Population in Human Hepatoma That Promotes Disease Progression.

UNLABELLED: B cells often constitute abundant cellular components in human tumors. Regulatory B cells that are functionally defined by their ability to produce IL10 downregulate inflammation and control T-cell immunity. Here, we identified a protumorigenic subset of B cells that constitutively expressed higher levels of programmed cell death-1 (PD-1) and constituted ∼10% of all B cells in advanced-stage hepatocellular carcinoma (HCC). These PD-1(hi) B cells exhibited a unique CD5(hi)CD24(-/+)CD27(hi/+)CD38(dim) phenotype different from the phenotype of conventional CD24(hi)CD38(hi) peripheral regulatory B cells. TLR4-mediated BCL6 upregulation was crucial for PD-1(hi) B-cell induction by HCC environmental factors, and that effect was abolished by IL4-elicited STAT6 phosphorylation. Importantly, upon encountering PD-L1(+) cells or undergoing PD-1 triggering, PD-1(hi) B cells acquired regulatory functions that suppressed tumor-specific T-cell immunity and promoted cancer growth via IL10 signals. Our findings provide significant new insights for human cancer immunosuppression and anticancer therapies regarding PD-1/PD-L1. SIGNIFICANCE: We identify a novel protumorigenic PD-1(hi) B-cell subset in human HCC that exhibits a phenotype distinct from that of peripheral regulatory B cells. TLR4-mediated BCL6 upregulation is critical for induction of PD-1(hi) B cells, which operate via IL10-dependent pathways upon interacting with PD-L1 to cause T-cell dysfunction and foster disease progression. Cancer Discov; 6(5); 546-59. ©2016 AACR.See related commentary by Ren et al., p. 477This article is highlighted in the In This Issue feature, p. 461.

[Effects of drought and waterlogging on carbohydrate contents of cotton boll and its relationship with boll biomass accumulation at the flowering and bolling stage].

Cotton cultivar NuCOTN 33B was planted in isolated pools treated with drought or waterlogging for 7 or 14 d to explore their effects on cotton boll carbohydrate content and its relationship with the biomass accumulation. The results showed that the drought treatment reduced the carbohydrate content of cotton boll shell on middle fruit branches, but had a weak effect on cotton boll shells on lower fruit branches. Soluble sugar, starch and sucrose contents of cotton boll shell on upper fruit branches under the drought condition and on whole plant branches under waterlogging treatment changed similarly, namely, the soluble sugar and starch content increased, while the sucrose content went down firstly and then increased later, which indicated that the exportation of sucrose from boll shell was inhibited and became worse with the increase of waterlogging duration. Compared with the boll shell, the carbohydrate contents of cotton seed were less affected by the drought and waterlogging treatments at the flowering and bolling stage. Under the treatments of drought and 7 d-waterlogging, the biomass accumulation of cotton bolls on the middle fruit branches initiated earlier but lasted less days, and the maximum speed at lower and upper fruit branches reduced, while the treatment of waterlogging for 14 d caused the decline of maximum speed of biomass accumulation of bolls on whole branches. On the other side, the correlation analysis showed the significant positive relationships among the boll biomass, the maximum speed and the contents of soluble sugar and sucrose in the boll shell respectively. In conclusion, the treatment of drought and waterlogging at the flowering and bolling stage retarded the outward transportation of sucrose from cotton bolls, changed the boll biomass accumulation characteristics, and therefore were detected as the important cause of cotton boll total biomass reduction.

B7-H1-expressing antigen-presenting cells mediate polarization of protumorigenic Th22 subsets.

Classical IL-22-producing T helper cells (Th22 cells) mediate inflammatory responses independently of IFN-γ and IL-17; however, nonclassical Th22 cells have been recently identified and coexpress IFN-γ and/or IL-17 along with IL-22. Little is known about how classical and nonclassical Th22 subsets in human diseases are regulated. Here, we used samples of human blood, normal and peritumoral liver, and hepatocellular carcinoma (HCC) to delineate the phenotype, distribution, generation, and functional relevance of various Th22 subsets. Three nonclassical Th22 subsets constituted the majority of all Th22 cells in human liver and HCC tissues, although the classical Th22 subset was predominant in blood. Monocytes activated by TLR2 and TLR4 agonists served as the antigen-presenting cells (APCs) that most efficiently triggered the expansion of nonclassical Th22 subsets from memory T cells and classical Th22 subsets from naive T cells. Moreover, B7-H1-expressing monocytes skewed Th22 polarization away from IFN-γ and toward IL-17 through interaction with programmed death 1 (PD-1), an effect that can create favorable conditions for in vivo aggressive cancer growth and angiogenesis. Our results provide insight into the selective modulation of Th22 subsets and suggest that strategies to influence functional activities of inflammatory cells may benefit anticancer therapy.

[Effects of nitrogen application rate on cotton leaf antioxidant enzyme activities and endogenous hormone contents under short-term waterlogging at flowering and boll-forming stage].

A pot experiment was conducted to study the effects of applying 0, 240, and 480 kg N x hm(-2) on the cotton leaf antioxidant enzyme activities and endogenous hormone contents under eight days waterlogging at flowering and boll-forming stage. Under this short-term waterlogging, the leaf soluble protein content and SOD and CAT activities decreased while the MDA content and POD activity increased, and in the meantime, the leaf ABA content increased while the ZR, GA, and IAA contents as well as the ZR/ABA, IAA/ABA, and GA/ABA ratios decreased, compared with those under normal watering. Treatment 240 kg N x hm(-2) had the lowest contents of leaf MDA and ABA, but the highest contents of leaf ZR, GA, and IAA, highest ratios of ZR/ABA, GA/ABA, and IAA/ABA, and highest net photosynthetic rate. At the 15th day after terminating waterlogging, no significant differences in the leaf antioxidant enzyme activities, MDA content, and endogenous hormone contents were observed between the short-term waterlogged and well-watered cotton. N application promoted the cotton leaf antioxidant enzyme activities, decreased the MDA and ABA contents, and increased the ZR, GA, and IAA contents as well as the ZR/ABA, IAA/ABA, and GA/ ABA ratios, with the highest cotton biomass and yield in treatment 240 kg N x hm(-2).

[Physiological responses of cotton plant to fertlizer nitrogen at flowering and boll-forming stages under soil drought].

With pot culture, the physiological responses of cotton plant to fertilizer nitrogen at flowering and boll-forming stages were studied under soil drought and after re-watering. The results showed that under soil drought, the net photosynthetic rate (Pn) and transpiration rate (Tr) declined rapidly with decreasing soil relative water content (SRWC). At the early stage of soil drought, owing to the declining degree of Tr was greater than that of Pn, the WUE had an increasing trend; but after then, the WUE decreased with declining SRWC and Pn. Soil drought altered the diurnal patters of Pn and Tr, i.e., decreased continuously from 8:00 to 16:00, while in CK, their peak values appeared at 10:00-11:00 and 12:00, respectively. The diurnal patterns of WUE in drought treatment and CK were the same, i.e., decreased first and increased then, with the bottom appeared at 12:00. Under soil drought, the Pn and Tr decreased with increasing fertilizer nitrogen level, while WUE was in adverse. After re-watering, the diurnal patterns of Pn, Tr and WUE were similar to CK, but their values were smaller than CK, especially under nitrogen application. It was suggested that under soil drought, nitrogen fertilization could be helpful to the increase of water use efficiency, but decreased the photosynthesis of cotton plant at its flowering and boll-forming stages.