Quantitative Proteomics Analysis of Lettuce (Lactuca sativa L.) Reveals Molecular Basis-Associated Auxin and Photosynthesis with Bolting Induced by High Temperature.

Bolting is a key process in the growth and development of lettuce (Lactuca sativa L.). A high temperature can induce early bolting, which decreases both the quality and production of lettuce. However, knowledge of underlying lettuce bolting is still lacking. To better understand the molecular basis of bolting, a comparative proteomics analysis was conducted on lettuce stems, during the bolting period induced by a high temperature (33 °C) and a control temperature (20 °C) using iTRAQ-based proteomics, phenotypic measures, and biological verifications using qRT-PCR and Western blot. The high temperature induced lettuce bolting, while the control temperature did not. Of the 5454 identified proteins, 619 proteins presented differential abundance induced by high-temperature relative to the control group, of which 345 had an increased abundance and 274 had a decreased abundance. Proteins with an abundance level change were mainly enriched in pathways associated with photosynthesis and tryptophan metabolism involved in auxin (IAA) biosynthesis. Moreover, among the proteins with differential abundance, proteins associated with photosynthesis and tryptophan metabolism were increased. These findings indicate that a high temperature enhances the function of photosynthesis and IAA biosynthesis to promote the process of bolting, which is in line with the physiology and transcription level of IAA metabolism. Our data provide a first comprehensive dataset for gaining novel understanding of the molecular basis underlying lettuce bolting induced by high temperature. It is potentially important for further functional analysis and genetic manipulation for molecular breeding to breed new cultivars of lettuce to restrain early bolting, which is vital for improving vegetable quality.

Comparative Proteomic Analysis of Posterior Silk Glands of Wild and Domesticated Silkworms Reveals Functional Evolution during Domestication.

The wild silkworm Bombyx mandarina was domesticated to produce silk in China approximately 5000 years ago. Silk production is greatly improved in the domesticated silkworm B. mori, but the molecular basis of the functional evolution of silk gland remains elusive. We performed shotgun proteomics with label-free quantification analysis and identified 1012 and 822 proteins from the posterior silk glands (PSGs) of wild silkworms on the third and fifth days of the fifth instar, respectively, with 128 of these differentially expressed. Bioinformatics analysis revealed that, with the development of the PSG, the up-regulated proteins were mainly involved in the ribosome pathway, similar to what we previously reported for B. mori. Additionally, we screened 50 proteins with differential expression between wild and domesticated silkworms that might be involved in domestication at the two stages. Interestingly, the up-regulated proteins in domesticated compared to wild silkworms were enriched in the ribosome pathway, which is closely related to cell size and translation capacity. Together, these results suggest that functional evolution of the PSG during domestication was driven by reinforcing the advantageous pathways to increase the synthesis efficiency of silk proteins in each cell and thereby improve silk yield.

Prevention of in-stent restenosis with endothelial progenitor cell (EPC) capture stent placement combined with regional EPC transplantation: An atherosclerotic rabbit model.

BACKGROUND: Even with drug-eluting stents, the risk of in-stent restenosis (ISR) remains high. The goal of this study was to investigate the use of an endothelial progenitor cell (EPC) capture stent plus regional EPC transplantation to reduce the ISR rate. METHODS: Endothelial progenitor cell capture stents were fabricated using fibrin gel and anti-CD34 plus anti-VEGFR-2 dual antibodies. Twenty male New Zealand white rabbits established as an atherosclerotic model were randomly divided into two groups: group 1 (n = 10), in which EPC capture stents were deployed into the right iliac artery; and group 2 (n = 10), in which sirolimus-eluting stents were placed. In both groups, EPCs were transplanted into target vessels beyond the stents, with outflow blocked. Radiologic-pathologic correlation outcomes were reviewed after 2 months. RESULTS: The technical success rate of EPC capture stent placement plus EPC transplantation was 100%. The ISR rate in group 1 was lower than in group 2 (1/10 vs. 4/10; p > 0.05). Minimal luminal diameters were larger in group 1 than in group 2 (computed tomographic angiography, 1.85 ± 0.15 mm vs. 1.50 ± 0.20 mm; duplex ultrasound, 1.90 ± 0.10 mm vs. 1.70 ± 0.30 mm; p > 0.05). Transplanted EPCs were tracked positively only in group 1. Pathologic analysis demonstrated neointimal hyperplasia thickness of 0.21 ± 0.09 mm in group 1 vs. 0.11 ± 0.07 mm in group 2 (p < 0.05). CONCLUSION: Endothelial progenitor cell capture stent placement plus local EPC transplant decreases the ISR rate through thrombosis reduction rather than through neointimal hyperplasia inhibition.

Real-time observation of nucleoplasmin-mediated DNA decondensation and condensation reveals its specific functions as a chaperone.

Fertilization requires decondensation of promatine-condensed sperm chromatin, a dynamic process serving as an attractive system for the study of chromatin reprogramming. Nucleoplasmin is a key factor in regulating nucleosome assembly as a chaperone during fertilization process. However, knowledge on nucleoplasmin in chromatin formation remains elusive. Herein, magnetic tweezers (MT) and a chromatin assembly system were used to study the nucleoplasmin-mediated DNA decondensation/condensation at the single-molecular level in vitro. We found that protamine induces DNA condensation in a stepwise manner. Once DNA was condensed, nucleoplasmin, polyglutamic acid, and RNA could remove protamine from the DNA at different rates. The affinity binding of the different polyanions with protamine suggests chaperone-mediated chromatin decondensation activity occurs through protein-protein interactions. After decondensation, both RNA and polyglutamic acid prevented the transfer of histones onto the naked DNA. In contrast, nucleoplasmin is able to assist the histone transfer process, even though it carries the same negative charge as RNA and polyglutamic acid. These observations imply that the chaperone effects of nucleoplasmin during the decondensation/condensation process may be driven by specific spatial configuration of its acidic pentamer structure, rather than by electrostatic interaction. Our findings offer a novel molecular understanding of nucleoplasmin in sperm chromatin decondensation and subsequent developmental chromatin reprogramming at individual molecular level.

Microsatellite analysis of royal jelly producing traits of Italian honeybee (Apis mellifera Liguatica).

Genetic variations at 10 microsatellite loci were surveyed to determine the evolutionary relationships and molecular characteristics of three different honeybee (Apis mellifera L.) populations from Italy and China, i. e., native Italian (Ee), Chinese-Italian (Eb) and selected high royal jelly producing bees (Ea). A total of 96 alleles,an average of 9.6 alleles per locus,were scored in Ee,Eb and Ea bees at 10 loci. Out of which 48 (5%) were different. This indicated a high degree of polymorphism and ever, some genetic differentiation among the three populations due to artificial selection and geographical isolation. The polymorphic information contents (PIC) and heterozyosity of the three populations at 10 loci were 0.57, 0.50, 0.57, and 0.60, 0.57, 0.61, for Ee, Eb, Ea populations respectively, neither of which were different. This indicated same gene diversity within the three populations. The genetic distance was shorter between Ee and Eb bees as well as between Eb and Ea bees. Whereas that between Ee and Eb bees was longer. Further analysis indicated that the allele frequency of seven alleles at six loci (159 bp at A29,100 bp and 104 bp at A24; 110 bp at A7; 126 bp at A43, 221 bp at A14 and 221 bp at A113) increased going from Ee to Eb to Ea bees. Paired tests showed significant higher allele frequency between Ea and Eb bees,as well as between Ea and Eb bees. This indicates that these seven alleles are likely molecular markers of the high royal jelly producing bees. In addition,the allele frequency of four alleles at four loci (106 bp at A24,140 bp at A43;215 bp at A113 and 219 bp at A14) decreased going from Ea bees to Eb to Ee. Paired tests indicated significant lower allele frequency between Ea and Ee bees,as well as between Ea and Eb bees. Those four alleles may be the genetic markers for low royal jelly production.

[Genetic analysis for developmental behavior of honeybee colony's royal jelly production traits in western honeybees].

Analyses of genotypic effects for colony's royal jelly (RJ) yields and RJ in each queen cell as well as acceptances of queen cell cups of three lines of Western honeybees (Apis melliffera Lingistica) were conducted by using a genotypic model for analyzing the genetic and non-genetic effects. Analytis approaches of conventional and conditional variance and correlation were employed to evaluate the developmental behavior of honeybee colony's RJ producing ability. The results indicated that significant variance due to genotypic effect was detected for colony's RJ yields and RJ in each queen cell cup at all stages, while the acceptance of queen cell cups was found variance significantly at 10 stages of its total 11. It meant that the three traits were dominated by genotypic effect. Significant conditional variances were found at some stages when no unconditional one being detected. Correlation analysis between same trait at different stages indicated that the significant coefficients always existed due to genotypic effects for RJ yields and RJ in each queen cell cup. Although the significant coefficients of the acceptance of queen cell cup at different stages were found at most of the stages, the coefficients were not found in come of the stages. These results indicated that the genotypic effects dominated in early stages of colony's RJ yield and RJ in each queen cell cup influencing its late stages in the same way, but the acceptance of queen cells cup was not. The analysis of correlation between different pair of traits showed that the coefficients between colony's RJ yields and RJ in each queen cell cup were significant at all stages, and while the coefficients between colony's RJ yields and the acceptance of queen cell cups were not always significant at all stages. It was because the genotypic effects dominated the former pair of traits had harmonious effects, while the later pair of traits were not.

Effects of sulfhydryl compounds, carbohydrates, organic acids, and sodium sulfite on the formation of lysinoalanine in preserved egg.

To identify inhibitors for lysinoalanine formation in preserved egg, sulfhydryl compounds (glutathione, L-cysteine), carbohydrates (sucrose, D-glucose, maltose), organic acids (L-ascorbic acid, citric acid, DL-malic acid, lactic acid), and sodium sulfite were individually added at different concentrations to a pickling solution to prepare preserved eggs. Lysinoalanine formation as an index of these 10 substances was determined. Results indicate that glutathione, D-glucose, maltose, L-ascorbic acid, citric acid, lactic acid, and sodium sulfite all effectively diminished lysinoalanine formation in preserved egg albumen and yolk. When 40 and 80 mmol/L of sodium sulfite, citric acid, L-ascorbic acid, and D-glucose were individually added into the pickling solution, the inhibition rates of lysinoalanine in the produced preserved egg albumen and yolk were higher. However, the attempt of minimizing lysinoalanine formation was combined with the premise of ensuring preserved eggs quality. Moreover, the addition of 40 and 80 mmol/L of sodium sulfite, 40 and 80 mmol/L of D-glucose, 40 mmol/L of citric acid, and 40 mmol/L of L-ascorbic acid was optimal to produce preserved eggs. The corresponding inhibition rates of lysinoalanine in the albumen were approximately 76.3% to 76.5%, 67.6% to 67.8%, 74.6%, and 74.6%, and the corresponding inhibition rates of lysinoalanine in the yolk were about 68.7% to 69.7%, 50.6% to 51.8%, 70.4%, and 57.8%. It was concluded that sodium sulfite, D-glucose, L-ascorbic, and citric acid at suitable concentrations can be used to control the formation of lysinoalanine during preserved egg processing.

Proteomics analysis of major royal jelly protein changes under different storage conditions.

Protein changes in fresh royal jelly (RJ) were compared when stored at -20, 4 degrees C, and room temperature (RT) for 12 months. Protein was partially identified using combinations of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS), gel filtration chromatography, nanoLC MS/MS, and a protein engine identification tool applied to the honeybee genome. Significantly more protein spots were found in fresh (85 spots) and -20 degrees C (81 spots) stored RJ than in samples stored at 4 degrees C (73 spots) and at RT (70 spots) for 1 year. Most identified spots, 56, 57, 51, 46, corresponding to RJ sample of the fresh, -20 degrees C, 4 degrees C, and RT, were assigned to major royal jelly proteins (MRJPs). Marked differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. The quantity of MRJP1 decreased significantly following the temperature trend in all images, but MRJP 2 and -3 did not increase or decrease following the temperature trend, thus, suggesting that MRJP 1-3 are sensitive to temperature. However, MRJP4, 5, glucose oxidase (GOD), peroxiredoxin (PRDX), and glutathione S-transferase (GST) S1 were clearly absent in all images in samples held at RT for 1 year. This indicates that they are the proteins most sensitive to storage temperature and protein markers for freshness of RJ. Combining chromatography and nanoLC MS/MS results, we tentatively conclude that MRJP5 is a reliable freshness marker and that the best way to maintain quality of RJ is under freezing conditions.

Possible effect of 30K proteins in embryonic development of silkworm Bombyx mori.

The silkworm Bombyx mori possesses a 30K protein family of 3x10(4) Da, the biological functions of which have not been fully identified. The relationship between the 30K protein family and the embryonic development of temperature sensitive sex-linked mutant strain of silkworm was investigated by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results show that protein spots 1-5 of the 30K protein family, mainly existing in normal strain, are possibly related to embryonic development. The early consumption of a 30K protein named 6G1-30K-1 and the accumulation of 30K proteins named 6G1-30K-3 and 6G1-30K-4 are likely caused by the destruction of physiological balance in normal embryonic development, which may lead to lower hatchability of the temperature sensitive strain. The results suggest that reasonable metabolism of 30K proteins is a prerequisite for the embryo's normal development.