蛹虫草原基分化的差异蛋白质组学研究

蛹虫草子实体形成及发育的蛋白分子机制尚不清楚,本研究引入SWATH非标记定量蛋白质组学技术,对蛹虫草Cordyceps militaris 905菌株的菌丝体（mycelium,My）、原基（primordium,Po）、生长期子实体（developmental fruiting body,DF）和成熟期子实体（mature fruiting body,MF）进行了比较蛋白质组学分析。经搜库比对,从蛹虫草的My、Po、DF和MF中依次鉴定蛋白1 136个、1 090个、1 018个和997个（global FDR 1%）,经维恩分析后获得C. militaris 905蛹虫草表达蛋白1 578个。在此基础上,SWATH非标记技术定量蛋白1 109个。本研究获得了蛹虫草Po期与My期、DF期与Po期、MF期与DF期的差异表达蛋白,依次为115个、352个和104个,并对菌丝体分化形成原基的差异表达蛋白进行了重点解析。GO注释结果表明,Po期与My期差异表达蛋白以有机含氮类化合物代谢为主,其中AMP（活性成分虫草素合成的中间产物）从头生物合成途径富集最为显著。约1/5的差异表达蛋白参与氧化还原反应,还原酶活性的蛋白在原基中几乎都上调表达,而氧化功能的蛋白受到抑制,表明蛹虫草原基分化可能受到氧化应激的诱导。蛋白互作网络分析结果进一步表明,氧化还原反应与核苷类物质代谢相关联,可能通过影响AMP从头生物合成途径来调控虫草素的生物合成。对蛹虫草子实体系统的蛋白质组学研究和解析有利于揭示子实体形成的蛋白分子机制,为蛹虫草的基础和栽培研究提供了理论支撑。     

Genetic control of multiple births in low ovulating mammalian species

In mammals, litter size is a highly variable trait. Some species such as humans or cattle are monotocous, with one or sometimes two newborns per birth, whereas others, the polytocous species such as mice or pigs, are highly prolific and often produce a dozen newborns at each farrowing. In monotocous species, however, two or three newborns per birth may sometime be unwanted. In more polytocous species such as sheep or pigs, litter size is studied in order to increase livestock prolificacy. By contrast, twinning rates in humans or cattle may increase birth difficulties and health problems in the newborns. In this context, the aim of our review was to provide a clearer understanding of the genetic and physiological factors that control multiple births in low-ovulating mammalian species, with particular focus on three species: sheep, cattle, and humans, where knowledge of the ovulation rate in one may enlighten findings in the others. This article therefore reviews the phenotypic and genetic variability observed with respect to ovulation and twinning rates. It then presents the QTL and major genes that have been identified in each species. Finally, we draw a picture of the diversity of the physiological mechanisms underlying multiple ovulation. Although several major genes have been discovered in sheep, QTL detection methods in humans or cattle have suggested that the determinism of litter size is complex and probably involves several genes in order to explain variations in the number of ovulations.     

Genetic control of multiple births in low ovulating mammalian species

In mammals, litter size is a highly variable trait. Some species such as humans or cattle are monotocous, with one or sometimes two newborns per birth, whereas others, the polytocous species such as mice or pigs, are highly prolific and often produce a dozen newborns at each farrowing. In monotocous species, however, two or three newborns per birth may sometime be unwanted. In more polytocous species such as sheep or pigs, litter size is studied in order to increase livestock prolificacy. By contrast, twinning rates in humans or cattle may increase birth difficulties and health problems in the newborns. In this context, the aim of our review was to provide a clearer understanding of the genetic and physiological factors that control multiple births in low-ovulating mammalian species, with particular focus on three species: sheep, cattle, and humans, where knowledge of the ovulation rate in one may enlighten findings in the others. This article therefore reviews the phenotypic and genetic variability observed with respect to ovulation and twinning rates. It then presents the QTL and major genes that have been identified in each species. Finally, we draw a picture of the diversity of the physiological mechanisms underlying multiple ovulation. Although several major genes have been discovered in sheep, QTL detection methods in humans or cattle have suggested that the determinism of litter size is complex and probably involves several genes in order to explain variations in the number of ovulations.

     

A high‐confidence reference dataset of differentially expressed proteins in elongating cotton fiber cells

In our previous study, we used a comparative proteomic approach based on 2DE to profile dynamic proteomes of cotton fibers and found 235 protein spots differentially expressed during the elongation process ranging from 5 to 25 days post‐anthesis. Of them, only 106 differentially expressed proteins (DEPs) were identified by MS due to database limitations at the time. In the present work, we successfully identified the remaining 129 DEPs from the same experimental system using high‐resolution MS with an updated database. Bioinformatic analysis revealed that proteins involved in carbohydrate and protein metabolism, transport, and redox homeostasis are the most abundant, and glycolysis was found to be the most significantly regulated process during fiber elongation. Our high‐confidence reference dataset, composed of 235 DEPs, provides a valuable resource for future studies on the molecular mechanism of cotton fiber elongation.     

Comparative Proteomic Analyses of the Liver in D‐Galactosamine‐Sensitized Mice Treated with Different Toll‐Like Receptor Agonists

Acute liver failure (ALF) is a severe consequence of abrupt hepatocyte injury and has lethal outcomes. Three toll‐like receptor agonists, including polyinosinic‐polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS), and cytosine‐phosphate‐guanine (CpG) DNA, cause acute and severe hepatitis, respectively, in D‐galactosamine (D‐GalN)‐sensitized mice. However, the molecular differences among three ALF models (LPS/D‐GalN, poly(I:C)/D‐GalN, and CpG DNA/D‐GalN), are unclear. Here, tandem mass tag based quantitative proteomic analyses of three ALF mouse models are performed. 52 common differentially expressed proteins (DEPs) are identified, in three ALF groups, compared to the control. Gene ontology analyses show that among the common DEPs, ten proteins are involved in immune system process, and 39 proteins in metabolic process. Among 80,195, and 23 specifically‐expressed proteins in poly(I:C)/D‐GalN, LPS/D‐GalN, and CpG DNA/D‐GalN groups, LPS/D‐GalN‐specific proteins are mostly distributed in the endoplasmic reticulum and more enriched in metabolic pathways, whereas poly (I:C)/D‐GalN‐specific proteins are mainly in the membrane and CpG DNA/D‐GalN‐specific proteins are related to the ribosome structural composition. In conclusion, the common and specific DEPs in three ALF mouse models at molecular level are identified; and determined a close‐to‐complete reference map of mouse liver proteins which will be useful for clinical diagnosis and treatment of liver failure in humans.     

Experimental demonstration that pre- and post-conceptional mechanisms influence sex ratio in mouse embryos

Previously we have demonstrated in two monotocous species (bovine and sheep), a relationship between time of insemination, moment of ovulation, and embryo sex ratio. Here, we have analyzed in a polytocous specie (mice) if in addition to pre-conceptional mechanisms, also post-conceptional ones affect the offspring sex ratio. To verify this hypothesis we carried out two experiments. In the first experiment, we analyzed the effect of mating dynamics on the sex ratio of mice with synchronic male and female embryo development. Females were mated before and after ovulation and sacrificed 13 days later for sex determination of embryos and reabsorptions. A decreased litter size, and an increased offspring sex ratio in matings occurring later in oestrus, supported the view that a biased sex ratio may occur as the result of behavioral differences between the populations of X- or Y-bearing spermatozoa. In the second experiment, embryos developmentally synchronic and asynchronic with the recipient female endometrium were transferred, and again, 13 days later, females were sacrificed for sex determination of embryos and reabsorptions. The male proportion per litter found, indicated that our developmentally asynchronic transfers favored a sex ratio disbalance at birth. When combined, these results become the first experimental evidence supporting the view that both pre- and post-conceptional mechanisms of sex ratio distortion in polytocous species are not mutually exclusive and both may explain, under different conditions, sex ratio deviations at birth.     

Quantitative proteomics identified 3 oxidative phosphorylation genes with clinical prognostic significance in gastric cancer

The aim of the present study was to explore the underlying mechanisms involved in gastric cancer (GC) formation using data‐independent acquisition (DIA) quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in biological metabolic processes in GC. Totally, 745 differentially expressed proteins (DEPs) were found in GC tissues vs. gastric normal tissues. Despite enormous complexity in the details of the underlying regulatory network, we find that clusters of proteins from the DEPs were mainly involved in 38 pathways. All of the identified DEPs involved in oxidative phosphorylation were down‐regulated. Moreover, GC possesses significantly altered biological metabolic processes, such as NADH dehydrogenase complex assembly and tricarboxylic acid cycle, which is mostly consistent with that in KEGG analysis. Furthermore the higher expression of UQCRQ, NDUFB7 and UQCRC2 were positively correlated with a better prognosis, implicating these proteins may as novel candidate diagnostic and prognostic biomarkers.     

金针菇成熟期和伸长期菌盖的转录组与蛋白组比较分析

菌盖是大型真菌的重要组成部分,也是其产生有性孢子的部位,但是其发育机制仍不明确。本研究以金针菇Flammulina filiformis为材料,采用转录组和蛋白组联合分析的方法,比较分析了金针菇成熟期和伸长期菌盖的差异基因与蛋白,并对其进行GO (gene ontology)功能聚类分析、KEGG (Kyoto encyclopedia of genes and genomes)富集分析和蛋白互作网络分析。本研究筛选到差异表达基因有1 391个,差异表达蛋白147个,均以上调表达为主。GO功能聚类分析结果表明,催化活性(catalytic activity)条目富集基因最多,其次是细胞组分(cell part)、细胞过程(cellular process)和细胞器(organelle)。KEGG富集分析结果表明,差异表达基因和蛋白主要富集在碳水化合物代谢通路(carbohydrate metabolism)和氨基酸代谢通路(amino acid metabolism)等。本研究选取了9个关键的差异表达基因,使用实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)对其表达量进行了验证。RT-qPCR验证结果与转录组测序结果相一致。蛋白互作网络分析表明,水解酶类、结构域类和转录调节类蛋白为互作网络的主要结点。本研究联合转录组、蛋白组测序数据,通过分析差异基因与蛋白,为深入了解金针菇菌盖发育机制提供数据参考。     

Isobaric tag for relative and absolute quantitation based quantitative proteomics reveals unique urinary protein profiles in patients with preeclampsia

Preeclampsia (PE) is one of the most significant pregnancy‐related hypertensive disorders. Currently, there are no useful markers to predict the onset of the condition in pregnant women. To provide further insights into the pathogenesis of PE and identify biomarkers of the condition, we used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics coupled with 2‐D LC‐MS/MS, to analyze urinary protein profiles from 7 PE patients and 7 normotensive pregnant women. A total of 294 proteins were abnormally expressed in PE patients. Of these, 233 were significantly down‐regulated and 61 proteins were significantly up‐regulated. Bioinformatics analysis using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, found that the most differentially expressed proteins (DEPs) were involved in coagulation and complement pathways, the renin‐angiotensin system and cell adhesion molecules (CAMs) pathways. We further validated three of the DEPs, including serotransferrin (TF) and complement factor B (CFB) by immunoblottingand serum paraoxonase/arylesterase 1 (PON1) by ELISA using 14 pairs of urine samples from PE patients and normal pregnant women. Taken together, our results provide the basis for further understanding the pathogenesis of PE and identifying predictive biomarkers.     

Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes

Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super‐SILAC‐based MS analysis on the exosomes secreted by three human HCC cell lines, including the non‐motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism‐centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism‐associated proteins via exosomes that differentiate them from non‐motile HCC cells.     

Human umbilical cord mesenchymal stem cells facilitate the up‐regulation of miR‐153‐3p,whereby attenuating MGO‐induced peritoneal fibrosis in rats

MiRNAs contribute greatly to epithelial to mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs), which is a crucial step in peritoneal fibrosis (PF). In this study, we tried to profile whether miRNA expression differences exist after human umbilical cord mesenchymal stem cells (hUCMSCs) treatment in PF rats and investigate the possible role of miR‐153‐3p involved in anti‐EMT process. We randomly assigned 34 rats into three groups: control group (Group Control), MGO‐induced PF rats (Group MGO) and hUCMSCs‐treated rats (Group MGO + hUCMSCs). MiRNA microarrays and real‐time PCR analyses were conducted in three groups. α‐SMA, Snail1 and E‐cadherin expression were detected by Western blot. Luciferase reporter assays were used to detect the effects of miR‐153‐3p overexpression on Snai1 in rat peritoneal mesothelial cells (RPMCs). We identified differentially expressed miRNAs related to EMT, in which miR‐153‐3p demonstrated the greatest increase in Group MGO + hUCMSCs. Transient cotransfection of miR‐153‐3p mimics with luciferase expression plasmids resulted in a significant repression of Snai1 3′‐untranslated region luciferase activity in RPMCs. These studies suggest that miR‐153‐3p is a critical molecule in anti‐EMT effects of hUCMSCs in MGO‐induced PF rats. MiR‐153‐3p might exert its beneficial effect through directly targeting Snai1.     

利用蛋白质组学研究新德里金属b-内酰胺酶1对鲍曼不动杆菌的影响

【目的】比较临床分离的亲缘关系近的多药耐药鲍曼不动杆菌MDR-ZJ06(blaNDM-1–)和ABC3229(blaNDM-1+)的差异蛋白质组,以期发现新德里金属?-内酰胺酶1(New Delhimetallo-β-lactamase-1,NDM-1)对鲍曼不动杆菌生长代谢的影响。【方法】利用2-DE联合MALDI-TOF MS/MS技术鉴定差异表达蛋白,并在GO注析的基础上,对差异蛋白进行通路分析、功能分类和富集分析,并作出蛋白与蛋白相互作用网络。【结果】发现ABC3299相对于MDR-ZJ06有51个差异表达蛋白,其中11个蛋白表达上调,40个蛋白表达下调,并且这些差异蛋白主要涉及降低碳代谢、氨基酸代谢、脂肪酸代谢和细胞壁合成,增加铁离子转运系统形成。【结论】这个结果揭示了NDM-1可能是通过减缓细菌自身的代谢,增加自身铁的摄取使细菌机体系统地抵抗抗生素从而达到耐药。     

Identification and functional analysis of proteins in response to light intensity,temperature and water potential in Brassica rapa hypocotyl

Hypocotyl elongation is an early event in plant growth and development and is sensitive to fluctuations in light, temperature, water potential and nutrients. Most research on hypocotyl elongation has focused on the regulatory mechanism of a single environment factor. However, information about combined effects of multi‐environment factors remains unavailable, and overlapping sites of the environmental factors signaling pathways in the regulation of hypocotyl elongation remain unclear. To identify how cross‐talks among light intensity, temperature and water potential regulate hypocotyl elongation in Brassica rapa L. ssp. chinesis, a comprehensive isobaric tag for relative and absolute quantitation‐based proteomic approach was adopted. In total, 7259 proteins were quantified, and 378 differentially expressed proteins (DEPs) were responsive to all three environmental factors. The DEPs were involved in a variety of biochemical processes, including signal transduction, cytoskeletal organization, carbohydrate metabolism, cell wall organization, protein modification and transport. The DEPs did not function in isolation, but acted in a large and complex interaction network to affect hypocotyl elongation. Among the DEPs, phyB was outstanding for its significant fold change in quantity and complex interaction networks with other proteins. In addition, changes of sensitivity to environmental factors in phyB‐9 suggested a key role in the regulation of hypocotyl elongation. Overall, the data presented in this study show a profile of proteins interaction network in response to light intensity, temperature and water potential and provides molecular basis of hypocotyl elongation in B. rapa.     

Proteomics analysis reveals protein expression differences for hypopharyngeal gland activity in the honeybee,Apis mellifera carnica Pollmann

Background

Most of the proteins contained in royal jelly (RJ) are secreted from the hypopharyngeal glands (HG) of young bees. Although generic protein composition of RJ has been investigated, little is known about how age-dependent changes on HG secretion affect RJ composition and their biological consequences. In this study, we identified differentially expressed proteins (DEPs) during HG development by using the isobaric tag for relative and absolute quantification (iTRAQ) labeling technique. This proteomic method increases the potential for new protein discovery by improving the identification of low quantity proteins.

Results

A total of 1282 proteins were identified from five age groups of worker bees, 284 of which were differentially expressed. 43 (15.1%) of the DEPs were identified for the first time. Comparison of samples at day 6, 9, 12, and 16 of development relative to day 3 led to the unambiguous identification of 112, 117, 127, and 127 DEPs, respectively. The majority of these DEPs were up-regulated in the older worker groups, indicating a substantial change in the pattern of proteins expressed after 3 days. DEPs were identified among all the age groups, suggesting that changes in protein expression during HG ontogeny are concomitant with different states of worker development. A total of 649 proteins were mapped to canonical signaling pathways found in the Kyoto Encyclopedia of Genes and Genomes (KEGG), which were preferentially associated with metabolism and biosynthesis of secondary metabolites. More than 10 key high-abundance proteins were involved in signaling pathways related to ribosome function and protein processing in the endoplasmic reticulum. The results were validated by qPCR.

Conclusion

Our approach demonstrates that HG experienced important changes in protein expression during its ontogenic development, which supports the secretion of proteins involved in diverse functions in adult workers beyond its traditional role in royal jelly production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-665) contains supplementary material, which is available to authorized users.     

Radioimmunoassay of 5-methoxy tryptophol in sheep plasma and pineal glands

No 5-methoxytryptophol (ML) could be detected in sheep plasma using a specific, sensitive radioimmunoassay developed for the purpose. Blood samples were collected from sheep during darkness and daylight and during various stages of the estrous cycle, but in no sample was the ML content above the detection limit of the method. Addition of 1 mM pargyline and neostigmine to blood immediately after collection to block metabolism did not result in a detectable ML concentration. The failure to detect ML was not due to degradation since added ML was not degraded by blood enzymes even after 16 hours incubation at 37 degrees C. Injection of 100 microgram and 1 mg ML sc in sheep resulted in a rapid rise of ML to 95-130pg/ml and 560-1000pg/ml respectively and disappearing with a half life of approximately 15-20 minutes. Sheep pineal glands collected during the light phase contained ML (51 +/- 5pg/mg tissue. X +/- SE, n = 7) which represents less than 6% of the melatonin content. It is concluded that if ML is present in sheep blood it is present at very low levels. It is thus unlikely to be a major circulating pineal hormone in this species, however, its role within the CNS as a local hormone cannot be excluded.