Second Order Normalization in the Generalized Photogravitational Restricted Three Body Prob
GeneOntologyAnalysis:基因本体论分析

17
GO tree example
GO tree: A child can have more than one parent
⎯ Standard assignment of genes into functional categories ⎯ Controlled vocabulary for describing biological meanings
u Gene Ontology or GO project at NCBI
2) Define controlled terms (ontologies) for description of gene products from 3 aspects:
u Biological process (DNA repair, mitosis) u Molecular function (protein serine/threonine kinase activity, transcription factor
Gene Ontology -Cellular Component
/GO_nature_genetics_2000.pdf
Any one gene can be a member of more than one GO classification
21
Temporal snapshots of Go terms and mappings are available in BioC (~700, April 2014)
N ENG DA-EPOCH-R

Dose-Adjusted EPOCH-Rituximab Therapy in Primary Mediastinal B-Cell LymphomaKieron Dunleavy, M.D., Stefania Pittaluga, M.D., Ph.D., Lauren S. Maeda, M.D., Ranjana Advani, M.D., Clara C. Chen, M.D., Julie Hessler, R.N., Seth M. Steinberg, Ph.D., Cliona Grant, M.D., George Wright, Ph.D., Gaurav Varma,M.S.P.H., Louis M. Staudt, M.D., Ph.D., Elaine S. Jaffe, M.D., and Wyndham H. Wilson, M.D., Ph.D.N Engl J Med 2013; 368:1408-1416April 11, 2013DOI: 10.1056/NEJMoa1214561Share:AbstractArticleReferencesCiting Articles (26)LettersKaplan–Meier Estimates of Event-free and Overall Survival of Patients with Primary Mediastinal B-CellLymphoma Receiving DA-EPOCH-R, According to Study Group.Cardiac Ejection Fraction after Treatment with DA-EPOCH-R in 42 Patients in the Prospective NCI Cohort.Primary mediastinal B-cell lymphoma is adistinct pathogenetic subtype of diffuse large-B-cell lymphoma that arises in the thymus.1,2Although it comprises only 10% of cases of diffuse large-B-cell lymphoma, primary mediastinal B-cell lymphoma, which predominantly affects young women,3 is aggressive and typically ismanifested by a localized, bulky mediastinal mass, often with pleural and pericardial effusions.Less commonly, the disease involves extranodal sites, including the lung, kidneys, gastrointestinal organs, or brain.4,5 This disease is clinically and biologically related to nodular sclerosingHodgkin's lymphoma; the putative cell of origin for both conditions is a thymic B cell.1,2The molecular features of primary mediastinal B-cell lymphoma, and its relationship to Hodgkin'slymphoma and other types of diffuse large-B-cell lymphoma, have been studied.1,2,6-8 Mostpatients with primary mediastinal B-cell lymphoma have mutations in the B-cell lymphoma 6 gene (BCL6), usually along with somatic mutations in the immunoglobulin heavy-chain gene, suggesting late-stage germinal-center differentiation.6,7 Unlike other types of diffuse large-B-cell lymphoma,primary mediastinal B-cell lymphoma involves defective immunoglobulin production despite theexpression of the B-cell transcription factors OCT-2, BOB.1, and PU.1. More than half of patients with the disease also have amplification of the REL proto-oncogene and the JAK2 tyrosine kinase gene, which frequently are found in patients with Hodgkin's lymphoma, suggesting that thesediseases are related.9,10 Furthermore, genes that are more highly expressed in primarymediastinal B-cell lymphoma than in other types of diffuse large-B-cell lymphoma arecharacteristically overexpressed in Hodgkin's lymphoma.2Prospective studies in primary mediastinal B-cell lymphoma are few, which has led to conflictingfindings and a lack of treatment standards.11-14 Nonetheless, several observations have emerged from the literature. First, in most patients, adequate tumor control is not achieved with standardimmunochemotherapy, necessitating routine mediastinal radiotherapy.13-15 Second, even withradiotherapy, which is associated with serious late side effects, 20% of patients have diseaseprogression.11,13 Third, more aggressive chemotherapy is associated with an improvedoutcome.12,13 Consistent with this observation, we found that the dose-intense chemotherapy regimen consisting of dose-adjusted etoposide, doxorubicin, and cyclophosphamide with vincristine and prednisone (DA-EPOCH) had a favorable overall survival rate (79%) without consolidation radiotherapy in patients with primary mediastinal B-cell lymphoma.16 On the basis of the hypothesis that rituximab may improve treatment, we undertook a phase 2, prospective study of DA-EPOCH plus rituximab (DA-EPOCH-R) to determine whether it would improve outcomes and obviate the need for radiotherapy.METHODSStudy ConductThe study was designed and the manuscript was written by the last author. All authors reviewed and approved the draft of the manuscript submitted for publication. All the authors vouch for the adherence of the study to the protocol (available with the full text of this article at ) and for the completeness and accuracy of the data and analysis. The prospective study was approved by the institutional review board of the National Cancer Institute (NCI). All patients provided written informed consent. The retrospective analysis was approved by the institutional review board at Stanford University.Filgrastim was provided to the NCI through an agreement with Amgen, which played no role in the study design, analysis, or data collection. No other commercial support was provided for the prospective study.Prospective NCI StudyPatientsFrom November 1999 through August 2012, we prospectively enrolled 51 patients with untreated primary mediastinal B-cell lymphoma in an uncontrolled phase 2 study of DA-EPOCH-R. The primary study objectives were the rate of complete response, the rate of progression-free survival, and the toxicity of DA-EPOCH-R.All eligible patients had not received any previous systemic chemotherapy, had adequate organ function, and had negative results on testing for the human immunodeficiency virus; among women with childbearing potential, a negative test for pregnancy was required. Any localized mediastinal masses (stage I) had to measure at least 5 cm in the greatest dimension. Evaluations included standard blood tests, whole-body computed tomography (CT), and bone marrow biopsy. Assessment of cardiac function, by means of echocardiography, and of central nervous system disease, with the use of CT or magnetic resonance imaging (MRI) and flow cytometry or cytologic analysis of cerebral spinal fluid, were performed if clinically indicated.Study TherapyPatients received chemotherapy consisting of DA-EPOCH-R with filgrastim for 6 to 8 cycles.17,18 Disease sites were evaluated after cycles 4 and 6. Patients with a reduction of more than 20% inthe greatest diameter of their tumor masses between cycles 4 and 6 received 8 cycles of treatment. Patients with a reduction of 20% or less between cycles 4 and 6 discontinued therapy after 6 cycles. The method of administering the DA-EPOCH-R is summarized in the Supplementary Appendix (available at ).We used standard criteria for tumor response to assess the study end points.19,20 We used 18F-fluorodeoxyglucose–positron-emission tomography–CT (FDG-PET-CT) after therapy to evaluate residual masses. Patients who had a maximum standardized uptake value greater than that of the mediastinal blood pool in the residual mediastinal mass underwent repeat scans at approximately 6-week intervals until normalization or stabilization. Mediastinal blood pool activity was defined as the maximum standardized uptake value over the great vessels and ranged from 1.5 to 2.5 in the study population. Tumor biopsy was performed as clinically indicated. Patients with evidence of thymic rebound underwent repeat CT at 6-week intervals until stabilization. All FDG-PET-CT scans were reviewed and scored by the same nuclear-medicine physician. No patients received radiation treatment during this prospective study.Independent, Retrospective Stanford StudyTo provide an independent assessment of DA-EPOCH-R, we collaborated with investigators at Stanford University Medical Center who had begun to use DA-EPOCH-R in 2007 to treat primary mediastinal B-cell lymphoma.21 They reviewed all charts from 2007 through 2012 and found 16 previously untreated patients who had been consecutively treated with DA-EPOCH-R; none required radiotherapy. NCI investigators confirmed the presence of primary mediastinal B-cell lymphoma in all 16 patients, according to the WHO [World Health Organization] Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th edition.3 Standard immunohistochemical studies were performed as indicated.3,18Other Comparative DataTo provide a long-term assessment of the DA-EPOCH platform, we reviewed the pathological data for all patients from our phase 2 study of DA-EPOCH in patients with diffuse large-B-cell lymphoma, which also did not permit radiotherapy, and identified 18 patients with primary mediastinal B-cell lymphoma.16Statistical AnalysisWe calculated the duration of overall survival from the date of enrollment until the time of death or last follow-up. The duration of event-free survival was calculated from the date of enrollment until the date of progression, radiotherapy, discovery of a second mass, or time of last follow-up. We used the Kaplan–Meier method to determine the probability of overall or event-free survival.22 Patients' characteristics were compared by means of Fisher's exact test for dichotomous variables and by means of the Wilcoxon rank-sum test for continuous variables. All P values are two-tailed.The median follow-up was calculated from the date of enrollment through November 2012, the date of the most recent update.RESULTSBaseline Characteristics and Clinical OutcomesThe 51 patients enrolled in the NCI phase 2 prospective study had a median age of 30 years (range, 19 to 52) and a median tumor diameter of 11 cm; 59% were women (Table 1TABLE 1Baseline Characteristics of the Study Patients.). Indicators of advanced disease included bulky tumor with a greatest diameter of 10 cm or more (in 65% of patients), an elevated lactate dehydrogenase level (in 78%), and stage IV disease (in 29%).The 16 patients identified in the retrospective Stanford study had baseline characteristics similar to those of our 51 patients (Table 1) except for a significantly lower frequency of extranodal disease and significantly older age; 56% of patients had bulky disease, and 44% of patients had stage IV disease.At a median follow-up of 63 months (range, 3 to 156), the event-free survival rate in the prospective NCI study was 93% (95% confidence interval [CI], 81 to 98), and the overall survivalrate was 97% (95% CI, 81 to 99) (Figure 1A and 1B FIGURE 1Kaplan–Meier Estimates of Event-free and Overall Survival of Patients with Primary Mediastinal B-Cell Lymphoma Receiving DA-EPOCH-R, According to Study Group.). Three patients had evidence of disease after DA-EPOCH-R treatment; two had persistent focal disease, as detected on FDG-PET-CT, and one had disease progression. Two of these patients underwent mediastinal radiotherapy, and one was observed after excisional biopsy. All three patients became disease-free. One later died from acute myeloid leukemia, while still in remission from his primary mediastinal B-cell lymphoma.In the retrospective Stanford cohort, over a median follow-up of 37 months (range, 5 to 53), 100% of patients (95% CI, 79 to 100) were alive and event-free (Figure 1C and 1D).Finally, we assessed the outcome for 18 patients with primary mediastinal B-cell lymphoma who were enrolled in our phase 2 study of DA-EPOCH.16 These patients had baseline characteristics similar to those in the prospective DA-EPOCH-R study (data not shown). Over a median follow-up of 16 years, the event-free and overall survival rates were 67% (95% CI, 44 to 84) and 78% (95% CI, 55 to 91), respectively. No cardiac failure or second tumors were observed.The event-free and overall survival rates were greater with the addition of rituximab in the NCI prospective cohort than in the cohort of 18 patients who received DA-EPOCH alone (P=0.007 andP=0.01, respectively). This finding suggests that the addition of rituximab may account for the improvement and is consistent with other reports.11FDG-PET-CT FindingsTo identify DA-EPOCH-R treatment failures early, the 36 patients who were found to have residual mediastinal masses in the prospective study underwent FDG-PET-CT in order to optimize curative radiotherapy. Half the patients had a maximum standardized uptake value that was no more than the value in the mediastinal blood pool, which represents the upper limit of the normal range ofuptake (Table 2TABLE 2FDG-PET-CT Findings after DA-EPOCH-R Therapy in the Prospective NCI Cohort.). The other half had a maximum standardized uptake value that was more than the value in the mediastinal blood pool. Although diffuse or focal uptake within the residual tumor mass that is higher than that in the mediastinal blood pool has been considered indicative of lymphoma,20 among these 18 patients, only 3 (with maximum standardized uptake values of 5.9, 10.2, and 14.5) were found to have residual lymphoma. Thus, FDG-PET-CT had a positive predictive value of 17% and a negative predictive value of 100%.Among the 15 patients with a maximum standardized uptake value greater than that in the mediastinal blood pool who did not have disease, 10 underwent repeat FDG-PET-CT; the other 5 did not undergo additional screening, because their initial FDG-PET-CT scans were interpreted as unlikely to represent disease. The 10 patients underwent 1 to 6 additional FDG-PET-CT scans (total, 26); all the findings were interpreted as false positive results on the basis of stabilization or improvement of the maximum standardized uptake value. None of the 10 patients had a recurrence of lymphoma during follow-up.Three patients underwent post-treatment biopsy. One, with a maximum standardized uptake value of 5.9, had a viable tumor of less than 1 cm in area. Owing to the uncertain importance of this finding, the patient was followed for 7 years without treatment, and the tumor did not recur during follow-up. Two patients, with maximum standardized uptake values of 4.6 and 6.4, had negative biopsy results and no tumor recurrence during 6 years of follow-up.In two patients, treatment failed but repeat biopsy was not performed. One patient had disease progression on CT during treatment, and the other had a post-treatment maximum standardized uptake value that increased from 10.2 to 19, consistent with disease progression.Dose and Toxicity of DA-EPOCH-R in the NCI StudyIn the NCI study, 90% of patients received six cycles, and 10% received eight cycles, of DA-EPOCH-R. More than half the 51 patients had an escalation to at least dose level 4, representing a 73% increase over dose level 1; 6% of patients did not have a dose escalation. More than half thepatients received 69 mg of doxorubicin per square meter of body-surface area for at least one cycle and cumulative doses of 345 to 507 mg per square meter. To assess cardiac toxic effects, ejection fractions were measured in 42 patients. All had normal ejection fractions up to 10 yearsafter treatment (Figure 2FIGURE 2Cardiac Ejection Fraction after Treatment with DA-EPOCH-R in 42 Patients in the Prospective NCI Cohort.). There was no significant relationship between the ejection fraction and the length of time since treatment (P=0.30) or between the ejection fraction and the cumulative doxorubicin dose (P=0.20), and no significant interaction between the dose and time interval (P=0.40).Toxicity was assessed during the administration of all 294 cycles of DA-EPOCH-R. The targeted absolute neutrophil count of less than 500 cells per cubic milliliter occurred during 50% of cycles. Thrombocytopenia (<25,000 platelets per cubic millimeter) occurred during 6% of cycles, and hospitalization for fever and neutropenia occurred during 13% of cycles. Nonhematopoietic toxic effects were similar to those that have been reported previously.17,18 One patient died from acute myeloid leukemia while in remission from his primary mediastinal B-cell lymphoma, 49 months after treatment. Owing to the unexpected severe neutropenia during treatment in this patient, we looked for a germline telomerase mutation, which is associated with chemotherapy intolerance and myeloid leukemia.23 Telomere shortening (length, 2.5 SD below the mean) and a heterozygous mutation for the telomerase reverse transcriptase gene (TERT) codon Ala1062Thr were identified.DISCUSSIONThe use of DA-EPOCH-R obviated the need for radiotherapy in all but 2 of 51 patients (4%) with primary mediastinal B-cell lymphoma in a prospective cohort, and no patients had recurring disease over a median follow-up of more than 5 years (maximum, >13). Furthermore, in an independent retrospective cohort, treatment with DA-EPOCH-R in patients with primary mediastinal B-cell lymphoma resulted in an event-free survival rate of 100%. Despite the limitations of the phase 2 study and the retrospective study, these findings suggest that DA-EPOCH-R is a therapeutic advance for this type of lymphoma. Our results suggest that rituximab significantly improves the outcome of chemotherapy in patients with primary mediastinal B-cell lymphoma.The toxicity of DA-EPOCH-R was similar to that reported previously.16 The use of neutrophil-based dose adjustment maximized the delivered dose and limited the incidence of fever and neutropenia to 13% of the cycles. The infusional schedule of doxorubicin allowed for the delivery of high maximal and cumulative doses of doxorubicin without clinically significant cardiac toxic effects.24,25We used post-treatment FDG-PET-CT to identify patients who had persistent disease and a possible need for radiotherapy. Unlike the high clinical accuracy of FDG-PET-CT in other aggressive lymphomas,20 we found the technique to have a poor positive predictive value in primary mediastinal B-cell lymphoma. We frequently observed residual mediastinal masses that continued to shrink for 6 months, suggesting that inflammatory cells might account for the FDG uptake. These findings indicate that FDG-PET-CT uptake alone is not accurate for determining the presence of disease in these patients.There is no established standard treatment for primary mediastinal B-cell lymphoma. Although R-CHOP chemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) has become a de facto standard, it is not universally accepted.11,12 Most strategies also incorporate consolidation radiotherapy to overcome the inadequacy of immunochemotherapy, although some observers have questioned its routine use.12,26 The most accurate assessment of R-CHOP and radiotherapy is a subgroup analysis of patients with primary mediastinal B-cell lymphoma in the Mabthera International Trial Group study of R-CHOP–based treatment.11 Among 44 patients, 73% received radiotherapy, with an event-free survival rate of 78% at 34 months.11 These results indicate that patients who receive R-CHOP–based treatment, most of whom are young women, may have serious long-term consequences of radiotherapy, including second tumors and the acceleration of atherosclerosis and anthracycline-mediated cardiac damage.27Current standard therapy is also inadequate for children with primary mediastinal B-cell lymphoma. In a recent subgroup analysis in the FAB/LMB96 international study, the event-free and overall survival rates were 66% and 73%, respectively, among children receiving a multiagent pediatric regimen.28Retrospective studies have long suggested that patients with primary mediastinal B-cell lymphoma have improved outcomes with the receipt of regimens of increased dose intensity.13 Dose intensity appears to be important in treating Hodgkin's lymphoma, a closely related disease.29 Indeed, outcomes associated with the use of DA-EPOCH-R may well be related to dose intensity as well as the continuous infusion schedule.30 DA-EPOCH therapy involves the administration of pharmacodynamic doses to normalize drug exposure among patients and maximizes the rate of administration. DA-EPOCH may also more effectively modulate the expression of BCL6,7 which encodes a key germinal-center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell-cycle arrest (p21 and p27Kip1), and response to DNA damage (p53 and ATR) and that is expressed by most primary mediastinal B-cell lymphomas (Table 1).31 The inhibition of topoisomerase II also leads to down-regulation of BCL6 expression, suggesting that regimens directed against topoisomerase II may have increased efficacy in treating primary mediastinal B-cell lymphoma. In this regard, DA-EPOCH-R was designed to inhibit topoisomerase II by including two topoisomerase II inhibitors, etoposide and doxorubicin, and maximizing topoisomerase II inhibition by way of extended drug exposure.16In conclusion, our results indicate that DA-EPOCH-R had a high cure rate and obviated the need for radiotherapy in patients with primary mediastinal B-cell lymphoma. To provide confirmatory evidence, an international trial of DA-EPOCH-R in children with primary mediastinal B-cell lymphoma has been initiated ( number, NCT01516567).。
VAM包:变异调整马哈拉诺比距方法版本1.1.0说明书

Package‘V AM’November5,2023Type PackageTitle Variance-Adjusted MahalanobisVersion1.1.0Author H.Robert FrostMaintainer H.Robert Frost<***********************>Description Contains logic for cell-specific gene set scoring of single cell RNA sequencing data. Depends R(>=3.6.0),MASS,MatrixImports methods(>=3.6.0)Suggests Seurat(>=4.0.0),SeuratObject(>=4.0.0),sctransform(>=0.3.2)License GPL(>=2)Copyright Dartmouth CollegeEncoding UTF-8NeedsCompilation noRepository CRANDate/Publication2023-11-0516:30:02UTCR topics documented:V AM-package (2)createGeneSetCollection (2)vam (3)vamForCollection (5)vamForSeurat (6)Index812createGeneSetCollection VAM-package Variance-Adjusted MahalanobisDescriptionImplementation of Variance-adjusted Mahalanobis(V AM),a method for cell-specific gene set scor-ing of scRNA-seq data.DetailsPackage:V AMType:PackageVersion: 1.0.0Date:2021License:GPL-2NoteThis work was supported by the National Institutes of Health grants K01LM012426,R21CA253408, P20GM130454and P30CA023108.Author(s)H.Robert FrostReferences•Frost,H.R.(2020).Variance-adjusted Mahalanobis(V AM):a fast and accurate method forcell-specific gene set scoring.biorXiv e-prints.doi:https:///10.1101/2020.02.18.954321createGeneSetCollectionUtility function to help create gene set collection list objectDescriptionUtility function that creates a gene set collection list in the format required by vamForCollection() given the gene IDs measured in the expression matrix and a list of gene sets as defined by the IDs of the member genes.vam3UsagecreateGeneSetCollection(gene.ids,gene.set.collection,min.size=1,max.size) Argumentsgene.ids Vector of gene IDs.This should correspond to the genes measured in the gene expression data.gene.set.collectionList of gene sets where each element in the list corresponds to a gene set andthe list element is a vector of gene IDs.List names are gene set names.Mustcontain at least one gene set.min.size Minimum gene set size afterfiltering out genes not in the gene.ids vector.Gene sets whose post-filtering size is below this are removed from thefinal collectionlist.Default is1and cannot be set to less than1.max.size Maximum gene set size afterfiltering out genes not in the gene.ids vector.Gene sets whose post-filtering size is above this are removed from thefinal collectionlist.If not specified,nofiltering is performed.ValueVersion of the input gene.set.collection list where gene IDs have been replaced by position indices, genes not present in the gene.ids vector have been removed and gene sets failing the min/max size constraints have been removed.See AlsovamExamples#Create a collection with two sets defined over3genescreateGeneSetCollection(gene.ids=c("A","B","C"),gene.set.collection=list(set1=c("A","B"),set2=c("B","C")),min.size=2,max.size=3)vam Variance-adjusted Mahalanobis(VAM)algorithmDescriptionImplementation of the Variance-adjusted Mahalanobis(V AM)method,which computes distance statistics and one-sided p-values for all cells in the specified single cell gene expression matrix.This matrix should reflect the subset of the full expression profile that corresponds to a single gene set.The p-values will be computed using either a chi-square distribution,a non-central chi-square distribution or gamma distribution as controlled by the center and gamma arguments for the one-sided alternative hypothesis that the expression values in the cell are further from the mean (center=T)or origin(center=F)than expected under the null of uncorrelated technical noise,i.e., gene expression variance is purely technical and all genes are uncorrelated.4vamUsagevam(gene.expr,tech.var.prop,gene.weights,center=FALSE,gamma=TRUE)Argumentsgene.expr An n x p matrix of gene expression values for n cells and p genes.tech.var.prop Vector of technical variance proportions for each of the p genes.If specified, the Mahalanobis distance will be computed using a diagonal covariance matrixgenerated using these proportions.If not specified,the Mahalanobis distanceswill be computed using a diagonal covariance matrix generated from the samplevariances.gene.weights Optional vector of gene weights.If specified,weights must be>0.The weights are used to adjust the gene variance values included in the computation of themodified Mahalanobis distances.Specifically,the gene variance is divided bythe gene weight.This adjustment means that large weights will increase theinfluence of a given gene in the computation of the modified Mahalanobis dis-tance.center If true,will mean center the values in the computation of the Mahalanobis statis-tic.If false,will compute the Mahalanobis distance from the origin.Default isF.gamma If true,willfit a gamma distribution to the non-zero squared Mahalanobis dis-tances computed from a row-permuted version of gene.expr.The estimatedgamma distribution will be used to compute a one-sided p-value for each cell.Iffalse,will compute the p-value using the standard chi-square approximation forthe squared Mahalanobis distance(or non-central if center=F).Default is T. ValueA data.frame with the following elements(row names will match row names from gene.expr):•"cdf.value":1minus the one-sided p-values computed from the squared adjusted Mahalanobis distances.•"distance.sq":The squared adjusted Mahalanobis distances for the n cells.See AlsovamForCollection,vamForSeuratExamples#Simulate Poisson expression data for10genes and10cellsgene.expr=matrix(rpois(100,lambda=2),nrow=10)#Simulate technical variance proportionstech.var.prop=runif(10)#Execute VAM to compute scores for the10genes on each cellvam(gene.expr=gene.expr,tech.var.prop=tech.var.prop)#Create weights that prioritize the first5genesgene.weights=c(rep(2,5),rep(1,5))vamForCollection5 #Execute VAM using the weightsvam(gene.expr=gene.expr,tech.var.prop=tech.var.prop,gene.weights=gene.weights)vamForCollection VAM method for multiple gene setsDescriptionExecutes the Variance-adjusted Mahalanobis(V AM)method(vam)on multiple gene sets,i.e.,a gene set collection.UsagevamForCollection(gene.expr,gene.set.collection,tech.var.prop,gene.weights,center=FALSE,gamma=TRUE)Argumentsgene.expr An n x p matrix of gene expression values for n cells and p genes.gene.set.collectionList of m gene sets for which scores are computed.Each element in the list cor-responds to a gene set and the list element is a vector of indices for the genes inthe set.The index value is defined relative to the order of genes in the gene.exprmatrix.Gene set names should be specified as list names.tech.var.prop See description in vamgene.weights See description in vam.If specified as a single vector of weights,weights must be specified for all p genes and the same weights are used for all gene sets.Touse different weights for each set,specify as a list of the same length as thegene.set.collection list.In this case,each list element should be a vector ofgene weights of the same length as the size of the corresponding gene set.center See description in vamgamma See description in vamValueA list containing two elements:•"cdf.value":n x m matrix of1minus the one-sided p-values for the m gene sets and n cells.•"distance.sq":n x m matrix of squared adjusted Mahalanobis distances for the m gene sets and n cells.See Alsovam,vamForSeuratExamples#Simulate Poisson expression data for10genes and10cellsgene.expr=matrix(rpois(100,lambda=2),nrow=10)#Simulate technical variance proportionstech.var.prop=runif(10)#Define a collection with two disjoint sets that span the10genescollection=list(set1=1:5,set2=6:10)#Execute VAM on both sets using default values for center and gammavamForCollection(gene.expr=gene.expr,gene.set.collection=collection,tech.var.prop=tech.var.prop)#Create weights that prioritize the first2genes for the first set#and the last2genes for the second setgene.weights=list(c(2,2,1,1,1),c(1,1,1,2,2))#Execute VAM using the weightsvamForCollection(gene.expr=gene.expr,gene.set.collection=collection,tech.var.prop=tech.var.prop,gene.weights=gene.weights)vamForSeurat VAM wrapper for scRNA-seq data processed using the Seurat frame-workDescriptionExecutes the Variance-adjusted Mahalanobis(V AM)method(vamForCollection)on normalized scRNA-seq data stored in a Seurat object.If the Seurat NormalizeData method was used for normalization,the technical variance of each gene is computed as the proportion of technical variance(from FindVariableFeatures)multiplied by the variance of the normalized counts.If SCTransform was used for normalization,the technical variance for each gene is set to1(the nor-malized counts output by SCTransform should have variance1if there is only technical variation). UsagevamForSeurat(seurat.data,gene.weights,gene.set.collection,center=FALSE,gamma=TRUE,sample.cov=FALSE,return.dist=FALSE)Argumentsseurat.data The Seurat object that holds the scRNA-seq data.Assumes normalization has already been performed.gene.weights See description in vamForCollectiongene.set.collectionList of m gene sets for which scores are computed.Each element in the listcorresponds to a gene set and the list element is a vector of indices for the genesin the set.The index value is defined relative to the order of genes in the relevantseurat.data Assay object.Gene set names should be specified as list names.center See description in vamgamma See description in vamsample.cov If true,will use the a diagonal covariance matrix generated from the sample vari-ances to compute the squared adjusted Mahalanobis distances(this is equivalentto not specifying tech.var for the vam method).If false(default),will use thetechnical variances as determined based on the type of Seurat normalization.return.dist If true,will return the squared adjusted Mahalanobis distances in a new Assayobject called"V AM.dist".Default is F.ValueUpdated Seurat object that hold the V AM results in one or two new Assay objects:•If return.dist is true,the matrix of squared adjusted Mahalanobis distances will be storedin new Assay object called"V AM.dist".•The matrix of CDF values(1minus the one-sided p-values)will be stored in new Assay objectcalled"V AM.cdf".See Alsovam,vamForCollectionExamples#Only run example code if Seurat package is availableif(requireNamespace("Seurat",quietly=TRUE)&requireNamespace("SeuratObject",quietly=TRUE)){ #Define a collection with one gene set for the first10genescollection=list(set1=1:10)#Execute on the pbmc_small scRNA-seq data set included with SeuratObject#See vignettes for more detailed Seurat examplesvamForSeurat(seurat.data=SeuratObject::pbmc_small,gene.set.collection=collection)}Index∗filecreateGeneSetCollection,2vam,3vamForCollection,5vamForSeurat,6∗packageVAM-package,2 createGeneSetCollection,2vam,3,3,5–7VAM-package,2vamForCollection,4,5,6,7vamForSeurat,4,5,68。
植物学专有名词

前沿·热点Research Hot/Frontiers种系发生Phylogeny生命起源Origins of Life群落多样性Community diversity生物多样性Biodiversity外来物种Alien species物种共存Species coexistence生态系统健康Ecosystem HealthDNA条形码DNA barcoding信号转导Signal transduction细胞周期调控Cell cycle regulation细胞增殖cell proliferation细胞凋亡Cell Apoptosis生物钟Biological clock生物适应性Biochemical adaptation网络生物学Network biology生物控制论Biological Cybernetics染色体重排Chromosomal rearrangements 进化论The theory of evolution自然选择natural selection人工选择artificial selection细胞分裂cell division选择育种selective breeding遗传漂变Genetic drift种系发生phylogeny脱氧核糖核酸DNA生命起源Origin of life古生物学paleontology种系遗传学phylogenetics表型学phenetics共同祖先common descent物种形成Speciation古生菌archaea灵长类primates板块构造论plate tectonics无树大草原savannah寄生parasitism共生symbiosis生命之树Tree of life遗传分类学cladistics开放系统open system动态平衡dynamic equilibrium查尔斯·达尔文Charles Darwin动物Animals植物plants真菌fungi原生生物protists细菌bacteria原核生物prokaryote昆虫类insects病毒viruses多细胞生物multicellular organism热血动物warm-blood生态系统ecosystems系统发生树Phylogenetic tree新陈代谢metabolism遗传学Genetics遗传heredity变异variation核苷酸nucleotides氨基酸amino acids遗传密码genetic code泛生论pangenesis染色体chromosomes遗传连锁genetic linkage螺旋结构helical structure信使RNAmessenger RNA氨基酸序列amino acid sequence遗传的特征 Features of inheritance孟德尔Gregor Mendel等位基因alleles纯合子homozygote杂合子heterozygote基因型genotype表现型phenotype显性dominance隐性recessiveness不完全显性incomplete dominance共显性codominance有性繁殖sexual reproduction谱系图pedigree charts异位显性epistasis遗传力heritability遗传的分子基础 Molecular basis for inheritance 腺嘌呤adenine胞嘧啶cytosine鸟嘌呤guanine胸腺嘧啶thymine双螺旋double helix碱基对base pairs染色质chromatin核小体nucleosomes组蛋白histone单倍体haploid二倍体diploid性连锁遗传病sex-linked disorder无性生殖asexual reproduction有丝分裂mitosis配子gametes生殖细胞germ cells连锁图linkage map分子生物学中心法则central dogma of molecular biology 镰状细胞性贫血sickle-cell anemia核糖体RNArRNA转移核糖核酸tRNA微RNAmicroRNA苯(丙)酮尿症phenylketonuria转录因子Transcription factors大肠杆菌Escherichia coli色氨酸tryptophan负反馈negative feedback阻遏物repressor胞间信号intercellular signals副突变paramutation遗传改变Genetic change诱变mutagenesis紫外辐射UV radiationDNA修复DNA repair减数分裂meiosis适合度fitness果蝇Drosophila群体遗传学Population genetics适应adaptation分子钟molecular clock进化树evolutionary trees模式生物model organisms医学遗传学Medical genetics孟德尔随机化Mendelian randomization同源基因orthologues药物遗传学pharmacogenetics连接酶ligase分子克隆molecular cloning重组DNArecombinant DNA遗传多样性genetic diversity生物量biomass分子生物学Molecular biology分子筛molecular sieve核糖核酸RNA蛋白质的生物合成protein biosynthesis 生物大分子biomolecules复制replication翻译translation转录transcription克隆clone聚合酶链式反应PCR质粒plasmid载体vector启动子元件promoter elements抗生素耐性antibiotic resistance结合conjugation转导transduction转染transfection电穿孔electroporation显微注射microinjection三级结构tertiary structure定量多聚酶链反应QPCR电泳electrophoresis毛细管作用capillary action胚胎干细胞株embryonic stem cell lines SDS-PAGEnorthern blot放射自显影autoradiography增强子Enhancer抗体antibody蔗糖梯度sucrose gradient粘度测定法viscometry保护生物学Conservation biology生物多样性biodiversity种群动态population dynamics稀有种rare species自然保护运动conservation movement 灭绝extinction脊椎动物vertebrates无脊椎动物invertebrates人口过剩overpopulation砍伐森林deforestation放牧过度overgrazing刀耕火种法slash and burn应用生态学Applied ecology濒危物种Endangered species环境保护论Environmentalism迁地保护Ex-situ conservation基因污染Genetic pollution就地保护In situ conservation发育生物学Developmental Biology细胞生长cell growth形态发生morphogenesis分化differentiation染色体畸变chromosomal aberrations个体发生ontogeny细胞凋亡apoptosis先天性疾病congenital disorders内稳态homeostasis神经胚形成neurulation胚胎发生embryogenesis性别决定sex determination原肠胚形成gastrulation适应能力adaptive capacity衰老senescence细胞信号传导Cell signaling信号转导Signal transduction器官发生organogenesis胚胎发生embryogenesis形态发生morphogenesis生态学Ecology次级生产力secondary productivity捕食predationecozones食物链food chains大气圈atmosphere生物群落biocoenosis分解者Decomposers互利共生mutualism光合作用photosynthesis初级生产者primary producers初级消费者primary consumers三级消费者tertiary consumers食肉动物carnivores杂食动物omnivores生境biotope生物地球化学循环biogeochemical cycle氮循环nitrogen cycle陆地生态系统terrestrial ecosystems森林生态系统forest ecosystems水圈hydrosphere生物圈biosphere岩石圈lithosphere生态位ecological niche海洋生态系统marine ecosystems生态危机Ecological crisis血亲consanguinity植物学Botany/Plant Biology(Plant Science)真菌类fungi被子植物angiosperms藻类algae双子叶植物dicotyledon单子叶植物monocot裸子植物gymnosperm分类学taxonomy形态学morphology藓类Mosses苔类liverworts园艺学Horticulture花粉Pollen植物病理学Phytopathology植物生理学Plant physiology草本植物Herbs温室气体greenhouse gas地衣类lichens水循环water cycle古植物学家Paleobotanists果菜类fruit vegetable食性层次trophic level食品安全food security作物育种plant breeding人类植物学Ethnobotany遗传学定律genetic laws四氢大麻酚tetrahydrocannabinol跳跃基因jumping genes细胞生物学Cell biology显微镜microscope核糖体ribosomes细胞质cytoplasm膜蛋白membrane proteins内质网endoplasmic reticulum高尔基体Golgi apparatus细胞骨架cytoskeletal线粒体mitochondria细胞核nucleus叶绿体chloroplasts溶酶体lysosomes主动运输Active transportmRNA剪接mRNA splicing自吞噬Autophagy纤毛Cilia微管microtubule鞭毛Flagella脂质双层Lipid bilayerImmunostaining原位杂交In situ hybridization免疫沉淀反应Immunoprecipitation免疫组化immunohistochemistry生命周期life cycles神经生物学Neurobiology计算神经科学computational neuroscience 认知神经科学cognitive neuroscience行为神经学behavioral neuroscience生物精神病学biological psychiatry神经病学neurology神经心理学neuropsychology神经心理学neuropsychology树状突dendrites光受体photoreceptors动作电位action potentialsquid giant axon突触synapses神经系统nervous system多巴胺Dopamine去甲肾上腺素Norepinephrine肾上腺素Epinephrine黑色素Melanin神经递质neurotransmitters褪黑激素MelatoninP物质Substance P兴奋性突触后电位EPSP抑制性突触后电位IPSP神经元neuron听力系统auditory system嗅觉系统olfactory system视觉系统visual system胶质细胞glial cell神经板neural plate系统生物学Systems BiologyDNA微阵列DNA microarrays蛋白质组学Proteomics质谱测定法mass spectrometry高性能液体色谱HPLC phosphoproteomics糖蛋白质组学glycoproteomics代谢物组学Metabolomics Biomicsprocess calculi信息提取information extraction转录组学transcriptomics代谢组学metabolomics酶动力学enzyme kinetics糖组学Glycomics生物学的其他学科Other fields太空生物学Astrobiology微生物学Microbiology古生物学Paleontology寄生虫学Parasitology生理学Physiology动物学Zoology。
SODA:主要效应和交互效应选择的R包说明书

Package‘sodavis’October14,2022Type PackageTitle SODA:Main and Interaction Effects Selection for LogisticRegression,Quadratic Discriminant and General Index ModelsVersion1.2Depends R(>=3.0.0),nnet,MASS,mvtnormDate2018-05-12Author Yang Li,Jun S.LiuMaintainer Yang Li<*********************>Description Variable and interaction selection are essential to classification in high-dimensional set-ting.In this package,we provide the implementation of SODA procedure,which is a forward-backward algorithm that selects both main and interaction effects under logistic regres-sion and quadratic discriminant analysis.We also provide an extension,S-SODA,for deal-ing with the variable selection problem for semi-parametric models with continuous responses. License GPL-2NeedsCompilation noRepository CRANDate/Publication2018-05-1321:24:03UTCR topics documented:mich_lung (2)pumadyn (2)soda (3)soda_trace_CV (4)s_soda (5)s_soda_model (6)s_soda_pred (7)s_soda_pred_grid (8)Index912pumadyn mich_lung Gene expression data for Michigan lung cancer study in Beer et al.(2002)DescriptionGene expression data of5217genes for n=86subjects,with62subjects in"good outcomes"(class1)and24subjects in"poor outcomes"(class2),from the microarray study of Beer et al.(2002).Usagedata(mich_lung)FormatResponse variable vector and design matrix on86observations for expression of5217genes.ReferencesBeer et al.(1999)Gene-expression profiles predict survival of patients with lung adenocarcinoma.Nature medicine,286(8):816-824.pumadyn Pumadyn datasetDescriptionThis is a dataset synthetically generated from a realistic simulation of the dynamics of a Unimation Puma560robot arm.Usagedata(pumadyn)FormatResponse variable vector and design matrix on4499in-sample and3693out-sample observations for32predictor variables.ReferencesCorke,P.I.(1996).A Robotics Toolbox for MATLAB.IEEE Robotics and Automation Magazine, 3(1):24-32.soda3 soda SODA algorithm for variable and interaction selectionDescriptionSODA is a forward-backward variable and interaction selection algorithm under logistic regression model with second-order terms.In the forward stage,a stepwise procedure is conducted to screen for important predictors with both main and interaction effects,and in the backward stage SODA remove insignificant terms so as to optimize the extended BIC(EBIC)criterion.SODA is appli-cable for variable selection for logistic regression,linear/quadratic discriminant analysis and other discriminant analysis with generative model being in exponential family.Usagesoda(xx,yy,norm=F,debug=F,gam=0,minF=3)Argumentsxx The design matrix,of dimensions n*p,without an intercept.Each row is an observation vector.yy The response vector of dimension n*1.norm Logicalflag for xx variable quantile normalization to standard normal,prior to performing SODA algorithm.Default is norm=FALSE.Quantile-normalizationis suggested if the data contains obvious outliers.debug Logicalflag for printing debug information.gam Tuning paramter gamma in extended BIC criterion.EBIC for selected set S:EBIC=-2*log-likelihood+|S|*log(n)+2*|S|*gamma*log(p) minF Minimum number of steps in forward interaction screening.Default is minF=3.ValueEBIC Trace of extended Bayesian information criterion(EBIC)score.Type Trace of step type("Forward(Main)","Forward(Int)","Backward").Var Trace of selected variables.Term Trace of selected main and interaction terms.final_EBIC Final selected term set EBIC score.final_Var Final selected variables.final_Term Final selected main and interaction terms.Author(s)Yang Li,Jun S.Liu4soda_trace_CVReferencesLi Y,Liu JS.(2017).Robust Variable and Interaction Selection for Logistic Regression and Multiple Index Models.Technical Report.Examples##(uncomment the code to run)##simulation study with1main effect and2interactions#N=250;#p=1000;#r=0.5;#s=1;#H=abs(outer(1:p,1:p,"-"))#S=s*r^H;#S[cbind(1:p,1:p)]=S[cbind(1:p,1:p)]*s#xx=as.matrix(data.frame(mvrnorm(N,rep(0,p),S)));#zz=1+xx[,1]-xx[,10]^2+xx[,10]*xx[,20];#yy=as.numeric(runif(N)<exp(zz)/(1+exp(zz)))#res_SODA=soda(xx,yy,gam=0.5);#cv_SODA=soda_trace_CV(xx,yy,res_SODA)#cv_SODA##Michigan lung cancer dataset#data(mich_lung);#res_SODA=soda(mich_lung_xx,mich_lung_yy,gam=0.5);#cv_SODA=soda_trace_CV(mich_lung_xx,mich_lung_yy,res_SODA)#cv_SODAsoda_trace_CV Calculate a trace of cross-validation error rate for SODA forward-backward procedureDescriptionThis function takes a SODA result variable as input,and calculates the cross-validation error for each step of the SODA procedure.Usagesoda_trace_CV(xx,yy,res_SODA)Argumentsxx The design matrix,of dimensions n*p,without an intercept.Each row is an observation vector.yy The response vector of dimension n*1.res_SODA SODA result varaible.See example below.s_soda5Author(s)Yang Li,Jun S.LiuExamples#Michigan lung cancer dataset(uncomment the code to run)#data(mich_lung);#res_SODA=soda(mich_lung_xx,mich_lung_yy,gam=0.5);#cv_SODA=soda_trace_CV(mich_lung_xx,mich_lung_yy,res_SODA)#cv_SODAs_soda S-SODA algorithm for general index model variable selectionDescriptionS-SODA is an extension of SODA to conduct variable selection for general index models with continuous response.S-SODAfirst evenly discretizes the continuous response into H slices,and then apply SODA on the discretized pared with existing variable selection methods based on the Sliced Inverse Regression(SIR),SODA requires neither the linearity nor the constant variance condition and is much more robust.Usages_soda(x,y,H=5,gam=0,minF=3,norm=F,debug=F)Argumentsx The design matrix,of dimensions n*p,without an intercept.Each row is an observation vector.y The response vector of dimension n*1.H The number of slices.gam EBIC penalization coefficient parameter for SODA.minF Minimum number of steps in forward interaction screening.Default is minF=3.norm If set as True,S-SODAfirst marginally quantile-normalize each predictor to the standard normal distribution.debug If print debug information.ValueBIC Trace of extended Bayesian information criterion(EBIC)score.Var Trace of selected variables.Term Trace of selected main and interaction terms.best_BIC Final selected term set EBIC score.best_Var Final selected variables.best_Term Final selected main and interaction terms.6s_soda_modelExamples##(uncomment the code to run)##Simulation:x1/(1+x2^2)example#N=500#x1=runif(N,-3,+3)#x2=runif(N,-3,+3)#x3=x1/exp(x2^2)+rnorm(N,0,0.2)#ss=s_soda_model(cbind(x1,x2),x3,H=25)###true surface in grid#MM=50#xx1=seq(-3,+3,length.out=MM)#xx2=seq(-3,+3,length.out=MM)#yyy=matrix(0,MM,MM)#for(i in1:MM)#for(j in1:MM)#yyy[i,j]=xx1[i]/exp(xx2[j]^2)###predicted surface#ppp=s_soda_pred_grid(xx1,xx2,ss,po=1)##par(mfrow=c(1,2),mar=c(1.75,3,1.25,1.5))#persp(xx1,xx2,yyy,theta=-45,xlab="X1",ylab="X2",zlab="Y")#persp(xx1,xx2,ppp,theta=-45,xlab="X1",ylab="X2",zlab="Pred")###Pumadyn dataset##data(pumadyn);##s_soda(pumadyn_isample_x,pumadyn_isample_y,H=25,gam=0)s_soda_model S-SODA model estimation.DescriptionS-SODA assumes within each slice the X vector follow multivariate normal distribution.This function estimates the mean vector and covariance matrix of X for each slice.Usages_soda_model(x,y,H=10)Argumentsx The design matrix,of dimensions n*p,without an intercept.Each row is an observation vector.y The response vector of dimension n*1.H The number of slices.Valueint_h Slice index.int_p Proportion of samples in each slice.int_l Length of each slice(max-min response).int_m Mean vector of covariates in each slice.int_v Covariance matrix of covariates in each slice.s_soda_pred Predict the response y using S-SODA model.DescriptionS-SODA assumes within each slice the X vector follow multivariate normal distribution.This function predicts the response y by reverting the P(X|slice(y))to P(slice(y)|X),and estimates the E(y|X)as sum_h E(y|slice(y)=h,X)P(slice(y)=h|X)Usages_soda_pred(x,model,po=1)Argumentsx The design matrix,of dimensions n*p,without an intercept.Each row is an observation vector.model S-SODA model estimated from s_soda_model function.po Order of terms in X to approximate E(y|slice(y)=h,X).If po=0,E(y|slice(y)=h, X)is the mean of y in slice h.If po=1,E(y|slice(y)=h,X)is the linear regressionof X to predict y in slice h.If po=2,the linear regression also include2nd orderterms of X.ValuePredicted response.s_soda_pred_grid Predict the response y using S-SODA model in a2-dimensional grid.DescriptionCalls function s_soda_pred in a2-dimensional grid defined by x1and x2.Usages_soda_pred_grid(xx1,xx2,model,po=1)Argumentsxx1Grid breakpoints for predictor1.xx2Grid breakpoints for predictor2.model S-SODA model estimated from s_soda_model.po Order of terms in X to approximate E(y|slice(y)=h,X).ValuePredicted response.Index∗Predictions_soda_pred,7s_soda_pred_grid,8∗S-SODAs_soda,5s_soda_model,6s_soda_pred,7s_soda_pred_grid,8∗SODAsoda,3soda_trace_CV,4∗cross-validationsoda_trace_CV,4∗datasetsmich_lung,2pumadyn,2∗general index models_soda,5∗interaction_selections_soda,5soda,3∗logistic_regressionsoda,3∗quadratic_discriminant_analysis soda,3mich_lung,2mich_lung_xx(mich_lung),2 mich_lung_yy(mich_lung),2 pumadyn,2pumadyn_isample_x(pumadyn),2 pumadyn_isample_y(pumadyn),2 pumadyn_osample_x(pumadyn),2 pumadyn_osample_y(pumadyn),2 s_soda,5s_soda_model,6s_soda_pred,7s_soda_pred_grid,8soda,3soda_trace_CV,4 9。
人肝细胞癌预后不良相关基因的生物信息学分析及其临床意义

人肝细胞癌预后不良相关基因的生物信息学分析及其临床意义席义博1,2+,张皓 1,2+,杨 波2,陈熙勐1,2,贺培凤1△,卢学春2,1△(1.山西医科大学管理学院,太原030001;2.解放军总医院南楼血液科,国家老年疾病临床医学研究中心北京100853)【摘要】 目的:筛选肝细胞癌(HCC)预后不良相关基因,并探讨其临床意义。
方法:在基因表达综合数据库(GEO)中获取符合分析条件的肝细胞癌全基因组表达谱数据并分析得到差异表达基因(DEGs),再运用生物学信息注释及可视化数据库(DAVID)和蛋白相互作用数据库(String)分别进行功能富集分析和蛋白质互作用网络的构建。
利用癌症基因组图谱数据库(TCGA)和Cox比例风险回归模型对相关差异基因进行预后分析。
结果:找到一个符合条件的人类HCC数据库(GSE84402),共筛选出1141个差异表达基因(DEGs),其中上调基因720个,下调基因421个。
基因功能富集分析和蛋白质互作用分析结果显示CDK1、CDC6、CCNA2、CHEK1、CENPE、PIK3R1、RACGAP1、BIRC5、KIF11和CYP2B6为HCC预后的关键基因。
TCGA数据库和Cox回归模型分析显示CDC6、PIK3R1、RACGAP1和KIF11的表达升高,CENPE的表达降低与HCC预后不良密切相关。
结论:CDC6、CENPE、PIK3R1、RACGAP1和KIF11可能和HCC的预后不良相关,可作为未来HCC预后研究的参考标志物。
【关键词】 肝细胞癌;预后不良基因;生物信息学;Cox比例风险回归模型【中图分类号】R73.3 【文献标识码】A 【文章编号】1000 6834(2019)01 090 009【DOI】10.12047/j.cjap.5764.2019.021BioinformaticsanalysisofgenesrelatedtopoorprognosisofhumanhepatocellularcarcinomaanditsclinicalsignificanceXIYi bo1,2+,ZHANGHao min1,2+,YANGBo2,CHENXi meng1,2,HEPei feng1△,LUXue chun2,1△(1.SchoolofManagement,ShanxiMedicalUniversity,Taiyuan030001;2.DepartmentofHematology,SouthBuilding,GeneralHospitalofthePeople'sLiberationArmy,NationalCenterforClinicalResearchofGeriatricDiseases,Beijing100853,China)【ABSTRACT】Objective:Toscreengenesassociatedwithpoorprognosisofhepatocellularcarcinoma(HCC)andtoexploretheclinicalsignificanceofthesegenes.Methods:TheproperexpressionprofiledataofHCCwasobtainedfromtheGeneExpressionOm nibus(GEO)database,andthedifferentiallyexpressedgenes(DEGs)wereidentifiedbydifferentialexpressionanalysis.TheDAVIDandStringdatabasewereusedforfunctionenrichmentanalysisandtoconstructtheprotein proteininteraction(PPI)networkrespec tively.TheCancerGenomeAtlas(TCGA)databaseandtheCoxProportionalHazardModelwereusedforprognosisanalysisoftheDEGs.Results:AeligiblehumanHCCdataset(GSE84402)mettherequirements.Atotalof1141differentiallyexpressedgeneswereidentified,including720up regulatedand421down regulatedgenes.TheresultsoffunctionenrichmentanalysisandPPInetworkperformedthatCDK1、CDC6、CCNA2、CHEK1、CENPE、PIK3R1、RACGAP1、BIRC5、KIF11andCYP2B6wereprognosiskeygenes.AndtheprognosisanalysisshowedthattheexpressionsofCDC6、PIK3R1、KIF11andRACGAP1wereincreased,andtheexpressionofCENPEwasdecreased,whichwascloselyrelatedtoprognosisofHCC.Conclusion:CDC6、CENPE、PIK3R1、KIF11andRACGAP1maybecloselyrelatedtopoorprognosisofHCC,andcanbeusedasmolecularbiomarkersforfutureresearchofHCCprognosis.【KEYWORDS】 hepatocellularcarcinoma; poorprognosisgenes; bioinformatics; CoxProportionalHazardModel【基金项目】2017年度国家老年疾病临床医学研究中心招标课题(NCRCG PLAGH 2017011);解放军总医院转化医学项目(2017TM 020);山西省重点研发计划项目(201803D31067)【收稿日期】2018 10 09【修回日期】2018 11 05 △【通讯作者】Tel:13241892863,13934569928;E mail:luxuechun@126.com,hepeifeng2006@126.com.+:共同第一作者 肝细胞癌(hepatocellularcarcinoma,HCC)是原发性肝癌中最常见的类型,占原发性肝癌的83%,也是全球癌症死亡的第二大主要原因[1]。
荧光定量pcr基因相对表达量计算方法

荧光定量pcr基因相对表达量计算方法Quantitative Polymerase Chain Reaction (qPCR) is a widely used technique in molecular biology to measure gene expression levels. It is often used to analyze the relative expression of genes in different samples. For researchers studying gene expression, calculating the relative gene expression levels is crucial for understanding the biological processes underlying various cellular activities.荧光定量PCR(qPCR)是分子生物学中广泛使用的一种技术,用于测量基因表达水平。
它通常用于分析不同样本中基因的相对表达量。
对于研究基因表达的研究人员来说,计算基因的相对表达水平对于理解各种细胞活动背后的生物过程至关重要。
One common method for calculating gene expression is the 2^-ΔΔCT method. This method involves first determining the threshold cycle (CT) values for both the gene of interest and the reference gene in each sample. The ΔCT value is then calculated by subtract ing the reference gene CT value from the gene of interest CT value. The ΔΔCT value is obtained by subtracting the ΔCT value of the control sample from the ΔCT value of the experimental sample.一种常见的计算基因表达的方法是2^-ΔΔCT方法。
毕赤酵母表达手册

Pichia Expression KitVersion M01110225-0043Pichia Expression KitA Manual of Methods for Expression of Recombinant Proteins in Pichia pastorisCatalog no. K1710-01tech_service@iiINDIVIDUAL PICHIA EXPRESSION KIT LICENSE AGREEMENTThe Pichia Expression Kit is based on the yeast Pichia pastoris. Pichia pastoris was developed into an expression system by scientists at Salk Institute Biotechnology/Industry Associates (SIBIA) for high-level expression of recombinant proteins. All patents for Pichia pastoris and licenses for its use as an expression system are owned by Research Corporation Technologies, Inc. Tucson, Arizona. Invitrogen has an exclusive license to sell the Pichia Expression Kit to scientists for research purposes only, under the terms described below. Use of Pichia pastoris by commercial corporations requires the user to obtain a commercial license as detailed below. Before using the Pichia Expression Kit, please read the following license a greement. If you do not agree to be bound by its terms, contact Invitrogen within 10 days for authorization to return the unused Pichia Expression Kit and to receive a full credit. If you do agree to the terms of this Agreement, please complete the User Registration Card and return it to Invitrogen before using the kit.INDIVIDUAL PICHIA EXPRESSION KIT LICENSE AGREEMENTInvitrogen Corporation (INVITROGEN) grants you a non-exclusive license to use the enclosed Pichia Expression Kit (EXPRESSION KIT) for academic research or for evaluation purposes only. The EXPRESSION KIT is being transferred to you in furtherance of, and reliance on, such license. You may not use the EXPRESSION KIT, or the materials contained therein, for any commercial purpose without a license for such purpose from RESEARCH CORPORATION TECHNOLOGIES, INC., Tucson, Arizona. Commercial purposes include the use in or sale of expressed proteins as a commercial product, or use to facilitate or advance research or development of a commercial product. Commercial entities may conduct their evaluation for one year at which time this license automatically terminates. Commercial entities will be contacted by Research Corporation Technologies during the evaluation period regarding the purchase of a commercial license.Access to the EXPRESSION KIT must be limited solely to those officers, employees and students of your institution who need access thereto in order to perform the above-described research or evaluation. You must inform each of such officer, employee and student of the provisions of this Agreement and require them to agree, in writing, to be bound by the provisions of this Agreement. You may not distribute the EXPRESSION KIT to others, even those within your own institution. You may transfer modified, altered or original material from the EXPRESSION KIT to a third party following notification of INVITROGEN such that the recipient can be licensed. You may not assign, sub-license, rent lease or otherwise transfer this License or any of the rights or obligation hereunder, except as expressly permitted.This License is effective until terminated. You may terminate it at any time by destroying all Pichia expression products in your control. It will also terminate automatically if you fail to comply with the terms and conditions of the Agreement. You shall, upon termination of the License, destroy all Pichia Expression Kits in your control, and so notify INVITROGEN in writing.This License Shall be governed in its interpretation and enforcement by the laws of the State of California.Product User Registration CardPlease complete and return the enclosed Product User Registration Card for each Pichia Expression Kit that you purchase. This will serve as a record of your purchase and registration and will allow Invitrogen to provide you with technical support and manual updates. It will also allow Invitrogen to update you on future developments of and improvements to the Pichia Expression Kit. The agreement outlined above becomes effective upon our receipt of your User Registration Card or 10 days following the sale of the Pichia Expression Kit to you. Use of the kit at any time results in immediate obligation to the terms and conditions stated in this Agreement.Technical ServicesInvitrogen provides Technical Services to all of our registered Pichia Expression Kit users. Please contact us if you need assistance with the Pichia Expression Kit.United States Headquarters:Japanese Headquarters European Headquarters:Invitrogen Corporation1600 Faraday AvenueCarlsbad, CA 92008 USATel: 1 760 603 7200Tel (Toll Free): 1 800 955 6288 Fax: 1 760 602 6500E-mail:tech_service@ Invitrogen Japan K.K.Nihonbashi Hama-Cho Park Bldg. 4F2-35-4, Hama-Cho, NihonbashiTel: 81 3 3663 7972Fax: 81 3 3663 8242E-mail: jpinfo@Invitrogen Ltd3 Fountain DriveInchinnan Business ParkPaisley PA4 9RF, UKTel (Free Phone Orders): 0800 269 210Tel (General Enquiries): 0800 5345 5345Fax: +44 (0) 141 814 6287E-mail: eurotech@iiiivTable of ContentsMaterials (vii)Purchaser Notification (x)Product Qualification (xii)Introduction (1)Overview (1)Experimental Outline (3)Recombination and Integration in Pichia (7)Methods (11)Pichia Strains (11)E. coli Strains (13)Selecting a Pichia Expression Vector (14)pHIL-D2 (16)pPIC3.5 (17)pHIL-S1 (18)pPIC9 (19)Signal Sequence Processing (20)Cloning into the Pichia Expression Vectors (21)Transformation into E. coli (26)Preparation of Transforming DNA (27)Growth of Pichia for Spheroplasting (30)Preparation of Spheroplasts (32)Transformation of Pichia (34)Screening for Mut+ and Mut S Transformants (36)PCR Analysis of Pichia Integrants (40)Expression of Recombinant Pichia Strains (42)Analysis by SDS-Polyacrylamide Gel Electrophoresis (45)Optimization of Pichia Protein Expression (47)Scale-up of Expression (49)Protein Purification and Glycosylation (51)Recipes (53)E. coli Media Recipes (53)Pichia Media Recipes (54)Appendix (59)Electroporation of Pichia (59)PEG 1000 Transformation Method for Pichia (60)Lithium Chloride Transformation Method (61)Total DNA Isolation from Pichia (62)Detection of Multiple Integration Events (63)Procedure for Total RNA Isolation from Pichia (64)β-Galactosidase Assay (65)Technical Service (67)References (69)vviMaterialsKit Contents Box 1: Spheroplast Module. Store at room temperature.Reagent Amount ComponentsSOS media 20 ml 1 M Sorbitol0.3X YPD10 mM CaCl2Sterile Water 2 x 125 ml Autoclaved, deionized waterSE 2 x 125 ml 1 M Sorbitol25 mM EDTA, pH 8.0SCE 2 x 125 ml 1 M Sorbitol10 mM Sodium citrate buffer, pH 5.81 mM EDTA1 M Sorbitol2 x 125 ml --CaS 2 x 60 ml 1 M Sorbitol10 mM Tris-HCl, pH 7.5;10 mM CaCl240% PEG 25 ml 40% (w/v) PEG 3350 (Reagent grade) in waterCaT 25 ml 20 mM Tris-HCl, pH 7.520 mM CaCl2Stab Vials: Pichia and E. coli stabs. Store at +4°C.Phenotype(Pichia only)GenotypeStrain Amountstab his4Mut+GS115 1stab arg4 his4 aox1::ARG4 Mut S, Arg+KM71 1GS115 Albumin 1 stab HIS4Mut SGS115 β-Gal 1 stab HIS4Mut+stab F´ {pro AB, lac I q, lac Z∆M15, Tn10 (Tet R)} mcr A,TOP10F´ 1∆(mrr-hsd RMS-mcr BC), φ80lac Z∆M15, ∆lac X74,deo R, rec A1, ara D139, ∆(ara-leu)7697, gal U,gal K, rps L (Str R), end A1, nup G λ-.Box 2: Spheroplast Module. Store at -20°C.ComponentsReagent AmountZymolyase 10 x 20 µl 3 mg/ml Zymolyase in water(100,000 units/g lytic activity)1 M DTT 10 x 1 ml 1 M dithiothreitol in watercontinued on next pageviiKit Contents,continuedVector Box. Store at -20°C.Reagent DescriptionpHIL-D210 µg, lyophilized in TE, pH 8.0Vector for intracellular expression in PichiapPIC3.510 µg, lyophilized in TE, pH 8.0Vector for intracellular expression in PichiapHIL-S110 µg, lyophilized in TE, pH 8.0 Vector for secreted expression in Pichia. Uses the PHO1 signal sequencepPIC910 µg, lyophilized in TE, pH 8.0 Vector for secreted expression in Pichia. Uses the α-factor signal sequencePrimer Box. Store at -20°C.5´ AOX1 sequencing primer2 µg (312 pmoles), lyophilized5´-GACTGGTTCCAATTGACAAGC-3´3´ AOX1 sequencing primer2 µg (314 pmoles), lyophilized5´-GCAAATGGCATTCTGACATCC-3´α-Factor sequencing primer2 µg (315 pmoles), lyophilized5´-TACTATTGCCAGCATTGCTGC-3´Media The following prepackaged media is included for your convenience. Instructions for use are provided on the package.Media Amount Yield YP Base Medium 2 pouches 2 liters of YP mediumYP Base Agar Medium 2 pouches 2 liters of YP mediumYeast Nitrogen Base 1 pouch 500 ml of 10X YNBFor transformation of Pichia by spheroplasting, the Pichia Spheroplast Module isavailable separately from Invitrogen (see below for ordering information).Product Reactions or Amount Catalog no.Pichia Spheroplast Module 10 spheroplast preparations(50 transformations)K1720-01continued on next pageviiiRequired Equip-ment and Supplies (not provided) • 30°C rotary shaking incubator• Water baths capable of 37°C, 45°C, and 100°C• Centrifuge suitable for 50 ml conical tubes (floor or table-top)• Baffled culture flasks with metal covers (50 ml, 250 ml, 500 ml, 1000 ml, and 3 L)• 50 ml sterile, conical tubes• 6 ml and 15 ml sterile snap-top tubes (Falcon 2059 or similar)• UVSpectrophotometer• Mini agarose gel apparatus and buffers• Polyacrylamide Gel Electrophoresis apparatus and buffers• Media for transformation, growth, screening, and expression (see Recipes, pages 53-58) • 5% SDS solution (10 ml per transformation)• Sterile cheesecloth or gauze• Breaking Buffer (see Recipes, page 58)• Acid-washed glass beads (available from Sigma)• Replica-plating equipment (optional)• BeadBreaker™ (optional)ixPurchaser NotificationIntroduction The Pichia Expression Kit is based on the yeast Pichia pastoris. Pichia pastoris wasdeveloped into an expression system by scientists at Salk Institute Biotechnology/ IndustryAssociates (SIBIA) and Phillips Petroleum for high-level expression of recombinantproteins. All patents for Pichia pastoris and licenses for its use as an expression system areowned by Research Corporation Technologies (RCT), Inc., Tucson, Arizona. Forinformation on commercial licenses, please see page x.The Nature of the Invitrogen License Invitrogen has an exclusive license to sell the Pichia Expression Kit to scientists for research purposes only, under the terms described below. Use of Pichia pastoris by commercial entities for any commercial purpose requires the user to obtain a commercial license as detailed below. Before using the Pichia Expression Kit, please read the following license agreement. If you do not agree to be bound by its terms, contact Invitrogen within 10 days for authorization to return the unused Pichia Expression Kit and to receive a full credit. If you do agree to the terms of this license agreement, please complete the User Registration Card and return it to Invitrogen before using the kit.Pichia pastoris Patents Pichia pastoris is covered by one or more of the following U.S. patents and corresponding foreign patents owned and licensed by Research Corporation Technologies:4,683,293 4,808,537 4,812,405 4,818,700 4,837,148 4,855,231 4,857,467 4,879,231 4,882,279 4,885,242 4,895,800 4,929,555 5,002,876 5,004,688 5,032,516 5,122,465 5,135,868 5,166,329Individual Pichia Expression Kit License Agreement Invitrogen Corporation ("Invitrogen") grants you a non-exclusive license to use the enclosed Pichia Expression Kit ("Expression Kit") for academic research or for evaluation purposes only. The Expression Kit is being transferred to you in furtherance of, and reliance on, such license. You may not use the Expression Kit, or the materials contained therein, for any commercial purpose without a license for such purpose from Research Corporation Technologies, Inc., Tucson, Arizona.Definition of Commercial Purpose Commercial purposes include:(a) any use of Expression Products in a Commercial Product(b) any use of Expression Products in the manufacture of a Commercial Product(c) any sale of Expression Products(d) any use of Expression Products or the Expression Kit to facilitate or advanceresearch or development of a Commercial Product(e) any use of Expression Products or the Expression Kit to facilitate or advance anyresearch or development program the results of which will be applied to thedevelopment of Commercial Products"Expression Products" means products expressed with the Expression Kit, or with the use of any vectors or host strains in the Expression Kit. "Commercial Product" means any product intended for sale or commercial use.Commercial entities may conduct their evaluation for one year at which time this license automatically terminates. Research Corporation Technologies will contact commercial entities during the evaluation period regarding their desire for a commercial license.continued on next pagexPurchaser Notification, continuedIndividual Responsibilities Access to the Expression Kit must be limited solely to those officers, employees and students of your institution who need access to perform the above-described research or evaluation. You must inform each such officer, employee and student of the provisions of this license agreement and require them to agree, in writing, to be bound by the provisions of this license agreement. You may not distribute neither the Expression Kit nor the vectors or host strains contained in it to others, even to those within your own institution. You may only transfer modified, altered, or original material from the Expression Kit to a third party following written notification of, and written approval from, Invitrogen so that the recipient can be licensed. You may not assign, sub-license, rent, lease or otherwise transfer this license agreement or any of the rights or obligation thereunder, except as expressly permitted by Invitrogen and RCT.Termination of License This license agreement is effective until terminated. You may terminate it at any time by destroying all Pichia expression products in your control. It will also terminate auto-matically if you fail to comply with the terms and conditions of the license agreement. You shall, upon termination of the license agreement, destroy all Pichia Expression Kits in your control, and so notify Invitrogen in writing.This License shall be governed in its interpretation and enforcement by the laws of the State of California.Contact for Commercial Licensing Bennett Cohen, Ph.D.Research Corporation Technologies 101 North Wilmot Road, Suite 600 Tucson, Arizona 85711-3335 Phone: (520) 748-4400Fax: (520)748-0025User Registration Card Please complete and return the enclosed User Registration Card for each PichiaExpression Kit that you purchase. This will serve as a record of your purchase and regis-tration and will allow Invitrogen to provide you with technical support and manualupdates. It will also allow Invitrogen to update you on future developments and improve-ments to the Pichia Expression Kit. The agreement outlined above becomes effectiveupon our receipt of your User Registration Card or 10 days following the sale of thePichia Expression Kit to you. Use of the kit at any time results in immediate obligation tothe terms and conditions stated in this license agreement.xiProduct QualificationIntroduction This section describes the criteria used to qualify the components in the PichiaExpression Kit.Vectors All expression vectors are qualified by restriction enzyme digestion. Restriction digests must demonstrate the correct banding pattern when electrophoresed on an agarose gel.Spheroplast Reagents The spheroplast reagents are qualified by spheroplast preparation of GS115 following the protocol provided in the Pichia Expression Kit manual. At least 70% of the Pichia pastoris cells must form spheroplasts in 30 minutes or less.Pichia Strains The Pichia strains are by demonstrating viability of the culture. Single colonies should arise within 48 hours after streaking on YPD medium from the stabPrimers Sequencing primers are lot tested by automated DNA sequencing experiments.Buffers andSolutionsAll buffers and solutions are extensively tested for sterility.Media All Pichia growth and expression media are qualified by growing the GS115 Pichiastrain.xiiIntroductionOverviewReview Articles The information presented here is designed to give you a concise overview of the Pichia pastoris expression system. It is by no means exhaustive. For further information, pleaseread the articles cited in the text along with recent review articles (Buckholz and Gleeson,1991; Cregg et al., 1993; Sreekrishna et al., 1988; Wegner, 1990). A general review offoreign gene expression in yeast is also available (Romanos et al., 1992).General Characteristics of Pichia pastoris As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression levels. As a yeast, it shares the advantages of molecular and genetic manipulations with Saccharomyces, and has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia very useful as a protein expression system.Similarity to Saccharomyces Many of the techniques developed for Saccharomyces may be applied to Pichia including: • transformation by complementation• genedisruption• genereplacementIn addition, the genetic nomenclature used for Saccharomyces has been applied to Pichia. For example, the HIS4 gene in both Saccharomyces and Pichia encodes histidinol dehydrogenase. There is also cross-complementation between gene products in both Saccharomyces and Pichia. Several wild-type genes from Saccharomyces complement comparable mutant genes in Pichia. Genes such as HIS4, LEU2, ARG4, TRP1, and URA3 all complement their respective mutant genes in Pichia.Pichia pastoris as a Methylotrophic Yeast Pichia pastoris is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular oxygen by the enzyme alcohol oxidase. This reaction generates both formaldehyde and hydrogen peroxide. To avoid hydrogen peroxide toxicity, methanol metabolism takes place within a specialized cell organelle called the peroxisome, which sequesters toxic by-products from the rest of the cell. Alcohol oxidase has a poor affinity for O2, and Pichia pastoris compensates by generating large amounts of the enzyme. The promoter regulating the production of alcohol oxidase drives heterologous protein expression in Pichia.Two Alcohol Oxidase Proteins The AOX1 and AOX2 genes code for alcohol oxidase in Pichia pastoris. The AOX1 gene product accounts for the majority of alcohol oxidase activity in the cell. Expression of the AOX1 gene is tightly regulated and induced by methanol to high levels, typically > 30% ofthe total soluble protein in cells grown with methanol as the carbon source. The AOX1 gene has been isolated and the AOX1 promoter is used to drive expression of the gene of interest (Ellis et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a). While AOX2 is about 97% homologous to AOX1, growth on methanol is much slower than with AOX1. This slowgrowth allows isolation of Mut S strains (aox1) (Cregg et al., 1989; Koutz et al., 1989).continued on next page1Overview, continuedExpression Expression of the AOX1 gene is controlled at the level of transcription. In methanol-grown cells approximately 5% of the polyA+ RNA is from the AOX1 gene. The regulation of theAOX1 gene is a two step process: a repression/derepression mechanism plus an inductionmechanism (e.g. GAL1 gene in Saccharomyces (Johnston, 1987)). Briefly, growth onglucose represses transcription, even in the presence of the inducer methanol. For thisreason, growth on glycerol is recommended for optimal induction with methanol. Pleasenote that growth on glycerol (derepression) is not sufficient to generate even minute levelsof expression from the AOX1 gene. The inducer, methanol, is necessary for detectablelevels of AOX1 expression (Ellis et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a).Phenotype of aox1 mutants Loss of the AOX1 gene, and thus a loss of most of the cell's alcohol oxidase activity, results in a strain that is phenotypically Mut S (Methanol utilization slow). This has in the past been referred to as Mut. The Mut S designation has been chosen to accurately describe the phenotype of these mutants. This results in a reduction in the cells' ability to metabolize methanol. The cells, therefore, exhibit poor growth on methanol medium. Mut+ (Methanol utilization plus) refers to the wild type ability of strains to metabolize methanol as the sole carbon source. These two phenotypes are used when evaluating Pichia transformants for integration of your gene (Experimental Outline, page 3).Intracellular and Secretory Protein Expression Heterologous expression in Pichia can be either intracellular or secreted. Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretory pathway. While several different secretion signal sequences have been used successfully, including the native secretion signal present on some heterologous proteins, success has been variable. The secretion signal sequence from the Saccharomyces cerevisiaeα factor prepro peptide has been used most successfully (Cregg et al., 1993; Scorer et al., 1993).The major advantage of expressing heterologous proteins as secreted proteins is that Pichia pastoris secretes very low levels of native proteins. That, combined with the very low amount of protein in the minimal Pichia growth medium, means that the secreted heterologous protein comprises the vast majority of the total protein in the medium and serves as the first step in purification of the protein (Barr et al., 1992). Note: If there are recognized glycosylation sites (Asn-X-Ser/Thr) in your protein's primary sequence, glycosylation may occur at these sites.Posttranslational Modifications In comparison to Saccharomyces cerevisiae, Pichia may have an advantage in the glyco-sylation of secreted proteins because it may not hyperglycosylate. Both Saccharomyces cerevisiae and Pichia pastoris have a majority of N-linked glycosylation of the high-mannose type; however, the length of the oligosaccharide chains added posttranslationally to proteins in Pichia (average 8-14 mannose residues per side chain) is much shorter than those in S. cerevisiae (50-150 mannose residues) (Grinna and Tschopp, 1989; Tschopp et al., 1987b). Very little O-linked glycosylation has been observed in Pichia.In addition, Saccharomyces cerevisiae core oligosaccharides have terminal α1,3 glycan linkages whereas Pichia pastoris does not. It is believed that the α1,3 glycan linkages in glycosylated proteins produced from Saccharomyces cerevisiae are primarily responsible for the hyper-antigenic nature of these proteins making them particularly unsuitable for therapeutic use. Although not proven, this is predicted to be less of a problem for glycoproteins generated in Pichia pastoris, because it may resemble the glycoprotein structure of higher eukaryotes (Cregg et al., 1993).2Experimental OutlineSelection of Vector and Cloning To utilize the strong, highly inducible P AOX1 promoter for expression of your protein, four expression vectors are included in this kit. pHIL-D2 and pPIC3.5 are used for intracellular expression while pHIL-S1 and pPIC9 are used for secreted expression (see pages 14-19 for more information). Before cloning your insert, you must...• decide whether you want intracellular or secreted expression.• analyze your insert for the following restriction sites: Sac I, Stu I, Sal I, Not I, and Bgl II. These sites are recommended for linearizing your construct prior to Pichiatransformation. If your insert has all of these sites, see pages 28-29 for alternate sites.Transformation and IntegrationTwo different phenotypic classes of His+ recombinant strains can be generated: Mut+ and Mut S. Mut S refers to the "Methanol utilization slow" phenotype caused by the loss of alcohol oxidase activity encoded by the AOX1 gene. A strain with a Mut S phenotype has a mutant aox1 locus, but is wild type for AOX2. This results in a slow growth phenotype on methanol medium. Transformation of strain GS115 can yield both classes of transformants, His+ Mut+ and His+Mut S, while KM71 yields only His+ Mut S since the strain itself is Mut S. Both Mut+ and Mut S recombinants are useful to have as one phenotype may favor better expression of your protein than the other. Due to clonal variation, you should test 6-10 recombinants per phenotype. There is no way to predict beforehand which construct or isolate will better express your protein. We strongly recommend that you analyze Pichia recombinants by PCR to confirm integration of your construct (see page 40).Once you have successfully cloned your gene, you will then linearize your plasmid to stimulate recombination when the plasmid is transformed into Pichia. The table below describes the types of recombinants you will get by selective digestion of your plasmid. RestrictionEnzymeIntegration Event GS115 Phenotype KM71 PhenotypeSal I or Stu I Insertion at his4His+ Mut+ His+ Mut SSac I Insertion at 5´AOX1 regionHis+ Mut+ His+ Mut SNot I or Bgl II Replacement atAOX1 locusHis+ Mut SHis+ Mut+His+ Mut S (notrecommended, see page 11)Expression and Scale-up After confirming your Pichia recombinants by PCR, you will test expression of both His+Mut+ and His+ Mut S recombinants. This will involve growing a small culture of each recombinant, inducing with methanol, and taking time points. If looking for intracellular expression, analyze the cell pellet from each time point by SDS polyacrylamide gel electrophoresis (SDS-PAGE). If looking for secreted expression, analyze both the cellpellet and supernatant from each time point. We recommend that you analyze your SDS-PAGE gels by both Coomassie staining and Western blot, if you have an antibody to your protein. We also suggest checking for protein activity by assay, if one is available. Not all proteins express to the level of grams per liter, so it is advisable to check by Western blotor activity assay, and not just by Coomassie staining of SDS-PAGE gels for production of your protein.Choose the Pichia recombinant strain that best expresses your protein and optimizeinduction based on the suggestions on pages 47-48. Once expression is optimized, scale-up your expression protocol to produce more protein.continued on next page3。
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In this paper we have performed Birkhoff’s normalization of the Hamiltonian. For this we have utilised Henrard’s method and expanded the coordinates of the third body in Double d’Alembert series. We have found the values of first and second order components. The second order components are obtained as solutions of the two partial differential equations. We have employed the first condition of KAM theorem in solving these equations. The first and second order components are affected by radiation pressure, oblateness 1/2 1/2 and P-R drag. Finaly we obtained the third order part H3 of the Hamiltonian in I1 I2 zero. 2. Location of Triangular Equilibrium Points Equations of motions are (1) (2) (3) x ¨ − 2 ny ˙ = Ux , ∂U1 W1 N1 − 2 ∂x r1 ∂U1 W1 N2 y ¨ + 2 nx ˙ = Uy , Uy = − 2 ∂y r1 n2 (x2 + y 2 ) (1 − µ)q1 µ µA2 U1 = + + + 3 2 r1 r2 2r2 where, Ux =
arXiv:math/0602528v3 [math.DS] 8 Mar 2006
Abstract. In this paper we have performed second order normalization in the generalized photogravitaional restricted three body problem with Poynting-Robertson drag. We have performed Birkhoff’s normalization of the Hamiltonian. For this we have utilised Henrard’s method and expanded the coordinates of the third body in Double d’Alembert series. We have found the values of first and second order components. The second order components are obtained as solutions of the two partial differential equations. We have employed the first condition of KAM theorem in solving these equations. The first and second order components are affected by radiation pressure, oblateness and P-R drag. Finaly we obtained the third order part H3 of the 1/2 1/2 Hamiltonian in I1 I2 zero.
r 2 −r 2
−q1 ) e p 1 2 , µ = m1m W1 = (1−µ)(1 cd +m2 ≤ 2 , m1 , m2 be the masses of the primaries, A2 = 5r 2 be the oblateness coefficient, re andrp be the equatorial and polar radii respectively r be the distance between primaries, Fp q = 1 − Fg be the mass reduction factor expressed in terms of the particle’s radius a, density ρ and radiation 10 χ . Assumption q = constant is pressure efficiency factor χ (in the C.G.S.system) i.e., q = 1 − 5.6×aρ equivalent to neglecting fluctuation in the beam of solar radiation and the effect of solar radiation, the effect of the planet’s shadow, obviously q ≤ 1. Triangular equilibrium points are given by Ux = 0, Uy = 0, z = 0, y = 0, then we have 5 A2 + µ(1 − nW1 (1 − µ) 1 + 2 A2 δ 2 2 ) 2
AMS Classification:70F15 Keywords: Second Order Normalization/Generalized Photogravitaional/RTBP/P-R drag.
1. Introduction The restricted three body problem describes the motion of an infinitesimal mass moving under the gravitational effect of the two finite masses, called primaries, which move in circular orbits around their centre of mass on account of their mutual attraction and the infinitesimal mass not influencing the motion of the primaries. The classical restricted three body problem is generalized to include the force of radiation pressure, the Poynting-Robertson effect and oblateness effect. J. H. Poynting (1903) considered the effect of the absorption and subsequent re-emission of sunlight by small isolated particles in the solar system. His work was later modified by H. P. Robertson (1937) who used a precise relativistic treatments of the first order in the ratio of he velocity of the particle to that of light. The effect of radiation pressure and P-R. drag in the restricted three body problem has been studied by Colombo et al. (1966), Chernikov Yu. A. (1970) and Schuerman (1980) who discussed the position as well as the stability of the Lagrangian equilibrium points when radiation pressure, P-R drag force are included. Murray C. D. (1994) systematically discussed the dynamical effect of general drag in the planar circular restricted three body problem, Liou J. C. et al. (1995) examined the effect of radiation pressure, P-R drag and solar wind drag in the restricted three body problem. Moser’s conditions (1962), Arnold’s theorem (1961) and Liapunov’s theorem (1956) played a significant role in deciding the nonlinear stability of an equilibrium point. Applying Arnold’s theorem (1961), Leontovic (1962) examined the nonlinear stability of triangular points. Moser gave some modifications in Arnold’s theorem. Then Deprit and Deprit (1967) investigated the nonlinear stability of triangular points by applying Moser’s modified version of Arnold’s theorem (1961). Bhatnagar and Hallan (1983) studied the effect of perturbations on the nonlinear stability of triangular points. Maciejewski and Gozdziewski (1991) described the normalization algorithms of Hamiltonian near an equilibrium point. Niedzielska (1994) investigated the nonlinear stability of the libration points in the photogravitational restricted three body problem. Mishra P. and Ishwar B.(1995) studied second order normalization in the generalized restricted problem of three bodies, smaller primary being an oblate spheroid. Ishwar B.(1997) studied nonlinear stability in the generalized restricted three body problem.