大鼠睾丸限制性表达新基因Rtap2a的生物信息学分析

目的为进一步研究我们克隆到的大鼠新基因Rtap2a,本研究采用多种生物信息学方法对该序列进行特征分析和功能预测,以获得该基因及其编码蛋白更多的功能提示。方法应用B1ast、ORFFinder、ScanProsite、TMpredServer和UniGene等软件或数据库,进行序列的相似性比较,开放读码框预测、染色体定位、电子表达谱和编码蛋白质的功能等分析。结果我们得到的Rtap2a序列是该基因的全长cDNA序列,在多物种间高度保守,该基因编码的蛋白可能为一N端在胞内的单次跨膜蛋白;其小鼠同源基因的电子表达谱提示Rtap2a特异地高表达于睾丸组织,在成年小鼠的睾丸中表达量最高。结论大鼠新基因Rtap2a是一睾丸限制性表达的新基因,其编码蛋白单次跨膜,进化上高度保守,可能在哺乳动物的睾丸发育和精子发生过程中具有重要功能,也有可能参与肿瘤的发生、发展等过程。因此,新基因Rtap2a有进一步研究的价值。     

Identification of Novel Reference Genes Using Multiplatform Expression Data and Their Validation for Quantitative Gene Expression Analysis

Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required.     

Laparotomic Approach for Collecting Serial Hepatic Biopsies in Rats (Rattus norvegicus) and Mice (Mus musculus)

Researchers often consult with laboratory animal veterinarians for suggestions on how to improve their protocols. We assisted a researcher in performing serial liver biopsies in rats (Rattus norvegicus) to assess the transport of iron on a cellular level. We developed a novel collection approach that used laparotomy through a midline abdominal incision and disposable biopsy punches to obtain liver samples at 3 different times at various intervals. We hypothesized the survival of the subjects undergoing the multiple survival procedures would be independent of the weight loss or gain sustained throughout the study. Although 2 rats died during the study, the results were statistically significant with regard to survival when comparing the Belgrade rats to the Sprague Dawley rats and Swiss Webster mice and were independent of the weight loss or gain incurred during the study. We also performed a pilot study in mice (Mus musculus), using the same method as in the rats, with equivalent results. Our study showed the survival of rodents that underwent multiple laparotomies and liver biopsies was independent of the weight gain or loss throughout the study.Laboratory animal veterinarians often are called on to assist principal investigators with their research projects and procedures. Because of our experiences and backgrounds, we can refine procedures to make them more efficient, more productive, and less intrusive. On one such occasion, a researcher asked our department to assist with the collection of liver biopsies from Belgrade rats (Rattus norvegicus) after they had undergone oral iron loading and treatment with a binding agent. Our review of the available literature revealed a lack of data that supported a single, standard method for collecting multiple liver biopsies in rodents, regardless of whether the technique was invasive or noninvasive. The current pilot study presents a method for collecting a series of 3 liver biopsies from rats and mice that results in minimal hemorrhage, adhesion formation, and scarring. The method uses an open laparotomy technique and a disposable biopsy punch.Our major concerns when we considered performing multiple major surgeries in a single animal were that we adhere to all regulatory documents, maintain the wellbeing of the animal subjects, and provide an adequate scientific justification for the procedures. According to the Guide for the Care and Use of Laboratory Animals,⁵ multiple major surgeries can be performed on a single animal only when they are an essential component of a research protocol, scientifically justified by the investigator, or necessary for clinical reasons.⁵ We ensured that this need was an integral part of the researcher''s protocol and that there was scientific justification for performing multiple procedures on the same subject. Our researchers had a limited number of subjects in their colonies that could be included in their studies, and the biopsies were used to show the effects of diet over time on the liver of each subject. When performing multiple, major surgeries on a single animal, appropriate measures to support the animal''s wellbeing must be implemented throughout the study.Standard technique for collecting liver samples from rodents requires either laparotomy or euthanasia.^4,10,12 Although considered invasive due to the need to incise the skin and musculature of the abdominal body wall to access the abdominal cavity, laparotomies offer better visualization of the tissue and permit the acquisition of larger and more diagnostically useful samples than those obtained by percutaneous needle aspirates.^1,2 In recent years, the use of percutaneous needle biopsies to obtain samples from the liver has increased in favor, but samples collected in this manner may be too small for diagnostic purposes, can be damaged during removal from the needle, and may originate from an inappropriate site.^1,2,6,8,13 Percutaneous needle biopsies can be obtained by using a blind procedure or with the aid of specialized techniques, such as ultrasonography. With a blind technique, the tissue is not visualized, and samples might be acquired from the sites of previous biopsies or from organs other than those intended to be sampled. Ultrasonic assistance is desirable but may not be available at many institutions. Even though laparotomy is invasive, it provides sufficient visualization of the liver so that tissue can be harvested from the most appropriate site, and the use of a biopsy punch to collect hepatic tissue samples provides adequate sample with minimal damage to the remaining liver lobes. Previously described techniques often encouraged the removal of an entire liver lobe or the use of cautery to achieve hemostasis.^1,10,14 However both partial hepatectomy and electrocautery decrease the quantity and quality of liver tissue available for subsequent biopsy procedures.The purpose of the current study was to establish a laparotomy-based approach for collecting liver samples of sufficient size and quality that could be performed multiple times on the same animal and that did not negatively affect the health of the rodents or the histologic quality of the collected tissue. Our results demonstrate that the use of a disposable biopsy punch to obtain liver biopsies in rats (Rattus norvegicus) and mice (Mus musculus) causes minimal damage to the liver, provides adequate tissue specimens to perform diagnostic testing, and does not negatively affect the health of the animal when performed multiple times, thereby enabling researchers to reduce the number of subjects required in their studies.     

RNA干扰是静止基因表达的新方法

RNA干扰（RNAi）是一种封闭基因表达的有效方法,通过小干扰RNA片段（siRNA）启动细胞内的RNAi反应,显著增加对目标mRNA的表达抑制.本文综述了RNAi机理,哺乳动物细胞siRNA靶点选择,以及siRNA载体构建和转染哺乳动物体细胞的研究现状.     

Distinct Patterns of Gene Expression Associated with Development of Fluconazole Resistance in Serial Candida albicans Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis

Resistance to fluconazole is becoming an increasing problem in the management of oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Strains obtained from five patients developed decreased fluconazole susceptibility over time. DNA strain typing confirmed the high degree of relatedness among isolates from one patient and the variability among isolates from different patients. Expression of genes involved in development of fluconazole resistance was monitored in each isolate using probes specific for ERG11 (lanosterol 14α-demethylase), MDR1 (a major facilitator), and CDR (ATP-binding cassette or ABC transporter) genes. Increased expression of CDR genes was detected in the series of isolates from two patients. Isolates from one of the two patients also demonstrated increased ERG11 expression, whereas isolates from the other patient did not. Increased levels of MDR1 mRNA correlated with increased resistance in sequential isolates from another patient. Initial overexpression of MDR1 with subsequent overexpression of CDR genes and a final isolate again overexpressing MDR1 were detected in serial isolates from another patient. In another patient, overexpression of these genes was not detected despite an eightfold increase in fluconazole MIC. In this patient, sequence data of the ERG11 gene revealed no point mutations associated with decreased susceptibility. Five different patterns of gene expression were observed in isolates recovered from five patients who developed resistance. Therefore, these experiments demonstrate that a variety of mechanisms or combinations of mechanisms are associated with the development of fluconazole drug resistance. Additional studies are needed to estimate the frequency and clinical impact of these mechanisms of resistance.     

Metagenomic Analysis of Lysogeny in Tampa Bay: Implications for Prophage Gene Expression

Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host's replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e≤0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9×10⁷, and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L⁻¹ respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L⁻¹. Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L⁻¹. In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny.     

LRP15基因的表达谱分析及其在白血病细胞中的表达

为检测LRP15基因在肿瘤细胞以及处于不同发育阶段造血细胞的表达状况 ,探讨其在肿瘤发生、发展以及在白血病分型预后中可能具有的作用 ,利用NCBI提供的SAGE文库及NCI提供的正常组织及肿瘤细胞基因表达数据库 ,分析比较了LRP15基因在多种肿瘤组织、细胞系和正常组织中的表达谱 ;采用RT PCR方法检测了该基因在正常血细胞及原代白血病细胞中的表达状况。结果显示 ,LRP15在人类多种正常组织及肿瘤细胞中有表达 ;在幼稚细胞中表达阳性率高于成熟细胞 (P <0 .0 1) ,在急性髓细胞白血病中M1、M2 和M3 表达的阳性率高于其它亚型 (P <0 .0 1) ;难治组LRP15表达的阳性率有高于初治组的趋势。结论 ,LRP15基因与急性白血病及其它多种肿瘤的发生、发展密切相关 ,对急性白血病的临床分型具有重要的意义 ,可能对判断急性白血病的预后具有一定价值。     

Imaging Cyclooxygenase-2 (Cox-2) Gene Expression in Living Animals with a Luciferase Knock-in Reporter Gene

The cyclooxygenase-2 (Cox-2) gene plays a role in a variety of normal and pathophysiological conditions. Expression of the Cox-2 gene is induced in a broad range of cells, in response to many distinct stimuli. The ability to monitor and quantify Cox-2 expression noninvasively in vivo may facilitate a better understanding of the role of Cox-2, both in normal physiology and in different diseases. We generated a “knock-in” mouse in which the firefly luciferase reporter enzyme is expressed at the start site of translation of the endogenous Cox-2 gene. Correlation of luciferase and Cox-2 expression was confirmed in heterozygous Cox-2 ^luc/+ mouse embryonic fibroblasts isolated from the knock-in mouse. In an acute sepsis model, following injection of interferon gamma and endotoxin, ex vivo imaging and Western blotting demonstrated coordinate Cox-2 and luciferase induction in multiple organs. Using both paw and air pouch inflammation models, we can monitor repeatedly localized luciferase expression in the same living mouse. Cox-2 ^luc/+ knock-in mice should provide a valuable tool to analyze Cox-2 expression in many disease models.     

Agrobacterium-Mediated Transient Gene Expression and Silencing: A Rapid Tool for Functional Gene Assay in Potato

Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.     

Constitutive and Inducible Expression of the rRNA Methylase Gene erm(B) in Campylobacter

Macrolides are the antimicrobials of choice for treating human campylobacteriosis. The recent emergence of erm(B) in Campylobacter bacteria threatens the utility of this class of antibiotics. Here we report the constitutive and inducible expression of erm(B) in Campylobacter isolates derived from diarrheal patients and food-producing animals. Constitutive expression of erm(B) was associated with insertion and deletion in the regulatory region of the gene, providing the first documentation of the differential expression of erm(B) in Campylobacter bacteria.     

A Novel Approach to Measuring Efficiency of Scientific Research Projects: Data Envelopment Analysis

Purpose

Measuring the efficiency of resource allocation for the conduct of scientific projects in medical research is difficult due to, among other factors, the heterogeneity of resources supplied (e.g., dollars or FTEs) and outcomes expected (e.g., grants, publications). While this is an issue in medical science, it has been approached successfully in other fields by using data envelopment analysis (DEA). DEA has a number of advantages over other techniques as it simultaneously uses multiple heterogeneous inputs and outputs to determine which projects are performing most efficiently, referred to as being at the efficiency frontier, when compared to others in the data set.

Method

This research uses DEA for the evaluation of supported translational science projects by the Oregon Clinical and Translational Research Institute (OCTRI), a NCATS Clinical & Translational Science Award (CTSA) recipient.

Results

These results suggest that the primary determinate of overall project efficiency at OCTRI is the amount of funding, with smaller amounts of funding providing more efficiency than larger funding amounts.

Conclusion

These results, and the use of DEA, highlight both the success of using this technique in helping determine medical research efficiency and those factors to consider when distributing funds for new projects at CTSAs.     

Alterations in Patterns of Gene Expression and Perturbed Pathways in the Gut-Brain Axis Are Associated With Chemotherapy-Induced Nausea

ContextDespite current advances in antiemetic treatments, approximately 50% of oncology patients experience chemotherapy-induced nausea (CIN).ObjectivesThe purpose of this study was to evaluate for differentially expressed genes and perturbed pathways associated with the gut-brain axis (GBA) across two independent samples of oncology patients who did and did not experience CIN.MethodsOncology patients (n = 735) completed study questionnaires in the week before their second or third cycle of chemotherapy. CIN occurrence was assessed using the Memorial Symptom Assessment Scale. Gene expression analyses were performed in two independent samples using ribonucleic acid sequencing (Sample 1, n = 357) and microarray (Sample 2, n = 352) methodologies. Fisher's combined probability method was used to determine genes that were differentially expressed and pathways that were perturbed between the two nausea groups across both samples.ResultsCIN was reported by 63.6% of the patients in Sample 1 and 48.9% of the patients in Sample 2. Across the two samples, 703 genes were differentially expressed, and 37 pathways were found to be perturbed between the two CIN groups. We identified nine perturbed pathways that are involved in mechanisms associated with alterations in the GBA (i.e., mucosal inflammation, disruption of gut microbiome).ConclusionPersistent CIN remains a significant clinical problem. Our study is the first to identify novel GBA-related pathways associated with the occurrence of CIN. Our findings warrant confirmation and suggest directions for future clinical studies to decrease CIN occurrence.