Increasing access for biochemistry research in undergraduate education: The malate dehydrogenase CURE community

Integrating research into the classroom environment is an influential pedagogical tool to support student learning, increase retention of STEM students, and help students identify as scientists. The evolution of course-based undergraduate research experiences (CUREs) has grown from individual faculty incorporating their research in the teaching laboratory into well-supported systems to sustain faculty engagement in CUREs. To support the growth of protein-centric biochemistry-related CUREs, we cultivated a community of enthusiastic faculty to develop and adopt malate dehydrogenase (MDH) as a CURE focal point. The MDH CURE Community has grown into a vibrant and exciting group of over 28 faculty from various institutions, including community colleges, minority-serving institutions, undergraduate institutions, and research-intensive institutions in just 4 years. This collective has also addressed important pedagogical questions on the impact of CURE collaboration and the length of the CURE experience in community colleges, undergraduate institutions, and research-intensive institutions. This work provided evidence that modular or partial-semester CUREs also support student outcomes, especially the positive impact it had on underrepresented students. We are currently focused on expanding the MDH CURE Community network by generating more teaching and research materials, creating regional hubs for local interaction and increasing mentoring capacity, and offering mentoring and professional development opportunities for new faculty adopters.     

Involving undergraduates in the annotation and analysis of global gene expression studies: creation of a maize shoot apical meristem expression database

Through a multi-university and interdisciplinary project we have involved undergraduate biology and computer science research students in the functional annotation of maize genes and the analysis of their microarray expression patterns. We have created a database to house the results of our functional annotation of >4400 genes identified as being differentially regulated in the maize shoot apical meristem (SAM). This database is located at http://sam.truman.edu and is now available for public use. The undergraduate students involved in constructing this unique SAM database received hands-on training in an intellectually challenging environment, which has prepared them for graduate and professional careers in biological sciences. We describe our experiences with this project as a model for effective research-based teaching of undergraduate biology and computer science students, as well as for a rich professional development experience for faculty at predominantly undergraduate institutions.     

Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction

Multidrug efflux pumps contribute to multiple antibiotic resistance in Pseudomonas aeruginosa. Pump expression usually has been quantified by Western blotting. Quantitative real-time polymerase chain reaction has been developed to measure mRNA expression for genes of interest. Whether this method correlates with pump protein quantities is unclear. We devised a real-time PCR for mRNA expression of MexAB-OprM and MexXY-OprM multidrug efflux pumps. In laboratory strains differing in MexB and MexY expression and in several clinical isolates, protein and mRNA expression correlated well. Quantitative real-time PCR should be a useful alternative in quantitating expression of multidrug efflux pumps by P. aeruginosa isolates in clinical laboratories.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Careers in Virology: Teaching at a Primarily Undergraduate Institution

A faculty position at a primarily undergraduate institution requires working with undergraduates in both the classroom and the research lab. Graduate students and postdoctoral fellows who are interested in such a career should understand that faculty at these institutions need to teach broadly and devise research questions that can be addressed safely and with limited resources compared to a research I university. Aspects of, and ways to prepare for, this career will be reviewed herein.     

Role of the undergraduate student research assistant in the new millennium

In this study, we analyze the contribution of the undergraduate student who participates in the process of generating scientific data and developing a research project using Brazilian research as an example. Historically, undergraduate students have performed the critical role of research assistants in developing countries. This aspect has been underappreciated as a means of generating scientific data in Brazilian research facilities. Brazilian educational institutions are facing major age-related generational changes among the science faculty within the next 5-10 yr. A lack of adequate support for graduate students leads to a concern that undergraduates will not be interested in choosing research assistant programs and, subsequently, academic research careers. To remedy this situation it is important to focus on ways to encourage new research careers and enhance university-industry collaborations.     

Battelle Primate Facility

The Battelle Primate Facility houses one of the largest collections of neotropical primates in the United States. The facility is a research resource for undergraduate and graduate students. Battelle staff, as well as staff and faculty from U.S. and international institutions. Researchers have access to the animals for a variety of studies encompassing several disciplines, a large collection of preserved tissues, and an extensive biomedical database. The facility is a World Health Organization Collaborative Center for Clinical Pathology of Neotropical Primates and is involved with the Peruvian Primatological Project in Iquitos, Peru, which provides opportunities for research in primatology and conservation.     

Two models for an effective undergraduate research experience in physiology and other natural sciences

68A realistic research experience is beneficial to undergraduate students, but it is often difficult for liberal arts colleges to offer this opportunity. We describe two approaches for developing and maintaining an interdisciplinary research program at small colleges. An active and continuing involvement of an individual with extensive research experience is an essential element in both. One model was developed by the faculty of Taylor University, Upland, IN and a research scientist who had retired from a major university to join the Taylor faculty as their first Research Professor. The school's Science Research Training Program was initially funded by a modest endowment provided by interested alumni and by extramural grants awarded to the Research Professor and to the institution; the program now enjoys significant funding from diverse sources. Taylor is not located near any large research university and consequently supplies all resources required for the experiments and stipends for students pursuing projects full-time during the summer. The second model was developed by the faculty at Asbury College in Wilmore, KY, working with a scientist having a full-time appointment at the University of Kentucky and a part-time appointment at the college. In this approach, Asbury faculty may place their students for a period of training, often during the summer, in a laboratory of a cooperating host faculty at the University of Kentucky or other institution. The host faculty funds the research and pays a stipend to those students who work full-time during the summer. Relationships established between faculty at the College and at the University of Kentucky have been mutually beneficial. The success of both programs is evidenced by the students' presenting their data at state and national scientific meetings, by their publishing their results in national journals, and by the undergraduate school faculty developing independent research programs.     

Quantification of thioredoxin mRNA expression in the rat hippocampus by real-time PCR following oxidative stress

Thioredoxin (Trx) is a multifunctional protein with a redox-active disulfide/dithiol in the active site. Thioredoxin, with its redox-regulating and reactive oxygen species (ROS) scavenging activities, plays several important biologic roles both in intracellular and extracellular compartments. The purpose of this report was to quantify the relative expression of Trx in rat hippocampus following an oxidative stress-involving treatment such as kainic acid (KA) using real-time PCR and the 2(-DeltaDeltaC(T)) method. The relative changes in expression of Trx mRNA in KA-treated and control animals were significantly different as 2.02 +/- 0.77 and 1.0 +/- 0.26, respectively (P<0.05). Minimum and maximum n-fold changes in Trx expression in KA-treated and control animals were determined as (1.4-5.2) and (0.8-1.3), respectively. Thus, real-time PCR and the 2(-DeltaDeltaC(T)) method for data analysis from real-time PCR were found to be an accurate and sensitive method for quantifying Trx mRNA levels.     

Genomic DNA functions as a universal external standard in quantitative real-time PCR

Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies.     

大鼠血管组织血管紧张素原基因表达的实时PCR定量分析

目的:探讨大鼠血管组织血管紧张素原(angiotensinogen,AGT)低丰度mRNA表达的实时PCR定量分析方法,并将其用于检测模拟失重大鼠基底和股动脉血管组织AGT mRNA的表达.方法:提取8周模拟失重(SUS)与对照组(CON)大鼠血管组织的总RNA,进行反转录后,对目的基因AGT与内参照基因GAPDH的mRNA进行实时PCR分析.应用TaqMan-MGB探针,测出上述mRNA实时PCR反应的放大效率(E)及阈循环数Ct,再依据一定数学模型由E与Ct得出经GAPDH归一化的AGT mRNA表达变化.结果:与CON相比,SUS大鼠基底动脉组织AGT mRNA表达增加240%,而股动脉组织则降低66%.结论:本工作为定量检测大鼠血管组织低丰度mRNA表达提示了一种特异、灵敏、精确、重复性好的简便方法.     

TaqMan荧光定量RT-PCR检测水稻RAcl基因mRNA方法的建立

构建包含RAcl基因cDNA片段的质粒,作为水稻肌动蛋白基因RAcl之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参。从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAcl基因中设计的目的片段,将纯化的目的片段与pMD19-T Simple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。建立了RAcl基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102—1护拷贝,阈值循环数（Ct）与PCR体系中起始模板量的对数值之间有着良好的线性关系（r=1．000）,扩增效率高（E=98．2％）。建立了基因RAcl实时定量PCR的质粒标准品。     

Assessing core manipulative skills in a large, first-year laboratory

Responding to the concern from our faculty that undergraduate students do not have robust laboratory skills, we designed and implemented a strategy to individually teach and assess the manipulative skills of students in first-year laboratories. Five core laboratory skills were selected for the course entitled Human Biology, a large, first-year class of students, most of whom were enrolled in Bachelor of Pharmacy and Human Movement Studies. Here, we report details for the 365 students enrolled primarily in Pharmacy and Human Movement Studies bachelor degree programs in semester 1 of 2006. We designed a specific strategy to assess five core laboratory skills: 1) accurate and precise use of a micropipette, 2) calculation of dilutions and preparation of diluted samples of saline, 3) accurate representation of data using a graph, 4) use of a light microscope, and 5) acquisition of digital data by measuring the latent period for the Achilles reflex. Graduate tutors were trained to teach and assess each student on each skill. The development of competency was tracked for all students across all five skills. Most students demonstrated proficiency on their first attempt. The development of proficiency across the core skills depended on both the skill and degree program. In semester 2 of 2006, 854 students mostly enrolled in the Bachelor of Science degree program and were similarly taught and assessed on the same five core skills. This approach was an effective teaching and assessment strategy that, when applied beyond first year, should increase the level of laboratory skills across undergraduate programs in physiology.     

New buffers to improve the quantitative real-time polymerase chain reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.     

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers^1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (˜$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.     

A Biochemistry of Human Disease course for undergraduate and graduate students

This paper describes the experience of members of a medical school faculty who have been offering for more than 10 years a two-course series in the biochemistry of human disease to undergraduate students majoring in biochemistry, biology, or chemistry. Each of the two 3-credit courses meets twice a week for 90 min per session. The courses are divided into five three-week blocs (total number of sessions per bloc, six), each of which is taught by a different instructor. The sixth and last class in each of the blocs is devoted to an exam; there is no cumulative final exam. The topics that are covered include the following: diabetes mellitus, alcoholism, Alzheimer's disease, trophoblastic diseases of pregnancy, molecular and cellular mechanisms of cancer (including chemical carcinogenesis), disorders of calcium metabolism, biochemical and nutritional causes of anemia, collagen diseases, and gene replacement therapy. The various teaching formats and kinds of reading assignments that are used are discussed, as are the reactions of selected faculty who have participated in these courses. The positive experience we have had with a bloc approach to topics-based, multi-instructor courses in human disease should encourage basic science faculty at other medical schools in the US and elsewhere to become involved in teaching specialized, advanced courses to undergraduate, pre-professional students.     

Solid-phase plate-reader quantification of specific PCR products by measurement of band-specific ethidium bromide fluorescence

Real-time PCR is widely employed to quantify PCR products across a range of applications. However, accurate real-time PCR is not always technically feasible, and alternative methods for PCR product quantification can be expensive and time consuming to validate. We have developed an inexpensive, rapid, and immediately accessible protocol to quantify PCR products, by measuring ethidium bromide fluorescence of PCR products excised from agarose gels. This protocol has relevance to a broad range of methods in molecular biology where quantification of PCR products is necessary.     

Magnetic scanometric DNA microarray detection of methyl tertiary butyl ether degrading bacteria for environmental monitoring

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications.