Improving human forensics through advances in genetics, genomics and molecular biology

Forensic DNA profiling currently allows the identification of persons already known to investigating authorities. Recent advances have produced new types of genetic markers with the potential to overcome some important limitations of current DNA profiling methods. Moreover, other developments are enabling completely new kinds of forensically relevant information to be extracted from biological samples. These include new molecular approaches for finding individuals previously unknown to investigators, and new molecular methods to support links between forensic sample donors and criminal acts. Such advances in genetics, genomics and molecular biology are likely to improve human forensic case work in the near future.     

Using a module-based laboratory to incorporate inquiry into a large cell biology course

Because cell biology has rapidly increased in breadth and depth, instructors are challenged not only to provide undergraduate science students with a strong, up-to-date foundation of knowledge, but also to engage them in the scientific process. To these ends, revision of the Cell Biology Lab course at the University of Wisconsin-La Crosse was undertaken to allow student involvement in experimental design, emphasize data collection and analysis, make connections to the big picture, and increase student interest in the field. Multiweek laboratory modules were developed as a method to establish an inquiry-based learning environment. Each module utilizes relevant techniques to investigate one or more questions within the context of a fictional story, and there is a progression during the semester from more instructor-guided to more open-ended student investigation. An assessment tool was developed to evaluate student attitudes regarding their lab experience. Analysis of five semesters of data strongly supports the module format as a successful model for inquiry education by increasing student interest and improving attitude toward learning. In addition, student performance on inquiry-based assignments improved over the course of each semester, suggesting an improvement in inquiry-related skills.     

Metabolic design: how to engineer a living cell to desired metabolite concentrations and fluxes

A biotechnological aim of genetic engineering is to increase the intracellular concentration or secretion of valuable compounds, while making the other concentrations and fluxes optimal for viability and productivity. Efforts to accomplish this based on over-expression of the enzyme, catalyzing the so-called rate-limiting step, have not been successful. Here we develop a method to determine the enzyme concentrations that are required to achieve such an aim. This method is called Metabolic Design Analysis and is based on the perturbation method and the modular (top-down) approach-formalisms that were first developed for the analysis of biochemical regulation such as, Metabolic Control Analysis. Contrary to earlier methods, the desired alterations of cellular metabolism need not be small or confined to a single metabolite or flux. The limits to the alterations of fluxes and metabolite concentrations are identified. To employ Metabolic Design Analysis, only limited kinetic information concerning the pathway enzymes is needed.     

Insufficient natural killer cell responses against retroviruses: how to improve NK cell killing of retrovirus-infected cells

Natural killer (NK) cells belong to the innate immune system and protect against cancers and a variety of viruses including retroviruses by killing transformed or infected cells. They express activating and inhibitory receptors on their cell surface and often become activated after recognizing virus-infected cells. They have diverse antiviral effector functions like the release of cytotoxic granules, cytokine production and antibody dependent cellular cytotoxicity. The importance of NK cell activity in retroviral infections became evident due to the discovery of several viral strategies to escape recognition and elimination by NK cells. Mutational sequence polymorphisms as well as modulation of surface receptors and their ligands are mechanisms of the human immunodeficiency virus-1 to evade NK cell-mediated immune pressure. In Friend retrovirus infected mice the virus can manipulate molecular or cellular immune factors that in turn suppress the NK cell response. In this model NK cells lack cytokines for optimal activation and can be functionally suppressed by regulatory T cells. However, these inhibitory pathways can be overcome therapeutically to achieve full activation of NK cell responses and ultimately control dissemination of retroviral infection. One effective approach is to modulate the crosstalk between NK cells and dendritic cells, which produce NK cell-stimulating cytokines like type I interferons (IFN), IL-12, IL-15, and IL-18 upon retrovirus sensing or infection. Therapeutic administration of IFNα directly increases NK cell killing of retrovirus-infected cells. In addition, IL-2/anti-IL-2 complexes that direct IL-2 to NK cells have been shown to significantly improve control of retroviral infection by NK cells in vivo. In this review, we describe novel approaches to improve NK cell effector functions in retroviral infections. Immunotherapies that target NK cells of patients suffering from viral infections might be a promising treatment option for the future.     

Learning cell biology as a team: a project-based approach to upper-division cell biology

To help students develop successful strategies for learning how to learn and communicate complex information in cell biology, we developed a quarter-long cell biology class based on team projects. Each team researches a particular human disease and presents information about the cellular structure or process affected by the disease, the cellular and molecular biology of the disease, and recent research focused on understanding the cellular mechanisms of the disease process. To support effective teamwork and to help students develop collaboration skills useful for their future careers, we provide training in working in small groups. A final poster presentation, held in a public forum, summarizes what students have learned throughout the quarter. Although student satisfaction with the course is similar to that of standard lecture-based classes, a project-based class offers unique benefits to both the student and the instructor.     

Teaching critical thinking in a developmental biology course at an American liberal arts college

We all expect our students to learn facts and concepts, but more importantly, we want them to learn how to evaluate new information from an educated and skeptical perspective; that is, we want them to become critical thinkers. For many of us who are scientists and teachers, critical thought is either intuitive or we learned it so long ago that it is not at all obvious how to pass on the skills to our students. Explicitly discussing the logic that underlies the experimental basis of developmental biology is an easy and very successful way to teach critical thinking skills. Here, I describe some simple changes to a lecture course that turn the practice of critical thinking into the centerpiece of the learning process. My starting point is the "Evidence and Antibodies" sidelight in Gilbert's Developmental Biology (2000), which I use as an introduction to the ideas of correlation, necessity and sufficiency, and to the kinds of experiments required to gather each type of evidence: observation ("show it"), loss of function ("block it") and gain of function ("move it"). Thereafter, every experiment can be understood quickly by the class and discussed intelligently with a common vocabulary. Both verbal and written reinforcement of these ideas dramatically improve the students' ability to evaluate new information. In particular, they are able to evaluate claims about cause and effect; they become experts at distinguishing between correlation and causation. Because the intellectual techniques are so powerful and the logic so satisfying, the students come to view the critical assessment of knowledge as a fun puzzle and the rigorous thinking behind formulating a question as an exciting challenge.     

Accessory cells, dendritic cells, or monocytes, are required for the lymphokine-activated killer cell induction from resting T cell but not from natural killer cell precursors

In this study we have investigated the role of accessory cells in the development of lymphokine-activated killer cells (LAK) from highly purified human NK and small resting T cell progenitors. As accessory cells we used autologous, as well as allogeneic, monocytes, and dendritic cell enriched cells. Both NK and T cells were able to generate LAK activity, but their activation requirements were different. NK cells were activated merely by IL-2, and accessory cells did not enhance their lytic activity in the presence or absence of IL-2. Conversely, T cells were practically unresponsive to even high concentrations of IL-2 having a strict requirement for accessory cells for the development of lytic activity and proliferation. Accessory cells differed in their ability to activate T cells presumably depending on their ability to induce IL-2 synthesis, allogeneic dendritic cells being the most effective accessory cells and IL-2 synthesis stimulators. Allogeneic accessory cells could induce lytic activity in T cells even in the absence of exogenous IL-2. Thus, accessory cells play a central role in expanding the LAK effector cell population.     

Maintaining a relevant, up-to-date capstone design course

Senior capstone design courses can be extremely helpful in preparing biomedical engineering students for careers in engineering and other fields. They allow students to develop communication, teamwork, and other transferable technical and nontechnical skills. They can also make students aware of the (1) legal, regulatory, economic, environmental, and social/political constraints of medical device design, (2) contemporary issues related to biomedical engineering and health care, and (3) the latest trends and tools in new product development and project management.     

Bacterial cyclostatin, or how do bacteria manipulate the eukaryotic cell cycle

Several bacterial proteins have been recently described that share the ability to inhibit the proliferation of cells in culture without causing early signs of cytotoxicity. Such observations suggest the existence of bacterial mechanisms of control of the eukaryotic cell cycle contributing to pathogenicity or adaptation to the host. This emerging concept of cellular microbiology is critically analyzed considering as a model the cytolethal distending toxins (CDT), a family of toxins whose mode of action on the cell cycle has been thoroughly studied over the last few years. CDTs activate a physiological G2 checkpoint in exposed cells, probably from an initial DNA alteration whose precise molecular nature has not yet been determined. Experimental data are lacking to extrapolate in vivo the antiproliferative effect of these bacterial proteins that we tentatively propose to call cyclostatins.     

Sorting through the cell biology of Alzheimer's disease: intracellular pathways to pathogenesis

During the first 100 years of Alzheimer's disease research, this devastating and intractable disorder has been characterized at the clinical, histological, and molecular levels. Nevertheless, many key mechanistic questions remain unanswered. Here we will emphasize the importance of the cell biology of Alzheimer's disease, reviewing the relevant literature that has expanded our mechanistic understanding, with a particular focus on pathways regulating protein sorting. Accumulated evidence indicates that sorting pathways may be uniquely vulnerable to disease pathogenesis, and recent studies have begun to reveal disease-related defects in the regulation of protein sorting.     

To sleep, to strive, or both: how best to optimize memory

While numerous studies have shown that a night of sleep profits memory relative to wake, we still have little understanding about what factors mediate this effect of sleep. A clear understanding of the dynamics of this effect of sleep beyond the initial night of sleep is also lacking. Here, we examined the effect of extrinsic rewards on sleep-dependent declarative memory processing across 12 and 24 hr training-retest intervals. Subjects were either paid based on their performance at retest ($1 for each correct answer), or received a flat fee for participation. After a 12 hr interval we observed pronounced benefits of both sleep and reward on memory. Over an extended 24 hr interval we found 1) that an initial night of sleep partially protects memories from subsequent deterioration during wake, and 2) that sleep blocks further deterioration, and may even have a restorative effect on memory, when it follows a full day of wake. Interestingly, the benefit imparted to rewarded (relative to unrewarded) stimuli was equal for sleep and wake subjects, suggesting that the sleeping brain may not differentially process rewarded information, relative to wake. However, looking at the overall impact of sleep relative to reward in this protocol, it was apparent that sleep both imparted a stronger mnemonic boost than reward, and provided a benefit to memory regardless of whether it occurred in the first or the second 12 hrs following task training.     

The influence of protein malnutrition on T cell, natural killer cell, and lymphokine-activated killer cell function, and on biological responsiveness to high-dose interleukin-2

Protein malnutrition is prevalent in cancer patients, however the influence of protein-calorie malnutrition on anti-tumor immune effector mechanisms is unclear. In addition, the effect of malnutrition on host immunological and biological responsiveness to recombinant interleukin-2 (rIL-2) is unknown. In Swiss mice (n = 100), we observed that T cell activation, T cell response to rIL-2, T suppressor cell generation, cytotoxic T lymphocyte development, and the cytolytic activity of LAK cells were not significantly impaired by two or three weeks of feeding with a 2.5% protein diet compared with mice fed an isocaloric diet in which protein calories constituted 24% of the total. In CBA/J mice (n = 100), we observed a significant (P less than 0.05) impairment of poly(I:C)-inducible natural killer cell function in mice ingesting the 2.5% diet. In both A/J (n = 40) and Swiss mice (n = 40), cytotoxic responses after 3 days treatment with rIL-2 (5 X 10(6) U/kg body wt. three times daily) were comparable in both dietary groups. These studies demonstrate that protein depletion is associated with impaired poly (I:C)-induced natural killer cell function in CBA/J mice. However, T cell function and biological responsiveness to high-dose rIL-2 were not significantly impaired.     

Regulation of natural killer cell-mediated swine endothelial cell lysis through genetic remodeling of a glycoantigen

The effect of remodeling of a glycoantigen such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes on natural killer (NK) cell-mediated direct cytotoxicity was investigated using human peripheral blood mononuclear cells (PBMC) or an NK-like cell line, YT cells, as an effector, and swine endothelial cells (SEC) as a target. Several SEC transfectants were established by transfection with the genes for beta1,4-N-acetylglucosaminyltransferase III, alpha2, 3-sialyltransferase and alpha1,2-fucosyltransferase. These transfections led to dramatic reductions in both direct and indirect NK cell-mediated cytotoxicity, by 72-94% in the case of PBMC and 27-72% in that of YT cells, in addition to an effective reduction in xenoantigenicity, which is substantially caused by the alpha-Gal epitope, to human natural antibodies. The NK cell-mediated direct cytotoxicity was remarkably blocked by an anti-alpha-Gal epitope monoclonal antibody or GSI lectin which preferentially binds to the epitope. Furthermore, treatment of the parental cells with alpha-galactosidase resulted in a significant reduction in cytotoxicity. These results suggest that the alpha-Gal epitope is involved not only in hyperacute rejection and acute vascular rejection, but also in NK cell-mediated direct cytotoxicity. Thus, the genetic remodeling of the alpha-Gal epitope and probably other glycoantigens as well can be expected to represent a new approach for overcoming not only indirect but also direct immunity to xenografts.     

Differential effects of BCNU on T cell,macrophage, natural killer and lymphokine-activated killer cell activities in mice bearing a syngeneic tumor

Summary Chloroethylnitrosoureas have been used widely to treat human and experimental animal tumors. We have earlier observed that >90% of the mice transplanted with syngeneic tumors survive following treatment with nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and furthermore, they resist subsequent challenge with the same tumor. The present investigation was initiated to determine the mechanism by which BCNU brings about this effect. Treatment of tumor cell targets in vivo or in vitro with BCNU, increased their susceptibility to macrophage (MØ)-mediated cytotoxicity as measured in a direct cytotoxicity assay or in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In contrast, the antitumor cytotoxicity caused by cytotoxic T lymphocytes (CTL), natural killer (NK) cells, or lymphokine-activated killer (LAK) cells, was not altered following BCNU treatment of tumor targets. Studies were also conducted to investigate the direct effect of BCNU in vivo on various cytotoxic effector cells. For this purpose, MØ, NK, LAK, and CTL activities from BCNU-treated-tumor-bearing mice were screened for cytotoxicity against untreated tumor targets in vitro. It was observed that tumor-specific CTL and LAK cell activity increased in BCNU-treated tumor-bearing mice when compared to untreated controls while the cytotoxic potential of NK cells and MØs was not altered. The present study suggests that antitumor drugs such as BCNU are not only tumoricidal but also selectively act in a variety of ways at both the effector and target cell level, leading to overall enhanced antitumor immunity and high rate of cures from the syngeneic tumor challenge.The work at Virginia Polytechnic Institute and State University was supported by NIH grants CA45009 and CA45010 and by a Biomedical Research Support Grant. The work at University of Kentucky was supported by NIH grants CA34052 and CA33629 and by a grant from the Tobacco and Health Institute