Growth mindset interventions improve academic performance but not mindset in biochemistry.

Growing evidence suggests that students' self-beliefs about altering their academic abilities can directly influence long-term achievement. These self-beliefs or mindsets can either be fixed (unchangeable) or growth oriented. Students with growth mindsets believe their academic abilities can change, which leads to higher grades and academic persistence in contrast to students with fixed mindsets. However, less is known about how these attributes affect student learning, particularly in college level biochemistry courses. In this study, we utilized metacognitive interventions to promote growth mindset among third and fourth year undergraduate students enrolled in a one semester Biochemistry survey course. Using a mixed-methods study design, we evaluated student mindset, attitudes toward learning, and academic performance over four semesters. Our results suggest that although students' mindsets did not change as a result of growth mindset interventions, their positive perceptions about learning versus performance did increase. Furthermore, students receiving growth mindset interventions significantly outperformed students who did not receive interventions on the final cumulative exam that assessed critical thinking skills. These results suggest that these types of metacognitive interventions can be an effective tool to improve student academic performance in a biochemistry course.

Integrating Bioinformatics Tools Into Inquiry-Based Molecular Biology Laboratory Education Modules.

The accelerating expansion of online bioinformatics tools has profoundly impacted molecular biology, with such tools becoming integral to the modern life sciences. As a result, molecular biology laboratory education must train students to leverage bioinformatics in meaningful ways to be prepared for a spectrum of careers. Institutions of higher learning can benefit from a flexible and dynamic instructional paradigm that blends up-to-date bioinformatics training with best practices in molecular biology laboratory pedagogy. At North Carolina State University, the campus-wide interdisciplinary Biotechnology (BIT) Program has developed cutting-edge, flexible, inquiry-based Molecular Biology Laboratory Education Modules (MBLEMs). MBLEMs incorporate relevant online bioinformatics tools using evidenced-based pedagogical practices and in alignment with national learning frameworks. Students in MBLEMs engage in the most recent experimental developments in modern biology (e.g., CRISPR, metagenomics) through the strategic use of bioinformatics, in combination with wet-lab experiments, to address research questions. MBLEMs are flexible educational units that provide a menu of inquiry-based laboratory exercises that can be used as complete courses or as parts of existing courses. As such, MBLEMs are designed to serve as resources for institutions ranging from community colleges to research-intensive universities, involving a diverse range of learners. Herein, we describe this new paradigm for biology laboratory education that embraces bioinformatics as a critical component of inquiry-based learning for undergraduate and graduate students representing the life sciences, the physical sciences, and engineering.

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress.

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress.

Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is integral in the response to DNA double-stranded breaks (DSBs). Cells and tissues lacking ATM are prone to tumor development and enhanced tumor cell migration and invasion. Interestingly, ATM-deficient cells exhibit high levels of oxidative stress; however, the direct mechanism whereby ATM-associated oxidative stress may contribute to the cancer phenotype remains largely unexplored. Rac1, a member of the Rho family of GTPases, also plays an important regulatory role in cellular growth, motility, and cancer formation. Rac1 can be activated directly by reactive oxygen species (ROS), by a mechanism distinct from canonical guanine nucleotide exchange factor-driven activation. Here we show that loss of ATM kinase activity elevates intracellular ROS, leading to Rac1 activation. Rac1 activity drives cytoskeletal rearrangements resulting in increased cellular spreading and motility. Rac1 siRNA or treatment with the ROS scavenger N-Acetyl-L-cysteine restores wild-type migration. These studies demonstrate a novel mechanism whereby ATM activity and ROS generation regulates Rac1 to modulate pro-migratory cellular behavior.

Making heads or tails: planarian stem cells in the classroom.

Stem cells hold great promise in the treatment of diseases ranging from cancer to dementia. However, as rapidly as the field of stem cell biology has emerged, heated political debate has followed, scrutinizing the ethical implications of stem cell use. It is therefore imperative to promote scientific literacy by educating students about stem cell biology. Yet, there is a definite lack of material to engage students in this subject at the basic science level. Therefore, we have developed and implemented a hands-on introductory laboratory module that introduces students to stem cell biology and can be easily incorporated into existing curricula. Students learn about stem cell biology using an in vivo planarian model system in which they down-regulate two genes important in stem cell differentiation using RNA interference and then observe the regenerative phenotype. The module was piloted at the high school, community college, and university levels. Here, we report that introductory biology students enrolled at a community college were able to demonstrate gains in learning after completion of a one-hour lecture and four 45-minute laboratory sessions over the course of three weeks. These gains in learning outcomes were objectively evaluated both before and after its execution using a student quiz and experimental results. Furthermore, students' self-assessments revealed increases in perceived knowledge as well as a general interest in stem cells. Therefore, these data suggest that this module is a simple, useful way to engage and to teach students about stem cell biology.

Tumor-selective, futile redox cycle-induced bystander effects elicited by NQO1 bioactivatable radiosensitizing drugs in triple-negative breast cancers.

AIMS: ß-Lapachone (ß-lap), a novel radiosensitizer with potent antitumor efficacy alone, selectively kills solid cancers that over-express NAD(P)H: quinone oxidoreductase 1 (NQO1). Since breast or other solid cancers have heterogeneous NQO1 expression, therapies that reduce the resistance (e.g., NQO1(low)) of tumor cells will have significant clinical advantages. We tested whether NQO1-proficient (NQO1(+)) cells generated sufficient hydrogen peroxide (H2O2) after ß-lap treatment to elicit bystander effects, DNA damage, and cell death in neighboring NQO1(low) cells. RESULTS: ß-Lap showed NQO1-dependent efficacy against two triple-negative breast cancer (TNBC) xenografts. NQO1 expression variations in human breast cancer patient samples were noted, where ~60% cancers over-expressed NQO1, with little or no expression in associated normal tissue. Differential DNA damage and lethality were noted in NQO1(+) versus NQO1-deficient (NQO1(-)) TNBC cells and xenografts after ß-lap treatment. ß-Lap-treated NQO1(+) cells died by programmed necrosis, whereas co-cultured NQO1(-) TNBC cells exhibited DNA damage and caspase-dependent apoptosis. NQO1 inhibition (dicoumarol) or H2O2 scavenging (catalase [CAT]) blocked all responses. Only NQO1(-) cells neighboring NQO1(+) TNBC cells responded to ß-lap in vitro, and bystander effects correlated well with H2O2 diffusion. Bystander effects in NQO1(-) cells in vivo within mixed 50:50 co-cultured xenografts were dramatic and depended on NQO1(+) cells. However, normal human cells in vitro or in vivo did not show bystander effects, due to elevated endogenous CAT levels. Innovation and Conclusions: NQO1-dependent bystander effects elicited by NQO1 bioactivatable drugs (ß-lap or deoxynyboquinone [DNQ]) likely contribute to their efficacies, killing NQO1(+) solid cancer cells and eliminating surrounding heterogeneous NQO1(low) cancer cells. Normal cells/tissue are protected by low NQO1:CAT ratios.

Inquiry into chemotherapy-induced p53 activation in cancer cells as a model for teaching signal transduction.

Intracellular and extracellular communication is conducted through an intricate and interwoven network of signal transduction pathways. The mechanisms for how cells speak with one another are of significant biological importance to both basic and industrial scientists from a number of different disciplines. We have therefore developed and implemented a new laboratory-intensive course that teaches students the theory and techniques used to study cell signaling pathways. Students learn these methodologies as they conduct a hypothesis-driven research project where they elucidate the mechanism of breast cancer cell death caused by a cancer chemotherapeutic agent. While each lab experiment can be conducted independently, the findings build upon one another to form the beginnings of a signaling pathway. In the lecture component of the course, students investigate different signaling pathways and the methods employed to study them. In addition, students actively participate in journal article discussions where they assess the primary scientific literature. We evaluated the course over two semesters and found that in both semesters learning outcomes were met by both undergraduate and graduate students. The evaluation of the course was based on a number of instructor assessments of student work, including lab reports, experimental results, journal article discussions, and a final cumulative exam. Furthermore, students' self-assessments revealed gains in perceived confidence in both conceptual knowledge and technical skills

Catalase abrogates ß-lapachone-induced PARP1 hyperactivation-directed programmed necrosis in NQO1-positive breast cancers.

Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. ß-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER-, PR-, Her2/Neu-)] breast cancers. To define cellular factors that influence the efficacy of ß-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where ∼120 moles of superoxide were formed per mole of ß-lapachone in 2 minutes. ß-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD(+)/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of ß-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. ß-Lapachone-induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of ß-lapachone and other NQO1 bioactivatable drugs.

Assessment of a novel group-centered testing schema in an upper-level undergraduate molecular biotechnology course.

Providing students with assignments that focus on critical thinking is an important part of their scientific and intellectual development. However, as class sizes increase, so does the grading burden, prohibiting many faculty from incorporating critical thinking assignments in the classroom. In an effort to continue to provide our students with meaningful critical thinking exercises, we implemented a novel group-centered, problem-based testing scheme. We wanted to assess how performing critical thinking problem sets as group work compares to performing the sets as individual work, in terms of student attitudes and learning outcomes. During two semesters of our recombinant DNA course, students had the same lecture material and similar assessments. In the Fall semester, student learning was assessed by two collaborative take-home exams, followed immediately by individual, closed-book in-class exams on the same content, as well as a final cumulative exam. Student teams on the take-home exams were instructor-assigned, and each team turned in one collaborative exam. In the Spring semester, the control group of students were required to turn in their own individual take-home exams, followed by the in-class exams and final cumulative exam. For the majority of students, learning outcomes were met, regardless of whether they worked in teams. In addition, collaborative learning was favorably received by students and grading was reduced for instructors. These data suggest that group-centered, problem-based learning is a useful model for achievement of student learning outcomes in courses where it would be infeasible to provide feedback on individual critical thinking assignments due to grading volume.

The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.

Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

The small GTPase RhoA localizes to the nucleus and is activated by Net1 and DNA damage signals.

BACKGROUND: Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells. PRINCIPAL FINDINGS: Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response.