Initial Development and Validation of the Plant Awareness Disparity Index.

Plant awareness disparity (PAD, formerly plant blindness) is the idea that students tend not to notice or appreciate the plants in their environment. This phenomenon often leads to naïve points of view, such as plants are not important or do not do anything for humans. There are four components of PAD: attitude (not liking plants), attention (not noticing plants), knowledge (not understanding the importance of plants), and relative interest (finding animals more interesting than plants). Many interventions have been suggested to prevent PAD, but without an instrument shown to demonstrate valid inferences to measure PAD, it is difficult to tell whether these interventions are successful or not. We have developed and validated the Plant Awareness Disparity Index (PAD-I) to measure PAD and its four components in undergraduate biology students. The study population was 74.32% female and 69.08% white, indicating that the need for further analysis is necessary if this instrument is to be used in a more diverse student population. We collected validity evidence based upon text content, response processes, and internal structure. Our findings demonstrate that our instrument generates reliable inferences regarding PAD with a Cronbach's alpha of 0.884 and a six-factor structure that aligns conceptually with the four components of PAD.

From genetics to biotechnology: Synthetic biology as a flexible course-embedded research experience.

The need for changing how science is taught and the expansion of undergraduate research experiences is essential to foster critical thinking in the Natural Sciences. Most faculty research programs only involve a small number of upper-level undergraduate students each semester. The course-based undergraduate research experience (CURE) model enables more students to take ownership over an independent project and experience authentic research. Further, by creating projects that fit into a curriculum's learning goals and student-oriented outcomes, departments help strengthen critical thinking skills in the classroom. Here, we report on the incorporation of a synthetic biology CURE into a mid-level cellular biology course and two advanced level genetics/molecular biology courses. Synthetic biology involves systematic engineering of novel organisms, such as bacteria and plants, to work as functional devices to solve problems in medicine, agriculture, and manufacturing. The value of synthetic biology and its ultimate utility as a teaching tool relies on reusable, standard genetic parts that can be interchanged using common genetic engineering principles. This Synthetic biology CURE effectively achieves five essential goals: (1) a sense of project ownership; (2) self-efficacy: mastery of a manageable number of techniques; (3) increased tolerance for obstacles through challenging research; (4) increased communication skills; and (5) a sense of belonging in a larger scientific community. Based upon our student assessment data, we demonstrate that this course-based synthetic biology laboratory engages students directly in an authentic research experience and models important elements of collaboration, discovery, iteration, and critical thinking.

Preparing for Class: Actions and Resources of Introductory Biology Students.

Instructors want students to be prepared for class. There are several different resources and activities available to help students prepare for class, but very little is known about how students choose to prepare for class in the context of undergraduate biology. In this study, we used content analysis to investigate what students do to prepare for an introductory biology course and if and how that preparation differed under two different conditions. Students were either directed to search out and choose their resources to prepare for class (choice treatment) or assigned specific pages from a textbook (text treatment). Students in the choice treatment reported preparing for class slightly more often than students in the text treatment, with both groups reporting that they prepared for over three-quarters of classes. However, students assigned specific textbook passages engaged more while preparing than students who had to find their resources. The textbook was a popular resource but second to websites for students who got to choose. Students in both groups performed similarly in the course. This work helps instructors understand what their students may be doing to prepare for class. We provide recommendations to guide instructors on how to help their students prepare for class.

Transitioning Cell Culture CURE Labs from Campus to Online: Novel Strategies for a Novel Time.

Course-based undergraduate research experiences (CUREs) provide a way for students to gain research experience in a classroom setting. Few examples of cell culture CUREs or online CUREs exist in the literature. The Cell Biology Education Consortium (CBEC) provides a network and resources for instructors working to incorporate cell-culture based research into the classroom. In this article, we provide examples from six instructors from the CBEC network on how they structure their cell-culture CUREs and how they transitioned the labs to online in the spring semester of 2020. We intend for these examples to provide instructors with ideas for strategies to set up cell culture CUREs, how to change that design mid-term, and for creating online CUREs in the future.

Introductory Biology Students' Use of Enhanced Answer Keys and Reflection Questions to Engage in Metacognition and Enhance Understanding.

Providing feedback to students as they learn to integrate individual concepts into complex systems is an important way to help them to develop robust understanding, but it is challenging in large, undergraduate classes for instructors to provide feedback that is frequent and directed enough to help individual students. Various scaffolds can be used to help students engage in self-regulated learning and generate internal feedback to improve their learning. This study examined the use of enhanced answer keys with added reflection questions and instruction as scaffolds for engaging undergraduate students in self-regulated learning within an introductory biology course. Study findings show that both the enhanced answer keys and reflection questions helped students to engage in metacognition and develop greater understanding of biological concepts. Further, students who received additional instruction on the use of the scaffolds changed how they used them and, by the end of the semester, were using the scaffolds in significantly different ways and showed significantly higher learning gains than students who did not receive the instruction. These findings provide evidence for the benefit of designing scaffolds within biology courses that will support students in engaging in metacognition and enhancing their understanding of biological concepts.

Maternal Interferon Regulatory Factor 6 is required for the differentiation of primary superficial epithelia in Danio and Xenopus embryos.

Early in the development of animal embryos, superficial cells of the blastula form a distinct lineage and adopt an epithelial morphology. In different animals, the fate of these primary superficial epithelial (PSE) cells varies, and it is unclear whether pathways governing segregation of blastomeres into the PSE lineage are conserved. Mutations in the gene encoding Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic forms of cleft lip and palate, consistent with a role for Irf6 in development of oral epithelia, and mouse Irf6 targeted null mutant embryos display abnormal differentiation of oral epithelia and skin. In Danio rerio (zebrafish) and Xenopus laevis (African clawed frog) embryos, zygotic irf6 transcripts are present in many epithelial tissues including the presumptive PSE cells and maternal irf6 transcripts are present throughout all cells at the blastula stage. Injection of antisense oligonucleotides with ability to disrupt translation of irf6 transcripts caused little or no effect on development. By contrast, injection of RNA encoding a putative dominant negative Irf6 caused epiboly arrest, loss of gene expression characteristic of the EVL, and rupture of the embryo at late gastrula stage. The dominant negative Irf6 disrupted EVL gene expression in a cell autonomous fashion. These results suggest that Irf6 translated in the oocyte or unfertilized egg suffices for early development. Supporting the importance of maternal Irf6, we show that depletion of maternal irf6 transcripts in X. laevis embryos leads to gastrulation defects and rupture of the superficial epithelium. These experiments reveal a conserved role for maternally-encoded Irf6 in differentiation of a simple epithelium in X. laevis and D. rerio. This epithelium constitutes a novel model tissue in which to explore the Irf6 regulatory pathway.

Characterization of modified antisense oligonucleotides in Xenopus laevis embryos.

A wide variety of modified oligonucleotides have been tested as antisense agents. Each chemical modification produces a distinct profile of potency, toxicity, and specificity. Novel cationic phosphoramidate-modified antisense oligonucleotides have been developed recently that have unique and interesting properties. We compared the relative potency and specificity of a variety of established antisense oligonucleotides, including phosphorothioates (PS), 2'-O-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), and neutral methoxyethyl (MEA) phosphoramidates with new cationic N,N-dimethylethylenediamine (DMED) phosphoramidate-modified antisense oligonucleotides. A series of oligonucleotides was synthesized that targeted two sites in the Xenopus laevis survivin gene and were introduced into Xenopus embryos by microinjection. Effects on survivin gene expression were examined using quantitative real-time PCR. Of the various modified oligonucleotide designs tested, LNA/PS chimeras (which showed the highest melting temperature) and DMED/phosphodiester chimeras (which showed protection of neighboring phosphate bonds) were potent in reducing gene expression. At 40 nM, overall specificity was superior for the LNA/PS-modified compounds compared with the DMED-modified oligonucleotides. However, at 400 nM, both of these compounds led to significant degradation of survivin mRNA, even when up to three mismatches were present in the heteroduplex.

Defective skeletogenesis with kidney stone formation in dwarf zebrafish mutant for trpm7.

Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as to global endocrine and physiological effectors. An excellent example of such coordination is the skeleton. Skeletal development depends on the differentiation and morphogenesis of multiple cell types to generate elements with distinct forms and functions throughout the body. We show that zebrafish touchtone/nutria mutants exhibit severe growth retardation and gross alterations in skeletal development in addition to embryonic melanophore and touch-response defects. These alterations include accelerated endochondral ossification but delayed intramembranous ossification, as well as skeletal deformities. We show that the touchtone/nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and kinase. We find trpm7 expression in the mesonephric kidney and show that mutants develop kidney stones, indicating renal dysfunction. These results identify a requirement for trpm7 in growth and skeletogenesis and highlight the potential of forward genetic approaches to uncover physiological mechanisms contributing to the development of adult form.

Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis.

The experimental manipulation of early embryologic events, resulting in the misexpression of the homeobox transcription factor pitx2, is associated with subsequent defects of laterality in a number of vertebrate systems. To clarify the role of one pitx2 isoform, pitx2c, in determining the left-right axis of amphibian embryos, we examined the heart and gut morphology of Xenopus laevis embryos after attenuating pitx2c mRNA levels using chemically modified antisense oligonucleotides. We demonstrate that the partial depletion of pitx2c mRNA in these embryos results in alteration of both cardiac morphology and intestinal coiling. The most common cardiac abnormality seen was a failure of rightward migration of the outflow tract, while the most common intestinal laterality phenotype seen was a full reversal in the direction of coiling, each present in 23% of embryos injected with the pitx2c antisense oligonucleotide. An abnormality in either the heart or gut further predisposed to a malformation in the other. In addition, a number of other cardiac anomalies were observed after pitx2c mRNA attenuation, including abnormalities of atrial septation, extracellular matrix restriction, relative atrial-ventricular chamber positioning, and restriction of ventricular development. Many of these findings correlate with cardiac defects previously reported in pitx2 null and hypomorphic mice, but can now be assigned specifically to attenuation of the pitx2c isoform in Xenopus.

Survivin mRNA is down-regulated during early Xenopus laevis embryogenesis.

One of the hallmarks of early development is the rapid proliferation of cells immediately after fertilization. Many of the rules that govern cell division in normal somatic cells, such as contact inhibition and apoptosis, seem temporarily suspended in the early embryo. A similar suspension of mechanisms normally regulating cell division occurs in the development of cancer. Survivin, an inhibitor of apoptosis and a positive regulator of progression through the cell cycle, localizes to the mitotic spindle and interacts with several proapoptotic caspases. Survivin protein expression has been studied during the development of the salivary gland in mouse. However, the regulation of survivin during the critical transitions defining oocyte maturation and the early restriction of developmental potential are not easily examined in the mouse. We therefore studied survivin mRNA expression during oogenesis and early embryogenesis in Xenopus laevis. We found that survivin mRNA is present in the earliest stages of Xenopus oocytes and that it accumulates during oogenesis. Progesterone-induced maturation of Xenopus oocytes leads to polyadenylation of the survivin transcript. Survivin mRNA is also present in early Xenopus embryos. After the onset of zygotic transcription, however, the amount of survivin mRNA declines rapidly to undetectable levels. This decrease in survivin mRNA correlates temporally with both the slowing of the cell cycle and the onset of endogenous embryonic apoptosis. With the exception of the ovary, survivin mRNA was undetectable in all adult Xenopus tissues examined.