Covariational reasoning and item context affect language in undergraduate mass balance written explanations.

Mass balance (MB) reasoning offers a rich topic for examination of students' scientific thinking and skills, as it requires students to account for multiple inputs and outputs within a system and apply covariational reasoning. Using previously validated constructed response prompts for MB, we examined 1,920 student-constructed responses (CRs) aligned to an emerging learning progression to determine how student language changes from low (1) to high (4) covariational reasoning levels. As students' abilities and thinking change with Context, we used the same general prompt in six physiological contexts. We asked how Level and Context affect student language and what language is conserved across Contexts at higher reasoning Levels. Using diversity methods, we found student language becomes more similar as covariational reasoning level increases. Using text analysis, we found context-dependent words at each Level; however, the type of context words changed. Specifically, at Level 1, students used context words that are tangential to MB reasoning, while Level 4 responses used words that specify inputs and outputs for the given Item Context. Further, at Level 4, students shared 30% of language across the six contexts and leveraged context-independent words including rate, equal, and some form of slower/lower/smaller. Together, these data demonstrate that Context affects undergraduate MB language at all covariational reasoning levels, but that the language becomes more specific and similar as Level increases. These findings encourage instructors to foster context-independent, comparative, and summative language during instruction to functionally build MB and covariational reasoning skills across contexts.NEW & NOTEWORTHY This article builds on the work of Scott et al. (Scott EE, Cerchiara J, McFarland JL, Wenderoth MP, Doherty JH. J Res Sci Teach 1: 37, 2023) and Shiroda et al. (Shiroda M, Fleming MP, Haudek KC. Front Educ 8: 989836, 2023) to quantitatively examine student language in written explanations of mass balance across six contexts using constructed response assessments. These results present an evaluation of student mass balance language and provide researchers and practitioners with tools to assist students in constructing scientific mass balance reasoning explanations.

What a Difference in Pressure Makes! A Framework Describing Undergraduate Students' Reasoning about Bulk Flow Down Pressure Gradients.

Pressure gradients serve as the key driving force for the bulk flow of fluids in biology (e.g., blood, air, phloem sap). However, students often struggle to understand the mechanism that causes these fluids to flow. To investigate student reasoning about bulk flow, we collected students' written responses to assessment items and interviewed students about their bulk flow ideas. From these data, we constructed a bulk flow pressure gradient reasoning framework that describes the different patterns in reasoning that students express about what causes fluids to flow and ordered those patterns into sequential levels from more informal ways of reasoning to more scientific, mechanistic ways of reasoning. We obtained validity evidence for this bulk flow pressure gradient reasoning framework by collecting and analyzing written responses from a national sample of undergraduate biology and allied health majors from 11 courses at five institutions. Instructors can use the bulk flow pressure gradient reasoning framework and assessment items to inform their instruction of this topic and formatively assess their students' progress toward more scientific, mechanistic ways of reasoning about this important physiological concept.

Oaks to arteries: the Physiology Core Concept of flow down gradients supports transfer of student reasoning.

The Physiology Core Concept of flow down gradients is a major concept in physiology, as pressure gradients are the key driving force for the bulk flow of fluids in biology. However, students struggle to understand that this principle is foundational to the mechanisms governing bulk flow across diverse physiological systems (e.g., blood flow, phloem sap flow). Our objective was to investigate whether bulk flow items that differ in scenario context (i.e., taxa, amount of scientific terminology, living or nonliving system) or in which aspect of the pressure gradient is kept constant (i.e., starting pressure or pressure gradient) influence undergraduate students' reasoning. Item scenario context did not impact the type of reasoning students used. However, students were more likely to use the Physiology Core Concept of "flow down [pressure] gradients" when the pressure gradient was kept constant and less likely to use this concept when the starting pressure was kept constant. We also investigated whether item scenario context or which aspect of the pressure gradient is kept constant impacted how consistent students were in the type of reasoning they used across two bulk flow items on the same homework. Most students were consistent across item scenario contexts (76%) and aspects of the pressure gradient kept constant (70%). Students who reasoned using "flow down gradients" on the first item were the most consistent (86, 89%), whereas students using "pressures indicate (but don't cause) flow" were the least consistent (43, 34%). Students who are less consistent know that pressure is somehow involved or indicates fluid flow but do not have a firm grasp of the concept of a pressure gradient as the driving force for fluid flow. These findings are the first empirical evidence to support the claim that using Physiology Core Concept reasoning supports transfer of knowledge across different physiological systems.NEW & NOTEWORTHY These findings are the first empirical evidence to support the claim that using Physiology Core Concept reasoning supports transfer of knowledge across different physiological systems.

Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

Automated Writing Assessments Measure Undergraduate Learning after Completion of a Computer-Based Cellular Respiration Tutorial.

The focus of biology education has shifted from memorization to conceptual understanding of core biological concepts such as matter and energy relationships. To examine undergraduate learning about matter and energy, we incorporated constructed-response (CR) questions into an interactive computer-based tutorial. The objective of this tutorial is to teach students about matter and energy and help dispel common misconceptions through the context of cellular respiration. We used a constructed-response classifier (CRC) tool to categorize ideas in responses to three CR questions and measure changes in student thinking about cellular respiration. Our data set includes 841 undergraduates from 19 geographically diverse institutions including two-year colleges, primarily undergraduate institutions, and research-intensive colleges and universities. We found students from all institution types included more scientific ideas in CRs post-tutorial. Students used an average of 2.1 ideas in CRs and frequently used both scientific and developing ideas. We found this mixed thinking persisted after the tutorial regardless of institution type. Students' multiple-choice (MC) selections were correlated with their CRs, but CRs revealed more mixed thinking than would be inferred from MC responses. Our study shows a CRC tool can measure student learning after a computer-based tutorial and provides more complete information than MC responses.

Introductory biology undergraduate students' mixed ideas about genetic information flow.

The core concept of genetic information flow was identified in recent calls to improve undergraduate biology education. Previous work shows that students have difficulty differentiating between the three processes of the Central Dogma (CD; replication, transcription, and translation). We built upon this work by developing and applying an analytic coding rubric to 1050 student written responses to a three-question item about the CD. Each response was previously coded only for correctness using a holistic rubric. Our rubric captures subtleties of student conceptual understanding of each process that previous work has not yet captured at a large scale. Regardless of holistic correctness scores, student responses included five or six distinct ideas. By analyzing common co-occurring rubric categories in student responses, we found a common pair representing two normative ideas about the molecules produced by each CD process. By applying analytic coding to student responses preinstruction and postinstruction, we found student thinking about the processes involved was most prone to change. The combined strengths of analytic and holistic rubrics allow us to reveal mixed ideas about the CD processes and provide a detailed picture of which conceptual ideas students draw upon when explaining each CD process.

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads.

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

A 10S galectin-3-U1 snRNP complex assembles into active spliceosomes.

In previous studies, we reported that fractionation of HeLa cell nuclear extracts on glycerol gradients revealed an endogenous ∼10S particle that contained galectin-3 and U1 snRNP and this particle was sufficient to load the galectin polypeptide onto a pre-mRNA substrate. We now document that this interaction between the galectin-3-U1 snRNP particle and the pre-mRNA results in a productive spliceosomal complex, leading to intermediates and products of the splicing reaction. Nuclear extracts were depleted of U1 snRNP with a concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3-U1 snRNP particle, isolated by immunoprecipitation of the 10S region (fractions 3-5) of the glycerol gradient with anti-galectin-3 antibodies. In contrast, parallel anti-galectin-3 immunoprecipitation of free galectin-3 molecules not in a complex with U1 snRNP (fraction 1 of the same gradient), failed to restore splicing activity. These results indicate that the galectin-3-U1 snRNP-pre-mRNA ternary complex is a functional E complex and that U1 snRNP is required to assemble galectin-3 onto an active spliceosome.

Examining the impact of question surface features on students' answers to constructed-response questions on photosynthesis.

One challenge in science education assessment is that students often focus on surface features of questions rather than the underlying scientific principles. We investigated how student written responses to constructed-response questions about photosynthesis vary based on two surface features of the question: the species of plant and the order of two question prompts. We asked four versions of the question with different combinations of the two plant species and order of prompts in an introductory cell biology course. We found that there was not a significant difference in the content of student responses to versions of the question stem with different species or order of prompts, using both computerized lexical analysis and expert scoring. We conducted 20 face-to-face interviews with students to further probe the effects of question wording on student responses. During the interviews, we found that students thought that the plant species was neither relevant nor confusing when answering the question. Students identified the prompts as both relevant and confusing. However, this confusion was not specific to a single version.

Examination of the role of galectins in pre-mRNA splicing.

Several lines of evidence have been accumulated to indicate that galectin-1 and galectin-3 are two of the many proteins involved in nuclear splicing of pre-mRNA. First, nuclear extracts, capable of carrying out splicing of pre-mRNA in a cell-free assay, contain both of the galectins. Second, depletion of the galectins from nuclear extracts, using either lactose affinity chromatography or immunoadsorption with antibodies, results in concomitant loss of splicing activity. Third, addition of either galectin-1 or galectin-3 to the galectin-depleted extract reconstitutes the splicing activity. Fourth, the addition of saccharides that bind to galectin-1 and galectin-3 with high affinity (e.g., lactose or thiodigalactoside) to nuclear extract results in inhibition of splicing whereas parallel addition of saccharides that do not bind to the galectins (e.g., cellobiose) fail to yield the same effect. Finally, when a splicing reaction is subjected to immunoprecipitation by antibodies directed against galectin-1, radiolabeled RNA species corresponding to the starting pre-mRNA substrate, the mature mRNA product, and intermediates of the splicing reaction are coprecipitated with the galectin. Similar results were also obtained with antibodies against galectin-3. This chapter describes two key assays used in our studies: one reports on the splicing activity by looking at product formation on a denaturing gel; the other reports on the intermediates of spliceosome assembly using non-denaturing or native gels.

What are they thinking? Automated analysis of student writing about acid-base chemistry in introductory biology.

Students' writing can provide better insight into their thinking than can multiple-choice questions. However, resource constraints often prevent faculty from using writing assessments in large undergraduate science courses. We investigated the use of computer software to analyze student writing and to uncover student ideas about chemistry in an introductory biology course. Students were asked to predict acid-base behavior of biological functional groups and to explain their answers. Student explanations were rated by two independent raters. Responses were also analyzed using SPSS Text Analysis for Surveys and a custom library of science-related terms and lexical categories relevant to the assessment item. These analyses revealed conceptual connections made by students, student difficulties explaining these topics, and the heterogeneity of student ideas. We validated the lexical analysis by correlating student interviews with the lexical analysis. We used discriminant analysis to create classification functions that identified seven key lexical categories that predict expert scoring (interrater reliability with experts = 0.899). This study suggests that computerized lexical analysis may be useful for automatically categorizing large numbers of student open-ended responses. Lexical analysis provides instructors unique insights into student thinking and a whole-class perspective that are difficult to obtain from multiple-choice questions or reading individual responses.

Harnessing technology to improve formative assessment of student conceptions in STEM: forging a national network.

Concept inventories, consisting of multiple-choice questions designed around common student misconceptions, are designed to reveal student thinking. However, students often have complex, heterogeneous ideas about scientific concepts. Constructed-response assessments, in which students must create their own answer, may better reveal students' thinking, but are time- and resource-intensive to evaluate. This report describes the initial meeting of a National Science Foundation-funded cross-institutional collaboration of interdisciplinary science, technology, engineering, and mathematics (STEM) education researchers interested in exploring the use of automated text analysis to evaluate constructed-response assessments. Participants at the meeting shared existing work on lexical analysis and concept inventories, participated in technology demonstrations and workshops, and discussed research goals. We are seeking interested collaborators to join our research community.

SR proteins and galectins: what's in a name?

Although members of the serine (S)- and arginine (R)-rich splicing factor family (SR proteins) were initially purified on the basis of their splicing activity in the nucleus, there is recent documentation that they exhibit carbohydrate-binding activity at the cell surface. In contrast, galectins were isolated on the basis of their saccharide-binding activity and cell surface localization. Surprisingly, however, two members (galectin-1 and galectin-3) can be found in association with nuclear ribonucleoprotein complexes including the spliceosome and, using a cell-free assay, have been shown to be required splicing factors. Thus, despite the difference in terms of their original points of interest, it now appears that members of the two protein families share four key properties: (a) nuclear and cytoplasmic distribution; (b) pre-mRNA splicing activity; (c) carbohydrate-binding activity; and (d) cell surface localization in specific cells. These findings provoke stimulating questions regarding the relationship between splicing factors in the nucleus and carbohydrate-binding proteins at the cell surface.

Dynamics of galectin-3 in the nucleus and cytoplasm.

This review summarizes selected studies on galectin-3 (Gal3) as an example of the dynamic behavior of a carbohydrate-binding protein in the cytoplasm and nucleus of cells. Within the 15-member galectin family of proteins, Gal3 (M(r) approximately 30,000) is the sole representative of the chimera subclass in which a proline- and glycine-rich NH(2)-terminal domain is fused onto a COOH-terminal carbohydrate recognition domain responsible for binding galactose-containing glycoconjugates. The protein shuttles between the cytoplasm and nucleus on the basis of targeting signals that are recognized by importin(s) for nuclear localization and exportin-1 (CRM1) for nuclear export. Depending on the cell type, specific experimental conditions in vitro, or tissue location, Gal3 has been reported to be exclusively cytoplasmic, predominantly nuclear, or distributed between the two compartments. The nuclear versus cytoplasmic distribution of the protein must reflect, then, some balance between nuclear import and export, as well as mechanisms of cytoplasmic anchorage or binding to a nuclear component. Indeed, a number of ligands have been reported for Gal3 in the cytoplasm and in the nucleus. Most of the ligands appear to bind Gal3, however, through protein-protein interactions rather than through protein-carbohydrate recognition. In the cytoplasm, for example, Gal3 interacts with the apoptosis repressor Bcl-2 and this interaction may be involved in Gal3's anti-apoptotic activity. In the nucleus, Gal3 is a required pre-mRNA splicing factor; the protein is incorporated into spliceosomes via its association with the U1 small nuclear ribonucleoprotein (snRNP) complex. Although the majority of these interactions occur via the carbohydrate recognition domain of Gal3 and saccharide ligands such as lactose can perturb some of these interactions, the significance of the protein's carbohydrate-binding activity, per se, remains a challenge for future investigations.

A mechanism for incorporation of galectin-3 into the spliceosome through its association with U1 snRNP.

Previously, we showed that galectin-1 and galectin-3 are redundant pre-mRNA splicing factors associated with the spliceosome throughout the splicing pathway. Here we present evidence for the association of galectin-3 with snRNPs outside of the spliceosome (i.e., in the absence of pre-mRNA splicing substrate). Immunoprecipitation of HeLa nuclear extract with anti-galectin-3 resulted in the coprecipitation of the five spliceosomal snRNAs, core Sm polypeptides, and the U1-specific protein, U1 70K. When nuclear extract was fractionated on glycerol gradients, some galectin-3 molecules cosedimented with snRNP complexes. This cosedimentation represents bona fide galectin-3--snRNP complexes as (i) immunoprecipitation of gradient fractions with anti-galectin-3 yielded several complexes with varying ratios of snRNAs and associated proteins and (ii) the distribution of galectin-3--snRNP complexes was altered when the glycerol gradient was sedimented in the presence of lactose, a galectin ligand. A complex at approximately 10S showed an association of galectin-3 with U1 snRNP that was sensitive to treatment with ribonuclease A. We tested the ability of this U1 snRNP to recognize an exogenous pre-mRNA substrate. Under conditions that assemble early splicing complexes, we found this isolated galectin-3--U1 snRNP particle was sufficient to load galectin-3 onto a pre-mRNA substrate, but not onto a control RNA lacking splice sites. Pretreatment of the U1 snRNP with micrococcal nuclease abolished the assembly of galectin-3 onto this early complex. These data identify galectin-3 as a polypeptide associated with snRNPs in the absence of splicing substrate and describe a mechanism for the assembly of galectin-3 onto the forming spliceosome.

Nucleocytoplasmic lectins.

This review summarizes studies on lectins that have been documented to be in the cytoplasm and nucleus of cells. Of these intracellular lectins, the most extensively studied are members of the galectin family. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus, in conjunction with their interacting ligand, Gemin4. Galectin-3, -7, and -12 regulate growth, cell cycle progression, and apoptosis. Bcl-2 and synexin have been identified as interacting ligands of galectin-3, involved in its anti-apoptotic activity in the cytoplasm. Although the annexins have been studied mostly as calcium-dependent phospholipid-binding proteins mediating membrane-membrane and membrane-cytoskeleton interactions, annexins A4, A5 and A6 also bind to carbohydrate structures. Like the galectins, certain members of the annexin family can be found both inside and outside cells. In particular, annexins A1, A2, A4, A5, and A11 can be found in the nucleus. This localization is consistent with the findings that annexin A1 possesses unwinding and annealing activities of a helicase and that annexin A2 is associated with a primer recognition complex that enhances the activity of DNA polymerase alpha. Despite these efforts and accomplishments, however, there is little evidence or information on an endogenous carbohydrate ligand for these lectins that show nuclear and/or cytoplasmic localization. Thus, the significance of the carbohydrate-binding activity of any particular intracellular lectin remains as a challenge for future investigations.