Chatbot responses suggest that hypothetical biology questions are harder than realistic ones.

The biology education literature includes compelling assertions that unfamiliar problems are especially useful for revealing students' true understanding of biology. However, there is only limited evidence that such novel problems have different cognitive requirements than more familiar problems. Here, we sought additional evidence by using chatbots based on large language models as models of biology students. For human physiology and cell biology, we developed sets of realistic and hypothetical problems matched to the same lesson learning objectives (LLOs). Problems were considered hypothetical if (i) known biological entities (molecules and organs) were given atypical or counterfactual properties (redefinition) or (ii) fictitious biological entities were introduced (invention). Several chatbots scored significantly worse on hypothetical problems than on realistic problems, with scores declining by an average of 13%. Among hypothetical questions, redefinition questions appeared especially difficult, with many chatbots scoring as if guessing randomly. These results suggest that, for a given LLO, hypothetical problems may have different cognitive demands than realistic problems and may more accurately reveal students' ability to apply biology core concepts to diverse contexts. The Test Question Templates (TQT) framework, which explicitly connects LLOs with examples of assessment questions, can help educators generate problems that are challenging (due to their novelty), yet fair (due to their alignment with pre-specified LLOs). Finally, ChatGPT's rapid improvement toward expert-level answers suggests that future educators cannot reasonably expect to ignore or outwit chatbots but must do what we can to make assessments fair and equitable.

Teaching science with the "universal language" of music: alignment with the Universal Design for Learning framework.

The idea of teaching science through music has undeniable appeal in implying that learning can be engaging and fun while also covering content efficiently. Indeed, there is little doubt that songs can be uniquely memorable, suggesting mnemonic options for core content. However, many classroom implementations of science music have limitations such as an overemphasis on rote memorization, rather than a constructivist building of understanding. In this brief review, we ask how music might facilitate the learning of science content in a manner consistent with the well-known pedagogical framework of Universal Design for Learning (UDL). In our view, UDL suggests certain distinct possible benefits of incorporating music into curricula, leading us to propose four models of practice. These four models are as follows: 1) students enjoy music together, 2) students critically analyze songs as texts, 3) students creatively augment existing songs, and 4) students create new songs. Model 1 can contribute to an inclusive learning environment, while models 2-4 can encourage cognitively rich active learning, and models 3-4 can additionally help students channel scientific understanding into the creation of authentic products. We conclude with comments on logistical issues that arise in implementing these four models, including the use of appropriate rubrics and the prioritization of artistic quality.NEW & NOTEWORTHY Instructors and students often find it fun to incorporate music into science classes. However, the casual usage of music in this context can unintentionally convey that science courses are mostly about memorizing scientific facts. In this article, the authors argue for a more nuanced approach to teaching science with music, rooted in Universal Design for Learning (UDL).

Student Perceptions of a Framework for Facilitating Transfer from Lessons to Exams, and the Relevance of This Framework to Published Lessons.

A main goal of academic courses is to help students acquire knowledge and skills that they can transfer to multiple contexts. In this article, we (i) examine students' responses to test question templates (TQTs), a framework intended to facilitate transfer, and (ii) determine whether similar transfer-promoting strategies are commonly embedded in published biology lessons. In study 1, in surveys administered over several academic quarters, students consistently reported that TQTs helped them transfer course content to exams and the real world; that multiple (two to five) examples were generally needed to understand a given TQT, leading >40% students to create their own additional examples; and that TQTs would be helpful in other science courses. In study 2, among 100 peer-reviewed lessons published by CourseSource or the National Center for Case Study Teaching in Science (NCCSTS), less than 5% of lessons gave students advice about exams or helped students create additional practice problems. The latter finding is not meant as criticism of these excellent lessons, which are a boon to the biology education community. However, with TQT-like prescriptions generally absent from peer-reviewed lessons, biology instructors may wish to supplement the lessons with TQT-like strategies to explicitly connect the material to subsequent exams.

Using Test Question Templates to teach physiology core concepts.

The past ∼15 years have seen increasing interest in defining disciplinary core concepts. Within the field of physiology, Michael, McFarland, Modell, and colleagues have published studies that defined physiology core concepts and have elaborated many of these as detailed conceptual frameworks. With such helpful definitions now in place, attention is turning to the related issue of how to maximize student understanding of the core concepts by linking these "big ideas" to concrete student-facing resources for active learning and assessment. Our practitioner-based view begins with the recognition that in many if not most undergraduate physiology courses assessment drives learning. We have therefore linked published conceptual frameworks to Test Question Templates (TQTs), whose structure promotes transparent assessments as well as the active learning needed to prepare for such assessments. We provide examples of conceptual framework-linked TQTs for the physiology core concepts of Homeostasis, Flow Down Gradients, the Cell Membrane, and Cell-Cell Communication. We argue that this deployment of TQTs has at least two distinct benefits for the teaching and learning of core concepts. First, documenting the connections between conceptual frameworks and TQTs may clarify coverage and assessment of the core concepts for both instructors and students. Second, misconceptions about core concepts may be directly targeted and dispelled via thoughtful construction, arrangement, and iteration of TQTs. We propose that the TQT framework or similar approaches may be applied fruitfully to any sufficiently articulated physiology core concept for high school, undergraduate, or graduate students.NEW & NOTEWORTHY Our students often focus on the grades they need to advance through academic programs. How can instructors harness this understandable interest in grades to help students gain a true understanding of core concepts? The new framework of Test Question Templates (TQTs) shows promise in linking student priorities like test scores to instructor priorities like core concepts.

Tunes in the Zoom Room: Remote Learning via Videoconference Discussions of Physiology Songs.

As most instruction has been forced online, biology instructors have become acutely aware of the many advantages and limitations of online teaching. Here, we investigate one possible advantage of online education: the ease of allowing remote guest speakers to interact with students in real time. In particular, we piloted a model in which guest speakers could facilitate direct music-related interactions with students, possibly benefiting students' content knowledge and sense of community. In the context of an undergraduate animal physiology course, face-to-face lessons on arterial blood gases and the renal system were supplemented with videoconferences with a guest speaker who presented relevant content-rich songs and led class discussions of the lyrics. Survey and test data suggested that, after each of the lessons, the students (i) had increased confidence in their understanding of the material, (ii) performed better on objective test questions, and (iii) attributed their learning chiefly to the musical intervention. While our approach awaits further exploration and testing, this report provides preliminary evidence of its feasibility and offers practical suggestions for others who may wish to give it a try.

Is memorization the name of the game? Undergraduates' perceptions of the usefulness of physiology songs.

The possible benefits of using music to enhance learning of STEM content are numerous, diverse, and largely unproven. We sought to determine which (if any) of these possible benefits are commonly experienced by undergraduate students and are thus especially worthy of further investigation. Four hundred ninety-three students in nine physiology courses at two midsized American universities rated the usefulness of short instructor-penned mathematical physiology songs and explained in their own words why each song would or would not be a useful study aid. The students collectively perceived the usefulness of each song to depend on both academic factors (e.g., the lyrics' clarity or relevance to the course) and aesthetic values (e.g., the appeal of the rhythm or the quality of the singing). Most strikingly, although students' free responses were brief (median length: 18 words in study phase 1, 16 words in study phase 2), nearly one-half of them (1,039 of 2,191) concerned memory, suggesting that many students see educational songs primarily as mnemonic devices. A second major theme of students' comments concerned the conciseness and information density of the songs. Though all 10 songs were brief, lasting 17-54 s, students seemed to prefer shorter songs (perhaps better called "jingles"). This first-of-its-kind data set on student perceptions of educational songs should inform the creation and usage of such songs, as well as further research on their possible value.

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry.

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds.

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds' mechanisms of action--i.e., the specific molecular targets by which they kill the parasite--would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children's Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 µM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.

Bioaffinity Mass Spectrometry Screening.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS or ESI-FTMS) was used to screen 192 natural product extracts and a 659-member natural product-based fragment library for bindings to a potential malaria drug target, Plasmodium falciparum Rab11a (PfRab11a, PF13_0119). One natural product extract and 11 fragments showed binding activity. A new natural product, arborside E, was identified from the active extract of Psydrax montigena as a weak binder. Its binding activity and inhibitory activity against PfRab11a were confirmed by ESI-FTMS titration experiments and an orthogonal enzyme assay.

High-throughput screening against thioredoxin glutathione reductase identifies novel inhibitors with potential therapeutic value for schistosomiasis.

BACKGROUND: Schistosomiasis, a parasitic disease also known as bilharzia and snail fever, is caused by different species of flatworms, such as Schistosoma mansoni (S. mansoni). Thioredoxin glutathione reductase (TGR) from S. mansoni (SmTGR) is a well-characterized drug target for schistosomiasis, yet no anti-SmTGR compounds have reached clinical trials, suggesting that therapeutic development against schistosomiasis might benefit from additional scaffolds targeting this enzyme. METHODS: A high-throughput screening (HTS) assay in vitro against SmTGR was developed and applied to a diverse compound library. SmTGR activity was quantified with ThioGlo®, a reagent that fluoresces upon binding to the free sulfhydryl groups of the reaction product GSH (reduced glutathione). RESULTS: We implemented an HTS effort against 59,360 synthetic compounds. In the primary screening, initial hits (928 or 1.56 %) showing greater than 90 % inhibition on SmTGR activity at a final concentration of 10 µM for each compound were identified. Further tests were carried out to confirm the effects of these hits and to explore the concentration-dependent response characteristics. As a result, 74 of them (0.12 %) representing 17 chemical scaffolds were confirmed and showed a great concentration-dependent inhibitory trend against SmTGR, including structures previously shown to be lethal to schistosomal growth. Of these, two scaffolds displayed a limited structure-activity relationship. When tested in cultured larvae, 39 compounds had cidal activity in 48 h, and five of them killed larvae completely at 3.125 µM. Of these, three compounds also killed adult worms ex vivo at concentrations between 5 µM and 10 µM. CONCLUSION: These confirmed hits may serve as starting points for the development of novel therapeutics to combat schistosomiasis.

The Bacterial Sec Pathway of Protein Export: Screening and Follow-Up.

Most noncytoplasmic bacterial proteins are exported through the SecYEG channel in the cytoplasmic membrane. This channel and its associated proteins, collectively referred to as the Sec pathway, have strong appeal as a possible antibiotic drug target, yet progress toward new drugs targeting this pathway has been slow, perhaps due partly to many researchers' focus on a single component, the SecA ATPase. Here we report on a pathway-based screen in which beta-galactosidase (ß-gal) activity is trapped in the cytoplasm of Escherichia coli cells if translocation through SecYEG is impaired. Several hit compounds passed a counterscreen distinguishing between ß-gal overexpression and impaired ß-gal export. However, the most extensively characterized hit gave limited E. coli growth inhibition (EC(50) ≥ 400 µM), and growth inhibition could not be unambiguously linked to the compound's effect on the Sec pathway. Our study and others underscore the challenges of finding potent druglike hits against this otherwise promising drug target.

Cofactor-independent phosphoglycerate mutase from nematodes has limited druggability, as revealed by two high-throughput screens.

Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z'-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a "druggability paradox" of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data.

Plasmodium gametocyte inhibition identified from a natural-product-based fragment library.

Fragment-based screening is commonly used to identify compounds with relatively weak but efficient localized binding to protein surfaces. We used mass spectrometry to study fragment-sized three-dimensional natural products. We identified seven securinine-related compounds binding to Plasmodium falciparum 2'-deoxyuridine 5'-triphosphate nucleotidohydrolase (PfdUTPase). Securinine bound allosterically to PfdUTPase, enhancing enzyme activity and inhibiting viability of both P. falciparum gametocyte (sexual) and blood (asexual) stage parasites. Our results provide a new insight into mechanisms that may be applicable to transmission-blocking agents.

A mechanism-based whole-cell screening assay to identify inhibitors of protein export in Escherichia coli by the Sec pathway.

More than 20% of bacterial proteins are noncytoplasmic, and most of these pass through the SecYEG channel en route to the periplasm, cell membrane, or surrounding environment. The Sec pathway, encompassing SecYEG and several associated proteins (SecA, SecB, YidC, SecDFYajC), is of interest as a potential drug target because it is distinct from targets of current drugs, is essential for bacterial growth, and exhibits dissimilarities in eukaryotes and bacteria that increase the likelihood of selectively inhibiting the microbial pathway. As a step toward validating the pathway as a drug target, we have adapted a mechanism-based whole-cell assay in a manner suitable for high-throughput screening (HTS). The assay uses an engineered strain of Escherichia coli that accumulates beta-galactosidase (ß-gal) in its cytoplasm if translocation through SecYEG is blocked. The assay should facilitate rapid identification of compounds that specifically block the Sec pathway because widely, toxic compounds and nonspecific protein synthesis inhibitors prevent ß-gal production and thus do not register as hits. Testing of current antibiotics confirmed that they do not generally act through the Sec pathway. A mini-screen of 800 compounds indicated the assay's readiness for larger screening projects.

TDR Targets: a chemogenomics resource for neglected diseases.

The TDR Targets Database (http://tdrtargets.org) has been designed and developed as an online resource to facilitate the rapid identification and prioritization of molecular targets for drug development, focusing on pathogens responsible for neglected human diseases. The database integrates pathogen specific genomic information with functional data (e.g. expression, phylogeny, essentiality) for genes collected from various sources, including literature curation. This information can be browsed and queried using an extensive web interface with functionalities for combining, saving, exporting and sharing the query results. Target genes can be ranked and prioritized using numerical weights assigned to the criteria used for querying. In this report we describe recent updates to the TDR Targets database, including the addition of new genomes (specifically helminths), and integration of chemical structure, property and bioactivity information for biological ligands, drugs and inhibitors and cheminformatic tools for querying and visualizing these chemical data. These changes greatly facilitate exploration of linkages (both known and predicted) between genes and small molecules, yielding insight into whether particular proteins may be druggable, effectively allowing the navigation of chemical space in a genomics context.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline.

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies.