What Do You Find Beautiful about Viruses? A Post-COVID Assessment Strategy.

In the age of an ongoing pandemic and the ungrading movement, many instructors have been taking a closer look at their assessment methods. Assessment in science courses typically focuses heavily on checking understanding of underlying vocabulary and processes rather than highlighting an emotional connection to the material. For the final exam in a senior-level virus biotechnology course in Spring 2021, Fall 2021, and Spring 2022, an additional assessment question asking students what they found beautiful about viruses was implemented. Students highlighted a surprisingly large range of concepts, and all students received full marks for their thorough descriptions of their chosen concept. These responses were also interesting and joyful to grade as the instructor. Examples of responses and potential benefits of this approach are provided and discussed.

Learning lab skills online: Lessons from implementing video-based instruction for a remote biotechnology lab.

The COVID-19 pandemic forced many courses to move online, presenting a particular challenge for hands-on laboratory courses. One such course in our Biotechnology track is an advanced Protein Interactions lecture/laboratory course. This 8-week course typically meets for 5 h a week in the laboratory space. For the Fall 2020 version of the course, first-person videos were produced for each of the laboratory experiments, and the corresponding experimental data produced by students in previous semesters were provided for the current students to analyze in their electronic lab notebooks and lab reports. Student perspectives and assessments were collected on course participants from Fall 2019 (in-person laboratories) and Fall 2020 (online laboratories) to compare experiences and outcomes. Analysis of the data shows that the online students appreciated the videos and gained self-confidence in the procedures, but maintained more misconceptions about the material. In addition to being unable to perform the hands-on experiments, other factors such as anxiety could also be interfering with the learning process under the pandemic conditions. The implementation process for the remote labs, student reactions, and lessons learned are discussed.

Change in cervical length and spontaneous preterm birth in nulliparous women with a history of loop electrosurgical excision procedure.

BACKGROUND: Prior studies have shown an association between history of loop electrode procedures (LEEP) and spontaneous preterm delivery (SPTD) independent of mid-trimester cervical length. These studies suggest that there may be other factors beyond an individual cervical length, which contribute to identifying at-risk pregnancies. OBJECTIVE: The objective of this study is to determine the association between change in cervical length and SPTD in women with a history of LEEP. STUDY DESIGN: This is a retrospective cohort study of singleton nulliparous women with a history of LEEP who received serial cervical length measurements at a single institution between 2012 and 2016. Women with serial cervical lengths and available outcome data were included. The cervical length at different gestational ages and the rate of change in length were compared with the risk for SPTD <37 weeks using Student's t-test. RESULTS: One-hundred-thirty subjects met the inclusion criteria for the study. The mean cervical length (35.3 versus 39.8 mm, p = .042 at 16 weeks; 32.2 versus 37.8 mm, p < .01 at 20 weeks; 29.9 versus 35.6 mm, p = .027 at 24 weeks; 21.6 versus 33.4 mm, p < .01 at 28 weeks) was significantly different between women who had an SPTD <37 weeks compared to women who did not. The average rate of change in transvaginal cervical length between 16 to 28 weeks was significantly different between women who had an SPTD <37 weeks compared to women who did not (-1.4 versus 0.4 mm/week, p < .01). CONCLUSION: Women with a history of LEEP who had an SPTD <37 weeks had a shorter cervical length at 16, 20, 24, and 28 weeks' gestation and a higher rate of change in cervical length between 16 and 28 weeks than women without a history of SPTD. Our findings support the concept of the preterm birth syndrome as an evolving biophysical process rather than a distinct event, suggesting improved prediction in the setting of prior history of a LEEP with serial imaging.

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.

Integrating research into a molecular cloning course to address the evolving biotechnology landscape.

The rapid development of molecular biotechnology presents a curricular challenge for educators trying to provide students with relevant coursework. A comprehensive biology education should also include opportunities for students to develop intellectual and technical skills through authentic research experiences. Integrating relevant and interesting research projects into their classes, however, can be a challenging task for instructors. To address these varied demands, we redesigned our existing molecular cloning course to incorporate an independent research project assessing calcium signaling. In the revised course, students use traditional and recombination-based cloning strategies to generate bacterial and mammalian expression vectors encoding CaMPARI, a novel fluorescent calcium indicator. Bacterially-expressed CaMPARI is used in protein quantification and purification assays. Students must also design their own research project evaluating the effect of chemotherapeutic agents on calcium signaling in a mammalian system. Revised and novel labs were designed to be modular, facilitating their integration into the course over 2 years. End-of-semester student evaluations were compared between years revealing a significant difference in students' perception of the course's difficulty between years. This change in attitude highlights potential pedagogical considerations that must be examined when introducing new material and activities into existing courses. Since calcium signaling is important for cellular process across diverse species, instructors may be able to develop research projects within their respective areas of interest. Integration of authentic research experiences into the curriculum is challenging; however, the framework described here provides a versatile structure that can be adapted to merge diverse instructor interests with evolving educational needs.

Resolving Toxic DNA repair intermediates in every E. coli replication cycle: critical roles for RecG, Uup and RadD.

DNA lesions or other barriers frequently compromise replisome progress. The SF2 helicase RecG is a key enzyme in the processing of postreplication gaps or regressed forks in Escherichia coli. A deletion of the recG gene renders cells highly sensitive to a range of DNA damaging agents. Here, we demonstrate that RecG function is at least partially complemented by another SF2 helicase, RadD. A ΔrecGΔradD double mutant exhibits an almost complete growth defect, even in the absence of stress. Suppressors appear quickly, primarily mutations that compromise priA helicase function or recA promoter mutations that reduce recA expression. Deletions of uup (encoding the UvrA-like ABC system Uup), recO, or recF also suppress the ΔrecGΔradD growth phenotype. RadD and RecG appear to avoid toxic situations in DNA metabolism, either resolving or preventing the appearance of DNA repair intermediates produced by RecA or RecA-independent template switching at stalled forks or postreplication gaps. Barriers to replisome progress that require intervention by RadD or RecG occur in virtually every replication cycle. The results highlight the importance of the RadD protein for general chromosome maintenance and repair. They also implicate Uup as a new modulator of RecG function.

Harnessing single-stranded DNA binding protein to explore protein-protein and protein-DNA interactions.

Proteins must interact with a variety of other cellular components to properly perform their functions. We have developed a series of five experiments based on the robust bacterial single-stranded DNA binding protein (SSB) to characterize both known and unknown protein-protein and protein-DNA interactions. Students work in groups to generate and process data from electrophoretic mobility shift assays (EMSA), yeast two-hybrid, far Western, chromatin immunoprecipitation (ChIP), and fluorescence microscopy experiments, including choosing a novel condition for each. A gamification approach was used to encourage student participation and laboratory safety. Student learning was assessed using pre- and post-surveys and course grade data. The results indicate a clear increase in both content knowledge and confidence in the topics presented. Ranking of course activities indicated that performing the hands-on laboratory exercises was the most valuable course component, and over half of the students would choose to take another course with a similar gamification component. Each of the five laboratory experiments can be performed in combination with each other or integrated separately into a related course, and the gamification structure can be applied to any course.

Frequent template switching in postreplication gaps: suppression of deleterious consequences by the Escherichia coli Uup and RadD proteins.

When replication forks encounter template DNA lesions, the lesion is simply skipped in some cases. The resulting lesion-containing gap must be converted to duplex DNA to permit repair. Some gap filling occurs via template switching, a process that generates recombination-like branched DNA intermediates. The Escherichia coli Uup and RadD proteins function in different pathways to process the branched intermediates. Uup is a UvrA-like ABC family ATPase. RadD is a RecQ-like SF2 family ATPase. Loss of both functions uncovers frequent and RecA-independent deletion events in a plasmid-based assay. Elevated levels of crossing over and repeat expansions accompany these deletion events, indicating that many, if not most, of these events are associated with template switching in postreplication gaps as opposed to simple replication slippage. The deletion data underpin simulations indicating that multiple postreplication gaps may be generated per replication cycle. Both Uup and RadD bind to branched DNAs in vitro. RadD protein suppresses crossovers and Uup prevents nucleoid mis-segregation. Loss of Uup and RadD function increases sensitivity to ciprofloxacin. We present Uup and RadD as genomic guardians. These proteins govern two pathways for resolution of branched DNA intermediates such that potentially deleterious genome rearrangements arising from frequent template switching are averted.

Shifting Faculty Approaches to Pedagogy through Structured Teaching Postdoc Experiences.

Many studies confirm the benefit of active learning in STEM teaching. However, many faculty have been slow to adopt such practices, perhaps due to limited time to learn and implement new approaches. One way to address this deficit is to offer structured teaching postdoctoral experiences to trained scientists who want to enter academia. We outline the benefits of providing pedagogical training at the postdoctoral level and present a framework for structuring an impactful teaching postdoc program.

What is Recklessness in Scientific Research? The Frank Sauer Case.

On May 22, 2017, administrative law Judge Leslie Rogall of the Department of Health and Human Services' Departmental Appeals Board, Civil Remedies Division, ruled in favor of the Office of Research Integrity (ORI) concerning its decision to charge former University of California at Riverside biochemistry professor Frank Sauer with research misconduct for fabricating or falsifying digital image data included in three papers and seven grant applications submitted to the National Institutes of Health. More specifically, Sauer was deemed responsible for manipulating, reusing, and falsely labeling images of autoradiograms and gels in his research in epigenetics. One month after this decision, ORI announced its final ruling concerning Sauer, which barred him from serving in any advisory capacity to the Public Health Services and required him to retract affected papers. The case raises some interesting and important questions concerning research integrity because it focused on the legal issue of what constitutes recklessness in scientific research.

Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein.

The bacterial single-stranded DNA binding protein (SSB) acts as an organizer of DNA repair complexes. The radD gene was recently identified as having an unspecified role in repair of radiation damage and, more specifically, DNA double-strand breaks. Purified RadD protein displays a DNA-independent ATPase activity. However, ATP hydrolytic rates are stimulated by SSB through its C terminus. The RadD and SSB proteins also directly interact in vivo in a yeast two-hybrid assay and in vitro through ammonium sulfate co-precipitation. Therefore, it is likely that the repair function of RadD is mediated through interaction with SSB at the site of damage.

Active displacement of RecA filaments by UvrD translocase activity.

The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with enhanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode.

Escherichia coli radD†(yejH) gene: a novel function involved in radiation resistance and double-strand break repair.

A transposon insertion screen implicated the yejH gene in the repair of ionizing radiation-induced damage. The yejH gene, which exhibits significant homology to the human transcription-coupled DNA repair gene XPB, is involved in the repair of double-strand DNA breaks. Deletion of yejH significantly sensitized cells to agents that cause double-strand breaks (ionizing radiation, UV radiation, ciprofloxacin). In addition, deletion of both yejH and radA hypersensitized the cells to ionizing radiation, UV and ciprofloxacin damage, indicating that these two genes have complementary repair functions. The ΔyejH ΔradA double deletion also showed a substantial decline in viability following an induced double-strand DNA break, of a magnitude comparable with the defect measured when the recA, recB, recG or priA genes are deleted. The ATPase activity and C-terminal zinc finger motif of yejH play an important role in its repair function, as targeted mutant alleles of yejH did not rescue sensitivity. We propose that yejH be renamed radD, reflecting its role in the DNA repair of radiation damage.

Escherichia coli genes and pathways involved in surviving extreme exposure to ionizing radiation.

To further an improved understanding of the mechanisms used by bacterial cells to survive extreme exposure to ionizing radiation (IR), we broadly screened nonessential Escherichia coli genes for those involved in IR resistance by using transposon-directed insertion sequencing (TraDIS). Forty-six genes were identified, most of which become essential upon heavy IR exposure. Most of these were subjected to direct validation. The results reinforced the notion that survival after high doses of ionizing radiation does not depend on a single mechanism or process, but instead is multifaceted. Many identified genes affect either DNA repair or the cellular response to oxidative damage. However, contributions by genes involved in cell wall structure/function, cell division, and intermediary metabolism were also evident. About half of the identified genes have not previously been associated with IR resistance or recovery from IR exposure, including eight genes of unknown function.

Top3α is required during the convergent migration step of double Holliday junction dissolution.

Although Blm and Top3α are known to form a minimal dissolvasome that can uniquely undo a double Holliday junction structure, the details of the mechanism remain unknown. It was originally suggested that Blm acts first to create a hemicatenane structure from branch migration of the junctions, followed by Top3α performing strand passage to decatenate the interlocking single strands. Recent evidence suggests that Top3α may also be important for assisting in the migration of the junctions. Using a mismatch-dHJ substrate (MM-DHJS) and eukaryotic Top1 (in place of Top3α), we show that the presence of a topoisomerase is required for Blm to substantially migrate a topologically constrained Holliday junction. When investigated by electron microscopy, these migrated structures did not resemble a hemicatenane. However, when Blm is together with Top3α, the dissolution reaction is processive with no pausing at a partially migrated structure. Potential mechanisms are discussed.

New mechanistic and functional insights into DNA topoisomerases.

DNA topoisomerases are nature's tools for resolving the unique problems of DNA entanglement that occur owing to unwinding and rewinding of the DNA helix during replication, transcription, recombination, repair, and chromatin remodeling. These enzymes perform topological transformations by providing a transient DNA break, formed by a covalent adduct with the enzyme, through which strand passage can occur. The active site tyrosine is responsible for initiating two transesterifications to cleave and then religate the DNA backbone. The cleavage reaction intermediate is exploited by cytotoxic agents, which have important applications as antibiotics and anticancer drugs. The reactions mediated by these enzymes can also be regulated by their binding partners; one example is a DNA helicase capable of modulating the directionality of strand passage, enabling important functions like reannealing denatured DNA and resolving recombination intermediates. In this review, we cover recent advances in mechanistic insights into topoisomerases and their various cellular functions.

Improved methods for creating migratable Holliday junction substrates.

Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying late stage homologous recombination in vitro, construction of the substrate requires significant effort. In particular, there are three bottlenecks: (i) production of large quantities of single-stranded DNA; (ii) the loss of a significant portion of the DNA following the recombination step; and (iii) the loss of DNA owing to inefficient gel extraction. To address these limitations, we have made the following changes to the protocol: (i) use of a helper plasmid, rather than exogenous helper phage, to produce single-stranded DNA; (ii) use of the unidirectional C31 integrase system in place of the bidirectional Cre recombinase reaction; and (iii) gel extraction by DNA diffusion. Here, we describe the changes made to the materials and methods and characterize the substrates that can be produced, including migratable single Holliday junctions, hemicatenanes and a quadruple Holliday junction substrate.

Essential functions of C terminus of Drosophila Topoisomerase IIIα in double holliday junction dissolution.

Topoisomerase IIIα (Top3α) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans, along with Blm and Rmi1/2. This important anti-recombinogenic function cannot be performed by Top3ß, the other type IA topoisomerase present in metazoans. The two share a catalytic core but diverge in their tail regions. To understand this difference in function, we investigated the role of the unique C terminus of Top3α. The Drosophila C terminus contains an insert region not conserved among metazoans. This insert contributes an independent interaction with Blm, which may account for the absence of Rmi1 in Drosophila. Mutant Top3α lacking this insert maintains the ability to perform dHJ dissolution but only partially rescues a top3α null fly line, indicating an in vivo role for the insert. Truncation of the C terminus has a minimal effect on the type IA relaxation activity of Top3α; however, dHJ dissolution is greatly reduced. The Top3α C terminus was found to strongly interact with both Blm and DNA, which are critical to the dissolution reaction; these interactions are greatly reduced in the truncated enzyme. The truncation mutant also cannot rescue the viability of top3α null flies, indicating an essential in vivo role. Our data therefore suggest that the Top3α C terminus has an important role in dHJ dissolution (by providing an interaction interface for Blm and DNA) and an essential function in vivo.