Using a decision tree to predict the number of COVID cases: a tutorial for beginners.

This manuscript describes the development of a module that is part of a learning platform named "NIGMS Sandbox for Cloud-based Learning" https://github.com/NIGMS/NIGMS-Sandbox . The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module delivers learning materials on machine learning and decision tree concepts in an interactive format that uses appropriate cloud resources for data access and analyses. Machine learning (ML) is an important tool in biomedical research and can lead to improvements in diagnosis, treatment, and prevention of diseases. During the COVID pandemic ML was used for predictions at the patient and community levels. Given its ubiquity, it is important that future doctors, researchers and teachers get acquainted with ML and its contributions to research. Our goal is to make it easier for everyone to learn about machine learning. The learning module we present here is based on a small COVID dataset, videos, annotated code and the use of Google Colab or the Google Cloud Platform (GCP). The benefit of these platforms is that students do not have to set up a programming environment on their computer which saves time and is also an important democratization factor. The module focuses on learning the basics of decision trees by applying them to COVID data. It introduces basic terminology used in supervised machine learning and its relevance to research. Our experience with biology students at San Francisco State University suggests that the material increases interest in ML.

SCIP: a self-paced summer coding program creates community and increases coding confidence.

In 2020, many students lost summer opportunities due to the COVID-19 pandemic. We wanted to offer students an opportunity to learn computational skills and be part of a community while stuck at home. Because the pandemic created an unexpected research and academic situation, it was unclear how to best support students to learn and build community online. We used lessons learned from literature and our own experience to design, run and test an online program for students called the Science Coding Immersion Program (SCIP). In our program, students worked in teams for 8 hours a week, with one participant as the team leader and Zoom host. Teams worked on an online R or Python class at their own pace with support on Slack from the organizing team. For motivation and career advice, we hosted a weekly webinar with guest speakers. We used pre- and post-program surveys to determine how different aspects of the program impacted students. We were able to recruit a large and diverse group of participants who were happy with the program, found community in their team, and improved their coding confidence. We hope that our work will inspire others to start their own version of SCIP.

Student-Authored Scientist Spotlights: Investigating the Impacts of Engaging Undergraduates as Developers of Inclusive Curriculum through a Service-Learning Course.

Scientist Spotlights-curricular materials that employ the personal and professional stories of scientists from diverse backgrounds-have previously been shown to positively influence undergraduate students' relatability to and perceptions of scientists. We hypothesized that engaging students in authoring Scientist Spotlights might produce curricular materials of similar impact, as well as provide a mechanism for student involvement as partners in science education reform. To test this idea and investigate the impact of student-authored Scientist Spotlights, we developed a service-learning course in which teams of biology students partnered with an instructor to develop and implement Scientist Spotlights in a biology course. Results revealed that exposure to three or four student-authored Scientist Spotlights significantly shifted peers' perceptions of scientists in all partner courses. Interestingly, student-authored Scientist Spotlights shifted peers' relatability to scientists similarly among both white students and students of color. Further, student authors themselves showed increases in their relatability to scientists. Finally, a department-wide survey demonstrated significant differences in students' perceptions of scientist representation between courses with and without student-authored Spotlights. Results suggest that engaging students as authors of inclusive curricular materials and partners in reform is a promising approach to promoting inclusion and addressing representation in science.

The Localization of Phytohormones within the Gall-inducing Insect Eurosta solidaginis (Diptera: Tephritidae).

The phytohormone production hypothesis suggests that organisms, including insects, induce galls by producing and secreting plant growth hormones. Auxins and cytokinins are classes of phytohormones that induce cell growth and cell division, which could contribute to the plant tissue proliferation which constitutes the covering gall. Bacteria, symbiotic with insects, may also play a part in gall induction by insects through the synthesis of phytohormones or other effectors. Past studies have shown that concentrations of cytokinins and auxins in gall-inducing insects are higher than in their host plants. However, these analyses have involved whole-body extractions. Using immunolocalization of cytokinin and auxin, in the gall inducing stage of Eurosta solidaginis, we found both phytohormones to localize almost exclusively to the salivary glands. Co-localization of phytohormone label with a nucleic acid stain in the salivary glands revealed the absence of Wolbachia sp., the bacterial symbiont of E. solidaginis, which suggests that phytohormone production is symbiont independent. Our findings are consistent with the hypothesis that phytohormones are synthesized in and secreted from the salivary glands of E. solidaginis into host-plant tissues for the purpose of manipulating the host plant.

Risk factors for abutment and implant fracture after loading.

PURPOSE: Implant component fractures are one of the most serious complications in implant treatment. With a better understanding of the risk factors for fracture in the preoperative, surgery, superstructure, and post-loading phases of implant treatment, low-risk treatment could reduce implant component fractures, leading to a better prognosis. The aim of this study was to clarify the risk factors for abutment and implant fractures that occur after loading, and to perform a retrospective, approximately 10-year follow-up study to explore the risk factors in each treatment phase. METHODS: Subjects were fitted with an implant prosthesis between January 2008 and December 2009. In total, 1,126 Ankylos implants in 430 patients were included for analysis. Binary logistic regression analysis was performed to extract factors related to non-fracture and fracture of the abutment or implant as a dependent variable. RESULTS: Gender (OR = 3.466, 95% CI 1.296-9.268, P = 0.013), gonial angle (OR = 3.420, 95% CI 1.308-8.945, P = 0.012), and splinting status of the superstructure (OR = 4.456, 95% CI 1.861-10.669, P = 0.001) were identified as significant risk factors. CONCLUSION: The risk of fracture is increased in males, especially those with a mandibular angle of less than 120° on panoramic radiographs, and those with a non-splinted superstructure.

Divergent mechanisms for regulating growth and development after imaginal disc damage in the tobacco hornworm, Manduca sexta.

Holometabolous insects have been able to radiate to vast ecological niches as adults through the evolution of adult-specific structures such as wings, antennae and eyes. These structures arise from imaginal discs that show regenerative capacity when damaged. During imaginal disc regeneration, development has been shown to be delayed in the fruit fly Drosophila melanogaster, but how conserved the delay-inducing mechanisms are across holometabolous insects has not been assessed. The goal of this research was to develop the hornworm Manduca sexta as an alternative model organism to study such damage-induced mechanisms, with the advantage of a larger hemolymph volume enabling access to the hormonal responses to imaginal disc damage. Upon whole-body X-ray exposure, we noted that the imaginal discs were selectively damaged, as assessed by TUNEL and Acridine Orange stains. Moreover, development was delayed, predominantly at the pupal-to-adult transition, with a concomitant delay in the prepupal ecdysteroid peak. The delays to eclosion were dose dependent, with some ability for repair of damaged tissues. We noted a shift in critical weight, as assessed by the point at which starvation no longer impacted developmental timing, without a change in growth rate, which was uncoupled from juvenile hormone clearance in the body. The developmental profile was different from that of D. melanogaster, which suggests species differences may exist in the mechanisms delaying development.

Investigating Instructor Talk in Novel Contexts: Widespread Use, Unexpected Categories, and an Emergent Sampling Strategy.

Instructor Talk-noncontent language used by instructors in classrooms-is a recently defined and promising variable for better understanding classroom dynamics. Having previously characterized the Instructor Talk framework within the context of a single course, we present here our results surrounding the applicability of the Instructor Talk framework to noncontent language used by instructors in novel course contexts. We analyzed Instructor Talk in eight additional biology courses in their entirety and in 61 biology courses using an emergent sampling strategy. We observed widespread use of Instructor Talk with variation in the amount and category type used. The vast majority of Instructor Talk could be characterized using the originally published Instructor Talk framework, suggesting the robustness of this framework. Additionally, a new form of Instructor Talk-Negatively Phrased Instructor Talk, language that may discourage students or distract from the learning process-was detected in these novel course contexts. Finally, the emergent sampling strategy described here may allow investigation of Instructor Talk in even larger numbers of courses across institutions and disciplines. Given its widespread use, potential influence on students in learning environments, and ability to be sampled, Instructor Talk may be a key variable to consider in future research on teaching and learning in higher education.

Collectively Improving Our Teaching: Attempting Biology Department-wide Professional Development in Scientific Teaching.

Many efforts to improve science teaching in higher education focus on a few faculty members at an institution at a time, with limited published evidence on attempts to engage faculty across entire departments. We created a long-term, department-wide collaborative professional development program, Biology Faculty Explorations in Scientific Teaching (Biology FEST). Across 3 years of Biology FEST, 89% of the department's faculty completed a weeklong scientific teaching institute, and 83% of eligible instructors participated in additional semester-long follow-up programs. A semester after institute completion, the majority of Biology FEST alumni reported adding active learning to their courses. These instructor self-reports were corroborated by audio analysis of classroom noise and surveys of students in biology courses on the frequency of active-learning techniques used in classes taught by Biology FEST alumni and nonalumni. Three years after Biology FEST launched, faculty participants overwhelmingly reported that their teaching was positively affected. Unexpectedly, most respondents also believed that they had improved relationships with departmental colleagues and felt a greater sense of belonging to the department. Overall, our results indicate that biology department-wide collaborative efforts to develop scientific teaching skills can indeed attract large numbers of faculty, spark widespread change in teaching practices, and improve departmental relations.

Classroom sound can be used to classify teaching practices in college science courses.

Active-learning pedagogies have been repeatedly demonstrated to produce superior learning gains with large effect sizes compared with lecture-based pedagogies. Shifting large numbers of college science, technology, engineering, and mathematics (STEM) faculty to include any active learning in their teaching may retain and more effectively educate far more students than having a few faculty completely transform their teaching, but the extent to which STEM faculty are changing their teaching methods is unclear. Here, we describe the development and application of the machine-learning-derived algorithm Decibel Analysis for Research in Teaching (DART), which can analyze thousands of hours of STEM course audio recordings quickly, with minimal costs, and without need for human observers. DART analyzes the volume and variance of classroom recordings to predict the quantity of time spent on single voice (e.g., lecture), multiple voice (e.g., pair discussion), and no voice (e.g., clicker question thinking) activities. Applying DART to 1,486 recordings of class sessions from 67 courses, a total of 1,720 h of audio, revealed varied patterns of lecture (single voice) and nonlecture activity (multiple and no voice) use. We also found that there was significantly more use of multiple and no voice strategies in courses for STEM majors compared with courses for non-STEM majors, indicating that DART can be used to compare teaching strategies in different types of courses. Therefore, DART has the potential to systematically inventory the presence of active learning with ∼90% accuracy across thousands of courses in diverse settings with minimal effort.

Central neural alterations predominate in an insect model of nociceptive sensitization.

Many organisms respond to noxious stimuli with defensive maneuvers. This is noted in the hornworm, Manduca sexta, as a defensive strike response. After tissue damage, organisms typically display sensitized responses to both noxious or normally innocuous stimuli. To further understand this phenomenon, we used novel in situ and in vitro preparations based on paired extracellular nerve recordings and videography to identify central and peripheral nerves responsible for nociception and sensitization of the defensive behavior in M. sexta. In addition, we used the in vivo defensive strike response threshold assayed with von Frey filaments to examine the roles that N-methyl-D-aspartate receptor (NMDAR) and hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels play in this nociceptive sensitization using the inhibitors MK-801 and AP5 (NMDAR), and ivabradine and ZD7288 (HCN). Using our new preparations, we found that afferent activity evoked by noxious pinch in these preparations was conveyed to central ganglia by axons in the anterior- and lateral-dorsal nerve branches, and that sensitization induced by tissue damage was mediated centrally. Furthermore, sensitization was blocked by all inhibitors tested except the inactive isomer L-AP5, and reversed by ivabradine both in vivo and in vitro. Our findings suggest that M. sexta's sensitization occurs through central signal amplification. Due to the relatively natural sensitization method and conserved molecular actions, we suggest that M. sexta may be a valuable model for studying the electrophysiological properties of nociceptive sensitization and potentially related conditions such as allodynia and hyperalgesia in a comparative setting that offers unique experimental advantages. J. Comp. Neurol. 525:1176-1191, 2017. © 2016 Wiley Periodicals, Inc.

Use of von Frey filaments to assess nociceptive sensitization in the hornworm, Manduca sexta.

BACKGROUND: The hornworm Manduca sexta exhibits a defensive strike to noxious assaults, a response that is robust and is easily observed by experimenters. Von Frey filaments and methods typical for studying nociception in other animals were used to assess the strike response in M. sexta. NEW METHODS: A series of von Frey filaments was applied to the body wall in ascending order and the data generated were used to determine the strike threshold by (i) the up-and-down method, (ii) the first response method, and (iii) the simplified up-and-down order method (SUDO). The effect of a noxious pinch on strike threshold was assessed. COMPARISON WITH EXISTING METHODS: To our knowledge none of these methods has been used on M. sexta previously, making the use of the up-and-down and SUDO methods the first in an invertebrate. The use of the first response method has been used in other invertebrates, and the method appears equally suited to M. sexta. RESULTS: All three methods were successful in monitoring the threshold sensitivity to touch, which was lowered (sensitized) by tissue damage induced with a pinch. Sensitization lasted 19h. CONCLUSIONS: All three methods of assessing nociception were successfully applied to quantify the defensive strike response in M. sexta, although the SUDO method required empirical assessment of which filament to start the test sequence with. The results revealed both short- and long-term sensitization. These methods should prove to be useful for quantifying sensitization in M. sexta.

Ecdysis behaviors and circadian rhythm of ecdysis in the stick insect, Carausius morosus.

Successful ecdysis in insects depends on proper timing and sequential activation of an elaborate series of motor programs driven by a relatively conserved network of neuropeptides. The behaviors must be activated at the appropriate times to ensure successful loosening and shedding of the old cuticle, and can be influenced by environmental cues in the form of immediate sensory feedback and by circadian rhythms. We assessed the behaviors, components of the neural network and the circadian basis of ecdysis in the stick insect, Carausius morosus. C. morosus showed many of the characteristic pre-ecdysis and ecdysis behaviors previously described in crickets and locusts. Ecdysis was described in three phases, namely the (i) preparatory or pre-ecdysis phase, (ii) the ecdysial phase, and (iii) the post-ecdysis or exuvial phase. The frequencies of push-ups and sways during the preparatory phase were quantified as well as durations of all the phases. The regulation of ecdysis appeared to act via elevation of cGMP, as described in many other insects, although eclosion hormone-like immunoreactivity was not noted using a lepidopteran antiserum. Finally, C. morosus showed a circadian rhythm to the onset of ecdysis, with ecdysis occurring just prior to or at lights on. Ecdysis could be induced precociously with mechanical stimulation.

Quantification and analysis of ecdysis in the hornworm, Manduca sexta, using machine vision-based tracking.

We have developed a machine vision-based method for automatically tracking deformations in the body wall to monitor ecdysis behaviors in the hornworm, Manduca sexta. The method utilizes naturally occurring features on the animal's body (spiracles) and is highly accurate (>95 % success in tracking). Moreover, it is robust to unanticipated changes in the animal's position and in lighting, and in the event tracking of specific features is lost, tracking can be reestablished within a few cycles without input from the user. We have paired our tracking technique with electromyography and have also compared our in vivo results to fictive motor patterns recorded from isolated nerve cords. We found no major difference in the cycle periods of contractions during naturally occurring ecdysis compared to ecdysis initiated prematurely through injection of the peptide ecdysis-triggering hormone, and we confirmed that the ecdysis period in vivo is statistically similar to that of the fictive motor pattern.

Different actions of ecdysis-triggering hormone on the brain and ventral nerve cord of the hornworm, Manduca sexta.

Ecdysis, or the shedding of the old cuticle, depends on coordinated stereotyped behaviors, regulated by a number of neuropeptides. In the hornworm, Manduca sexta, two neuropeptides interact, namely ecdysis-triggering hormone (ETH) and eclosion hormone. We looked at the effects of ETH in vivo and in vitro, on the brain and the ventral nerve cord to determine the roles played by these hormones. We monitored ecdysis onset and the presence of cGMP and eclosion hormone immunoreactivity. In vivo, only a fraction of larvae lacking the cell bodies containing eclosion hormone, and injected with ETH, were able to undergo ecdysis, with a delayed response. These animals showed strongest cGMP immunoreactivity in the subesophageal and thoracic ganglia, with concomitant reductions in eclosion hormone immunoreactivity in descending axons in comparison with animals not undergoing ecdysis. Animals lacking the brain showed reduced to no cGMP levels in all ganglia. In vitro, isolated CNS preparations lacking the brain initiated ecdysis motor programs after incubation in ETH, with faster onset times than controls, and with reduced cGMP immunoreactivity. If ETH was applied only to the brain of the isolated CNS, cGMP immunoreactivity was noted primarily in the subesophageal and thoracic ganglia, with a decrease in eclosion hormone immunoreactivity in descending axons. ETH addition to the rest of the nerve cord showed reduced eclosion hormone immunoreactivity but little to no cGMP immunoreactivity in any ganglion. Controls showed strong cGMP immunoreactivity in all ganglia, and even greater reductions in eclosion hormone staining after ETH application. These results support previous suggestions that eclosion hormone is required for a positive feedback loop with ETH as well as onset of an inhibitory component, but also suggest that ETH stimulates eclosion hormone release at multiple spike initiation zones. The resultant up regulation of cGMP does not appear to be required for onset of ecdysis. A new model for ecdysis regulation is considered.

Molecular characterization and cell-specific expression of an ion transport peptide in the tobacco hornworm, Manduca sexta.

The crustacean hyperglycemic hormone (CHH) peptides regulate diverse physiological processes from reproduction to metabolism and molting in arthropods. In insects, the ion transport peptides (ITP), also members of the CHH family, have only been implicated in ion transport. In this study, we sequenced a nucleotide fragment spanning the conserved A1/A2 region of the putative CHH/ITP gene. This fragment was amplified from larval cDNA of the tobacco hornworm, Manduca sexta and showed a high degree of sequence conservation with the same region from other insects and, to a lesser degree, with that of crustacean species, suggesting the presence of a Manduca-specific CHH/ITP mRNA (MasITP mRNA). CHH-like immunocytochemical analyses with two crustacean antisera (from Carcinus maenas and Cancer pagurus) identified the presence of CHH-like immunoreactivity in nervous tissue of all developmental stages, but not in the gut of M. sexta. Specifically, CHH-like peptides localized to paired type IA(2) neurosecretory cells of the pars lateralis of the brain (projecting ipsilaterallly to the corpora cardiaca-allata complex) and to neurosecretory cells and transverse nerves of the ventral nerve cord in larvae, pupae, and adults. The distribution of the putative MasITP peptide shifted during development in a manner consistent with metamorphic reorganization. A comparison of hemolymph equivalents of CHH detected by enzyme-linked immunosorbent assay with CHH-like immunoreactivity in transverse nerves provided evidence for the release of MasITP from the transverse nerves into the hemolymph at insect ecdysis. These data suggest the presence of an insect ITP in M. sexta and a role for this hormone during ecdysis.

Structure-activity relationship of ETH during ecdysis in the tobacco hornworm, Manduca sexta.

In insects, ecdysis or shedding of the old cuticle, consists of a series of behaviors that are regulated by the coordinated actions of a number of neuropeptides, one of which is ecdysis triggering hormone (ETH). ETH acts directly on central pattern generators of the abdominal ganglia to trigger onset of pre-ecdysis behaviors, as well as indirectly to activate release of eclosion hormone, thereby inducing onset of ecdysis behaviors through a cGMP-mediated mechanism. We assessed the minimal C-terminal amino acids required for biological activity of ETH, by assessing: (i) onset of pre-ecdysis and ecdysis behaviors in vivo, after injection of peptide analogs, (ii) onset of fictive pre-ecdysis and ecdysis motor patterns in vitro, as recorded extracellularly, after incubation of the CNS with the peptide analogs, and (iii) accumulation of cGMP within cells of the abdominal ganglia, as assessed immunohistochemically. Amidation of ETH at the C-terminus was required to elicit a biological response in vivo and in vitro, as well as an accumulation of cGMP within the CNS. The five amino acid amidated C-terminus of ETH (NIPRMamide) was the minimal moiety able to induce a robust pre-ecdysis response in vivo and in vitro, while a seven amino acid core (NKNIPRMa) was required for induction of ecdysis, including accumulation of cGMP immunoreactivity within the CNS. Analogs smaller than 12 amino acids in length were only active at very high concentrations in vivo, suggesting that smaller fragments might be susceptible to hemolymph degradation. Some alanine substitutions or removal of internal amino acids altered the activity of ETH, as well as the time of onset of ecdysis behaviors, suggesting that internal amino acids play a role in maintaining proper folding of the peptide for successful binding or activity at the ETH receptor.

Modulation of ecdysis in the moth Manduca sexta: the roles of the suboesophageal and thoracic ganglia.

The sequential behaviours shown by insects at ecdysis are due to the sequential release of various hormones, but the transition from one phase to the next can be fine-tuned by inhibitory influences. The ecdysis sequence in the moth Manduca sexta was initiated by injecting sensitive animals with the neuropeptide ecdysis-triggering hormone (ETH). Exposure to ETH stimulates the release of eclosion hormone (EH) which, in turn, activates a set of neurons containing crustacean cardioactive peptide (CCAP) by elevating their levels of intracellular cyclic GMP. We characterized a set of non-CCAP containing neurons that also appear to be EH targets because of their response to cyclic GMP at ecdysis. The neurons did not display leucokinin-, diuretic-hormone- or FMRFamide-like immunoreactivity. They are probably the bursicon-containing cells described previously. After release of EH, there is a transient inhibition of the abdominal centers responsible for ecdysis. Transection experiments suggested that this suppression is via descending inhibitory units from the suboesophageal and thoracic ganglia. The duration of this inhibition appears to depend on the levels of cyclic GMP and can be extended by pharmacologically suppressing cyclic GMP breakdown. We further found that brief exposure to CO(2) caused premature ecdysis. Since the CO(2) treatment was effective only after EH release, it probably acts by suppressing descending inhibition. Studies on adult eclosion suggest that CO(2), given at the appropriate time, can uncouple the basic larval motor program from modulatory influences provided by the adult pterothoracic ganglion. CO(2) therefore appears to be a novel and non-invasive tool for studies of ecdysis behavior in insects.