"They Have Shown Me It Is Possible to Thrive within STEM": Incorporating Learning Assistants in General Chemistry Enhances Student Belonging and Confidence.

Students often experience social and psychological barriers to success in General Chemistry, which is a key gateway to many students' science pathways. Learning assistants (LAs) have the potential to reduce these barriers and to strengthen students' sense of belonging in General Chemistry and STEM more broadly. Here, we used a 17-item Likert scale to determine whether incorporating LAs into General Chemistry I and II enhances students' sense of belonging in these courses. The incorporation of LAs into General Chemistry I had a significant positive effect and a medium to large effect size for students in all student groups examined: women and men; students in both racially and ethnically underrepresented and well-represented groups; first- and continuing-generation students. In General Chemistry II, similar results were observed for women and men; students in well-represented racial and ethnic groups; continuing-generation students. Further, we asked students to reflect on the impact that working with LAs had on their sense of belonging in STEM and confidence in talking about science. Sixty percent of students indicated that working with LAs had a positive impact on their STEM belonging, with five themes describing LA impacts: reducing isolation, serving as inspirational role models, providing mentoring, increasing opportunities for engagement and confidence building, and serving as accessible and approachable sources of support. Sixty-one percent of students also indicated that working with LAs increased their confidence in talking about science, with three themes emerging: fostering an environment with a lower risk of negative judgment, providing increased opportunities for feedback, and supporting students as they practiced their growing skills. Together, these results indicate that LAs can be an important means to reduce social and psychological barriers for students in gateway science courses, increasing their sense that they belong to the class and STEM more broadly.

AP-4 loss in CRISPR-edited zebrafish affects early embryo development.

Mutations in the heterotetrametric adaptor protein 4 (AP-4; ε/ß4/µ4/σ4 subunits) membrane trafficking coat complex lead to complex neurological disorders characterized by spastic paraplegia, microcephaly, and intellectual disabilities. Understanding molecular mechanisms underlying these disorders continues to emerge with recent identification of an essential autophagy protein, ATG9A, as an AP-4 cargo. Significant progress has been made uncovering AP-4 function in cell culture and patient-derived cell lines, and ATG9A trafficking by AP-4 is considered a potential target for gene therapy approaches. In contrast, understanding how AP-4 trafficking affects development and function at the organismal level has long been hindered by loss of conserved AP-4 genes in key model systems (S. cerevisiae, C. elegans, D. melanogaster). However, zebrafish (Danio rerio) have retained AP-4 and can serve as an important model system for studying both the nervous system and overall development. We undertook gene editing in zebrafish using a CRISPR-ExoCas9 knockout system to determine how loss of single AP-4, or its accessory protein tepsin, genes affect embryo development 24 h post-fertilization (hpf). Single gene-edited embryos display abnormal head morphology and neural necrosis. We further conducted the first exploration of how AP-4 single gene knockouts in zebrafish embryos affect expression levels and patterns of two autophagy genes, atg9a and map1lc3b. This work suggests zebrafish may be further adapted and developed as a tool to uncover AP-4 function in membrane trafficking and autophagy in the context of a model organism.

A guide to COVID-19 antiviral therapeutics: a summary and perspective of the antiviral weapons against SARS-CoV-2 infection.

Antiviral therapies are integral in the fight against SARS-CoV-2 (i.e. severe acute respiratory syndrome coronavirus 2), the causative agent of COVID-19. Antiviral therapeutics can be divided into categories based on how they combat the virus, including viral entry into the host cell, viral replication, protein trafficking, post-translational processing, and immune response regulation. Drugs that target how the virus enters the cell include: Evusheld, REGEN-COV, bamlanivimab and etesevimab, bebtelovimab, sotrovimab, Arbidol, nitazoxanide, and chloroquine. Drugs that prevent the virus from replicating include: Paxlovid, remdesivir, molnupiravir, favipiravir, ribavirin, and Kaletra. Drugs that interfere with protein trafficking and post-translational processing include nitazoxanide and ivermectin. Lastly, drugs that target immune response regulation include interferons and the use of anti-inflammatory drugs such as dexamethasone. Antiviral therapies offer an alternative solution for those unable or unwilling to be vaccinated and are a vital weapon in the battle against the global pandemic. Learning more about these therapies helps raise awareness in the general population about the options available to them with respect to aiding in the reduction of the severity of COVID-19 infection. In this 'A Guide To' article, we provide an in-depth insight into the development of antiviral therapeutics against SARS-CoV-2 and their ability to help fight COVID-19.

"It made me feel like a bigger part of the STEM community": Incorporation of Learning Assistants Enhances Students' Sense of Belonging in a Large Introductory Biology Course.

Large introductory science courses are a particularly important and challenging target for creating inclusive learning environments. In this study, we examined the impact of incorporating learning assistants (LAs) on the learning environment in an introductory biology course taught with two different structures: an in-person lecture with intermittent active-learning components and an online setting taught with a flipped instructional approach. Using a survey that measured sense of belonging in a single class, we found that students in sections with LAs reported greater sense of belonging than students in sections without LAs in both class structures. Further, student focus groups revealed that LAs promoted learning and engagement in the class by answering questions and providing clarity; allowing more use of active- and interactive-learning structures; and serving as accessible, approachable, and immediate sources of help. Student responses also indicated that LAs promoted a sense of belonging in science, technology, engineering, and mathematics (STEM) by decreasing feelings of isolation, serving as inspirational role models, clarifying progression through the STEM educational system, and helping students become more engaged and confident in their STEM-related knowledge and skills. These findings indicate that LAs can support multiple elements of inclusive STEM learning environments.

A Guide to COVID-19: a global pandemic caused by the novel coronavirus SARS-CoV-2.

The emergence of the SARS-CoV-2 strain of the human coronavirus has thrown the world into the midst of a new pandemic. In the human body, the virus causes COVID-19, a disease characterized by shortness of breath, fever, and pneumonia, which can be fatal in vulnerable individuals. SARS-CoV-2 has characteristics of past human coronaviruses, with close genomic similarities to SARS-CoV, the virus that causes the disease SARS. Like these related coronaviruses, SARS-CoV-2 is transmitted through the inhalation of droplets and interaction with contaminated surfaces. Across the world, laboratories are developing candidate vaccines for the virus - with vaccine trials underway in the United States and the United Kingdom - and considering various drugs for possible treatments and prophylaxis. Here, we provide an overview of SARS-CoV-2 by analyzing its virology, epidemiology, and modes of transmission while examining the current progress of testing procedures and possible treatments through drugs and vaccines.

AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate.

Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here, we report that a somite-derived prohematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch up-regulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing (RNA-seq), and assay for transposase-accessible chromatin using sequencing (ATAC-seq) indicate that Srebp2 transregulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.

RICE CRISPR: Rapidly increased cut ends by an exonuclease Cas9 fusion in zebrafish.

Application of CRISPR-Cas9 technology in diverse organisms has resulted in an explosion of genome modification efforts. To expand the toolbox of applications, we have created an E. coli Exonuclease I (sbcB)-Cas9 fusion that has altered enzymatic activity in zebrafish embryos. This Cas9 variant has increased mutation efficiency and favors longer deletions relative to wild-type Cas9. We anticipate that this variant will allow for more efficient screening for F0 phenotypes and mutation of a larger spectrum of genomic targets including deletion of regulatory regions and creating loss of function mutations in transcription units with poor sequence conservation such as lncRNAs where larger deletions may be required for loss of function.

Neuropilin 1 balances ß8 integrin-activated TGFß signaling to control sprouting angiogenesis in the brain.

Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that ß8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. ß8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor ß (TGFß) ligands and stimulates TGFß receptor signaling in endothelial cells. Nrp1 in endothelial cells suppresses TGFß activation and signaling by forming intercellular protein complexes with ß8 integrin. Cell type-specific ablation of ß8 integrin, Nrp1, or canonical TGFß receptors results in pathological angiogenesis caused by defective neuroepithelial cell-endothelial cell adhesion and imbalances in canonical TGFß signaling. Collectively, these data identify a paracrine signaling pathway that links the neuroepithelium to blood vessels and precisely balances TGFß signaling during cerebral angiogenesis.

Acinetobacter baumannii Repeatedly Evolves a Hypermutator Phenotype in Response to Tigecycline That Effectively Surveys Evolutionary Trajectories to Resistance.

The evolution of hypermutators in response to antibiotic treatment in both clinical and laboratory settings provides a unique context for the study of adaptive evolution. With increased mutation rates, the number of hitchhiker mutations within an evolving hypermutator population is remarkably high and presents substantial challenges in determining which mutations are adaptive. Intriguingly however, hypermutators also provide an opportunity to explore deeply the accessible evolutionary trajectories that lead to increased organism fitness, in this case the evolution of antibiotic resistance to the clinically relevant antibiotic tigecycline by the hospital pathogen Acinetobacter baumannii. Using a continuous culture system, AB210M, a clinically derived strain of A. baumannii, was evolved to tigecycline resistance. Analysis of the adapted populations showed that nearly all the successful lineages became hypermutators via movement of a mobile element to inactivate mutS. In addition, metagenomic analysis of population samples revealed another 896 mutations that occurred at a frequency greater than 5% in the population, while 38 phenotypically distinct individual colonies harbored a total of 1712 mutations. These mutations were scattered throughout the genome and affected ~40% of the coding sequences. The most highly mutated gene was adeS, a known tigecycline-resistance gene; however, adeS was not solely responsible for the high level of TGC resistance. Sixteen other genes stood out as potentially relevant to increased resistance. The five most prominent candidate genes (adeS, rpsJ, rrf, msbA, and gna) consistently re-emerged in subsequent replicate population studies suggesting they are likely to play a role in adaptation to tigecycline. Interestingly, the repeated evolution of a hypermutator phenotype in response to antibiotic stress illustrates not only a highly adaptive strategy to resistance, but also a remarkably efficient survey of successful evolutionary trajectories.

The ribosomal S10 protein is a general target for decreased tigecycline susceptibility.

Tigecycline is a translational inhibitor with efficacy against a wide range of pathogens. Using experimental evolution, we adapted Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, and Staphylococcus aureus to growth in elevated tigecycline concentrations. At the end of adaptation, 35 out of 47 replicate populations had clones with a mutation in rpsJ, the gene that encodes the ribosomal S10 protein. To validate the role of mutations in rpsJ in conferring tigecycline resistance, we showed that mutation of rpsJ alone in Enterococcus faecalis was sufficient to increase the tigecycline MIC to the clinical breakpoint of 0.5 µg/ml. Importantly, we also report the first identification of rpsJ mutations associated with decreased tigecycline susceptibility in A. baumannii, E. coli, and S. aureus. The identified S10 mutations across both Gram-positive and -negative species cluster in the vertex of an extended loop that is located near the tigecycline-binding pocket within the 16S rRNA. These data indicate that S10 is a general target of tigecycline adaptation and a relevant marker for detecting reduced susceptibility in both Gram-positive and -negative pathogens.