Mutation of hmgA, encoding homogentisate 1,2-dioxygenase, is responsible for pyomelanin production but does not impact the virulence of Burkholderia cenocepacia in a chronic granulomatous disease mouse lung infection.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals; pigment production has been reported to enable Bcc strains to overcome the host cell oxidative burst. In this work, we investigated the role of pyomelanin in resistance to oxidative stress and virulence in strains J2315 and K56-2, two epidemic CF isolates belonging to the Burkholderia cenocepacia ET-12 lineage. We previously reported that a single amino acid change from glycine to arginine at residue 378 in homogentisate 1,2-dioxygenase (HmgA) affects the pigment production phenotype: pigmented J2315 has an arginine at position 378, while non-pigmented K56-2 has a glycine at this position. Herein, we performed allelic exchange to generate isogenic non-pigmented and pigmented strains of J2315 and K56-2, respectively, and tested these to determine whether pyomelanin contributes to the protection against oxidative stress in vitro as well as in a respiratory infection in CGD mice in vivo. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist oxidative stress with H2O2 and NO in vitro and did not change the virulence and infection outcome in CGD mice in vivo suggesting that other factors besides pyomelanin are contributing to the pathophysiology of these strains.IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria that are often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals and overcoming the host cell oxidative burst. We investigated the role of pyomelanin in Burkholderia cenocepacia strains J2315 (pigmented) and K56-2 (non-pigmented) and performed allelic exchange to generate isogenic non-pigmented and pigmented strains, respectively. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist H2O2 or NO in vitro and did not alter the outcome of a respiratory infection in CGD mice in vivo. These results suggest that pyomelanin may not always constitute a virulence factor and suggest that other features are contributing to the pathophysiology of these strains.

Increasing Student Confidence in Writing: Integrating Authentic Manuscript Writing into an Online 8-Week Research Program.

In various formats, students at the secondary and postsecondary levels participate in multiweek authentic science research projects. There have been many papers explaining the operations of such programs, but few have provided explicit instruction on how to incorporate authentic communication practices into the student research process. In this paper, we describe how we integrated primary literature into an 8-week online research program for 8th to 11th graders. Each week, students were introduced to a specific section of a primary research article reflecting different stages of their research project, and they were guided on how to write that specific section for their own research paper. By the end of the program, students had an outline or first draft of a primary research paper based on their research. Following completion of the program, student participants reported greater self-efficacy and confidence in scientific writing. Here, we describe our approach and provide an adaptable framework for integrating primary literature into research projects.

No Science Fair? No Problem. Engaging Students in Science Communication through Peer Review and Publication in a Remote World.

Since March 2020, in-person science competitions have been cancelled or moved to a virtual space. This reality has encouraged teachers and students to find alternative ways to disseminate student research and participate in a scientific community. Participating in the peer review and publication of one's research offers one such alternative. The Journal of Emerging Investigators (JEI) is a free, online, peer-reviewed science journal specifically for middle school and high school students. JEI provides students the opportunity to engage with professional scientists through the peer review process and share their research with a broad audience, all on a remote platform. This article describes resources that are freely available to help teachers navigate the peer review and publication processes and guide their students through the successful completion of submission and publication of their research papers. Overall, students perceive the experience as attainable and found the JEI resources useful in completing their papers. Importantly, students expressed that the experience of publication increased their confidence and interest in STEM.

Characterizing changes in soil microbiome abundance and diversity due to different cover crop techniques.

Soil-based microorganisms assume a direct and crucial role in the promotion of soil health, quality and fertility, all factors known to contribute heavily to the quality and yield of agricultural products. Cover cropping, used in both traditional and organic farming, is a particularly efficient and environmentally favorable tool for manipulating microbiome composition in agricultural soils and has had clear benefits for soil quality and crop output. Several long-term investigations have evaluated the influence of multi-mix (multiple species) cover crop treatments on soil health and microbial diversity. The present study investigated the short-term effects of a seven species multi-mix cover crop treatment on soil nutrient content and microbial diversity, compared to a single-mix cover crop treatment and control. Analysis of 16S sequencing data of isolated soil DNA revealed that the single-mix cover crop treatment decreased overall microbial abundance and diversity, whereas the control and multi-mix treatments altered the overall microbial composition in similar fluctuating trends. Furthermore, we observed significant changes in specific bacteria belonging to the phyla Acidobacteria, Actinobacteria, Planctomycetes, Proteobacteria and Verrucombicrobia for all treatments, but only the single-mix significantly decreased in abundance of the selected bacteria over time. Our findings indicate that the control and multi-mix treatments are better at maintaining overall microbial composition and diversity compared to the single-mix. Further study is required to elucidate the specific difference between the treatment effect of the multi-mix treatment and the control, given that their microbial composition changes over time were similar but they diverge into two populations of unique bacterial types by the end of this short-term study.

Engaging in the Publication Process Improves Perceptions of Scientific Communication, Critique, and Career Skills Among Graduate Students.

Reading and critiquing primary scientific literature is an important skill for graduate students, as reviewing literature is critical to advancing science. Prior research indicates that graduate students lack understanding of effective communication as well as basic experimental design, but also that graduate students are capable of growth in their experimental design abilities when given proper opportunities. The Journal of Emerging Investigators (JEI) provides graduate students with the opportunity to review and edit original research papers submitted by middle and high school student-authors. The purpose of this project was to determine whether participation in the primary literature process through JEI effectively aids in developing graduate students' perceived abilities in the domains of communication, scientific critique, and career preparation. A 12-question survey was distributed using SurveyMonkey to 215 JEI reviewers and editors. Editors, whose role involves the synthesis of feedback from multiple reviewers and interaction with papers in their earliest stages, perceived that they benefited more than did reviewers in every domain assessed by the survey. Perceived impact on critiquing skills was only rated more highly by reviewers than by editors once the graduate students in question had reviewed 10 or more papers. The results of this research suggest that graduate students should participate early and often in the reading and reviewing of primary literature; furthermore, the study of flawed science writing can help to improve experimental design, critique, and science communication skills.

Incorporating Primary Scientific Literature in Middle and High School Education.

Primary literature is the most reliable and direct source of scientific information, but most middle school and high school science is taught using secondary and tertiary sources. One reason for this is that primary science articles can be difficult to access and interpret for young students and for their teachers, who may lack exposure to this type of writing. The Journal of Emerging Investigators (JEI) was created to fill this gap and provide primary research articles that can be accessed and read by students and their teachers. JEI is a non-profit, online, open-access, peer-reviewed science journal dedicated to mentoring and publishing the scientific research of middle and high school students. JEI articles provide reliable scientific information that is written by students and therefore at a level that their peers can understand. For student-authors who publish in JEI, the review process and the interaction with scientists provide invaluable insight into the scientific process. Moreover, the resulting repository of free, student-written articles allows teachers to incorporate age-appropriate primary literature into the middle and high school science classroom. JEI articles can be used for teaching specific scientific content or for teaching the process of the scientific method itself. The critical thinking skills that students learn by engaging with the primary literature will be invaluable for the development of a scientifically-literate public.

Using Scavenger Hunts to Familiarize Students with Scientific Journal Articles.

Primary scientific literature can be difficult to navigate for anyone unfamiliar with its foreign, formal structure. We sought to create a fun, easy learning tool to help familiarize students of all ages with the structure of a scientific article. Our main learning objective was for the student to realize that science writing is formulaic-that specific information is found in predictable locations within an article-and that, with an understanding of the formula, anyone can comfortably navigate any journal article and accurately predict what to expect to find in each section. To this end, we designed a Journal Article Scavenger Hunt that requires the user to find and identify a series of commonplace features of a primary research article. The scavenger hunt activity is quick and easy to implement, and is adaptable to various ages and settings, including the classroom, lab, and at outreach events. The questions in the scavenger hunt can be scaled in difficulty and specificity to suit the instructor's needs. Over many years of using this activity, we have received positive feedback from students of all ages, from elementary school students to lay adult-learners as well as science teachers themselves. By making the unknown seem predictable and approachable, the scavenger hunt helps a variety of audiences feel more comfortable with science and more confident in their ability to engage directly with the scientific literature. Journal of Microbiology & Biology Education.

Single amino acid substitution in homogentisate 1,2-dioxygenase is responsible for pigmentation in a subset of Burkholderia cepacia complex isolates.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacilli that are ubiquitous in the environment and have emerged over the past 30 years as opportunistic pathogens in immunocompromised populations, specifically individuals with cystic fibrosis (CF) and chronic granulomatous disease. This complex of at least 18 distinct species is phenotypically and genetically diverse. One phenotype observed in a subset of Burkholderia cenocepacia (a prominent Bcc pathogen) isolates is the ability to produce a melanin-like pigment. Melanins have antioxidant properties and have been shown to act as virulence factors allowing pathogens to resist killing by the host immune system. The melanin-like pigment expressed by B. cenocepacia is produced through tyrosine catabolism, specifically through the autoxidation and polymerization of homogentisate. Burkholderia cenocepacia J2315 is a CF clinical isolate that displays a pigmented phenotype when grown under normal laboratory conditions. We examined the amino acid sequences of critical enzymes in the melanin synthesis pathway in pigmented and non-pigmented Bcc isolates, and found that an amino acid substitution of glycine for arginine at amino acid 378 in homogentisate 1,2-dioxygenase correlated with pigment production; we identify this as one mechanism for expression of pigment in Bcc isolates.

PD-L1 limits the mucosal CD8+ T cell response to Chlamydia trachomatis.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Repeated infections with C. trachomatis lead to serious sequelae, such as infertility. It is unclear why the adaptive immune system, specifically the CD8(+) T cell response, is unable to protect against subsequent C. trachomatis infections. In this article, we characterize the mucosal CD8(+) T cell response to C. trachomatis in the murine genital tract. We demonstrate that the immunoinhibitory ligand, PD-L1, contributes to the defective CD8(+) T cell response. Deletion or inhibition of PD-L1 restores the CD8(+) T cell response and enhances C. trachomatis clearance.