Cutaneous wound healing is an intricate and multifaceted process. Despite these complexities, the distinct phases of wound healing provide a unique opportunity to evaluate the roles of different targets in these coordinated responses. This protocol details an in vivo wound healing assay to study the intersection of cellular, molecular, and systemic effector pathways. The role of certain proteins in the wound healing process can be efficiently explored in vivo through the generation of tissue-specific deficient mice. This approach, although optimized for use with animal models displaying epithelial deficiencies, can be used for other tissue-specific deficiencies, and utilizes simple and cost-effective methods, allowing investigators to precisely devise their experimental design. The coordination of immunological, epithelial, vascular, and microenvironmental factors in wound healing makes this technique a valuable tool for investigators across fields.
The goal of this research was to determine the effects of the growth of invasive plant Amur Honeysuckle (Lonicera maackii) on the rhizosphere bacterial community composition, and diversity in an urban wetland forest ecosystem. Bacterial communities from the rhizosphere of 5 L. maackii plants and control bulk soils that did not have any L. maackii were investigated at Nina Mason Pulliam EcoLab (NMPE) using a culture-independent pipeline. Bacterial communities were characterized by PCR amplification and cloning 16S rRNA gene fragments following total DNA isolation from the soil samples. Microbial communities associated with both L. maackii rhizosphere and control sites showed high bacterial diversity within each site and taxa unique to individual sites were observed. Phylogenetic analyses revealed 80% of 400 16S rDNA clones were classified as α-, β- and γ-Proteobacteria, Acidobacteria, Actinobacteria, Cytophaga-Flexibacter-Bacteroides (CFB) group, and Verrucomicrobia. Members of the Proteobacteria and Acidobacterium represented 66.5% and 14.5% of the clone library, respectively, whereas the remaining bacterial divisions each comprised less than 7% of the clone library. Twenty-five 16S rDNA clones could not be classified into any known bacterial divisions. Statistical analyses showed significant differences in the presence of L. maackii on the proportions of 16S rDNA clones affiliated with Proteobacteria and Acidobacterium, suggesting bacterial community composition and structure does significantly change in the presence of L. maackii. However, sequence-based community analysis and the corresponding lack of intact microbial cultures limit understanding of the potential influences of enriched microbial taxa on plant hosts and their roles in ecosystem functioning.
The increase in our aging population in combination with the growing incidence of late-life conditions presents a need for research on healthy aging. Here, we present work examining the roles of sphingolipid metabolism on aging. Sphingolipids play important roles in stress response, cell survival, cell signaling, and aging. Specifically, ceramide responds to oxidative stress and recruits apoptotic proteins to the cell membrane thereby causing cell cycle arrest. Furthermore, ceramide levels increase with age, making it an important lipid mediator of aging. The objective of this study is to examine the physiological role of ceramide metabolism on aging and lipid metabolism pathways in the roundworm, Caenorhabditis elegans. To assess these mechanisms, we utilized C. elegans’ mutants lacking acid sphingomyelinase (asm-3) and ceramide synthase (hyl-2) which we previously found to be long- and short-lived respectively. We performed a lipidomic analysis to explore longitudinal changes in lipid concentrations in wild type (N2), asm-3/acid sphingomyelinase, and hyl-2/ceramide synthase worms at 1, 5, and 10 days of age. Among our samples, we detected 700 different lipids that were abundant enough in concentration for analysis. Interestingly, 10 day old hyl-2 mutants, which have a reduced life-span, showed an increased concentration of eicosapentaenoic acid (EPA), an omega-3 fatty acid that has been shown to increase with longevity in worms. Conversely, asm-3 mutants, which are long-lived animals, have reduced levels of EPA. To expand upon our lipidomic data with enzymatic findings, we utilized RT-qPCR analysis to longitudinally analyze fatty acid desaturases (fat-1 and fat-4) involved in EPA metabolism in worms. We found that fat-4 expression is reduced in 1, 5, and 10 day hyl-2 animals, suggesting that ceramide metabolism may impact fatty acid genes involved in aging. Future work will examine the role of stress response genes including superoxide dismutase (sod-3) and fatty acid tail elongases (elo-5 and elo-6) in 1, 5, and 10 day old hyl-2 and asm-3 mutants. Finally, to expand upon the current knowledge of ceramide's role in lifespan, we present data on the RNAi mediated suppression of hyl-2 in long-lived mutants including mitochondrial respiration (clk-1/coenzyme Q), insulin-like signaling (daf-2/insulin-like receptor), and dietary starvation (eat-2/mAChR). Our goal is to identify whether ceramide metabolism works within these known pathways that mediate aging. With these assays, we hope to better understand the intrinsic biochemical lipid processes associated with ceramide metabolism in aging animals.
Many C. elegans aging studies use the compound 5-fluro-2ʹ-deoxyuridine (FUdR) to produce a synchronous population of worms. However, the effects of FUdR on the bacterial gene expression of OP50 E. coli, the primary laboratory C. elegans food source, is not fully understood. This is particularly relevant as studies suggest that intestinal microbes can affect C. elegans physiology. Therefore, it is imperative that we understand how exposure to FUdR can affect gene expression changes in OP50 E. coli.
An RNAseq dataset comprised of expression patterns of 2900 E. coli genes in the strain OP50, which were seeded on either nematode growth media (NGM) plates or on FUdR (50 µM) supplemented NGM plates, was analyzed. Analysis showed differential gene expression in genes involved in general transport, amino acid biosynthesis, transcription, iron transport, and antibiotic resistance. We specifically highlight metabolic enzymes in the l-histidine biosynthesis pathway as differentially expressed between NGM and FUdR exposed OP50. We conclude that OP50 exposed to FUdR results in differential expression of many genes, including those in amino acid biosynthetic pathways.
We conducted a study of the diet of the federally endangered Indiana bat (Myotis sodalis) at an urban/rural interface near Indianapolis International Airport in summer 2004. We used two 1-m2quadrats covered with window screening to collect guano under a known roost tree. We then examined 20 fecal pellets/week until the bats abandoned the roost (i.e., 13 weeks). The most common orders of insects eaten were: Lepidoptera (35.3% volume, 84.6% frequency), Diptera (27.9%, 73.2%), Coleoptera (16.9%, 62.9%), and Hymenoptera (10.9%, 45.9%). Components of the diet at the ordinal level varied significantly over time. Despite the developed nature of the site, the diet consisted of the same components reported in earlier studies.
G-protein coupled receptors (GPCRs), which activate heterotrimeric G proteins, are an essential class of transmembrane receptors that are responsible for a myriad of signaling events in normal and pathologic conditions. Two members of the G protein family, Gaq and Ga-11, activate one of the main GPCR pathways and function as oncogenes by integrating mitogen-stimulated signaling cascades that are active under malignant conditions. Recently, it has been shown that targeted deletion of Ga-11 and Gaq from endothelial cells impairs the Rho -mediated formation of focal adherens junctions, suggesting that Gai vg signaling may also play a significant role in cytoskeletal-mediated cellular responses in epithelial cells. Indeed, combined deletion of Ga-11 and Gaq confers a significant migratory defect in keratinocytes that delays cutaneous wound healing in an in vivo setting. This delay can be attributed to a defect during the reepithelialization phase due to significantly attenuated migratory capacity of Gaq-null keratinocytes under combined Ga-11 deficiency. In fact, cells lacking Gaivg demonstrate a severely reduced ability to respond to mitogenic and migratory signals in the microenvironment, leading to inappropriate and premature terminal differentiation. These results suggest that Gaivg signaling pathways may be critical for integrating mitogenic signals and cytoskeletal function to achieve normal physiological responses. Emergence of a malignant phenotype may therefore arise from both under- and overexpression of Gai vg signaling, implicating its upstream regulation as a potential therapeutic target in a host of pathologic conditions.
We join our voices with countless others across the country in an unequivocal condemnation of police brutality, institutionalized racism, and all forms of anti-black violence. As a scientific community, we acknowledge that our own past is filled with examples where we have brought violence against Black, Indigenous, and People of Color (BIPOC), from histories as overt as the Tuskegee Syphilis trials ( www.history.com, 2019a) or the unethical use of Puerto Rican women as subjects in birth control trials ( www.history.com, 2019b) to the more pernicious acts of everyday racism that occur in our colleges and universities. Unfortunately, we are still not far from this history. This past week, George Floyd was killed in Minneapolis, and as COVID-19 ravages the globe and affects all of us in deeply painful ways, our BIPOC communities have disproportionately carried the burden ( www.embracerace.org). However, insofar as we have been complicit in racism, we also hold the tools for a robust participation in the work of anti-racism. What we do with students in our classrooms and in our labs is not separate from the work of racial justice. We all have our part to play. Indeed, we as scientists must marshal all our skills (yes, even pipetting!) toward creating a brighter future and more equitable world. As scholar-teachers, we must create neuroscience as an inclusive space for our students, especially for our students of color. FUN commits to taking concrete steps to further educate, equip, and heal our community.
SEMA3F is an antilymphangiogenic signaling molecule expressed in the proliferating basal layer of normal epithelium. In orthotopic models of head and neck squamous cell carcinoma, loss of SEMA3F or its receptors results in enhanced tumor growth and locoregional metastasis, leading to speculation that SEMA3F may also prevent cancer initiation. To determine whether the loss of SEMA3F enhances susceptibility to cancer development, we employed a two‐step carcinogenesis model in a robustly immunogenic outbred strain of wild‐type (WT) or Sema3F knockout (KO) mice. Animals were treated with a single application of 7,12‐Dimethylbenz(a)anthracene (DMBA) followed by twice‐weekly applications of 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA) for 24 weeks. Sema3F KO animals had a modest but statistical decrease in tumor latency compared with WT. Additionally, both the number and size of lesions was significantly decreased in KO animals. Together, this suggested that loss of Sema3F may be influencing cancer development and progression primarily through modulation of the tumor microenvironment. By histochemical staining, KO animals demonstrated much higher percentage of immune infiltrate within the tumors. To investigate whether immunosurveillance may contribute to the diminished cancer progression in KO animals, we analyzed the mRNA expression of inflammatory cytokines from these animals. Analysis of the inflammatory tumor microenvironment in these lesions revealed a strong induction of both IL‐12α and IL‐1β in KO animals. Primarily produced by macrophages, IL‐12α has been shown to suppress the development of skin papillomas and suppress tumor growth and development. Compellingly, macrophages but not leukocytes express Sema3F receptors. Consequently, we propose that expression of Sema3F in the context of chemical carcinogenesis functions as a macrophage chemorepellent and antilymphangiogenic molecule, thus delaying the onset of an adaptive immune response and increasing the latency and tumor burden. Further, Sema3F may be an attractive target for cancer immunotherapies given its autocrine and paracrine signaling roles.
This laboratory experiment, designed for undergraduate nonmajors or advanced high school students, attempts to demystify the technique of spectrophotometry by utilizing a hands-on, real-world approach. Modification of the reaction for the formation of Fe[(bpy)3]2+ allows students to interact with vitamin C, a compound with which they are familiar from daily life, and observe its concentration in an approachable experimental framework. These connections are emphasized in a protocol designed to help students correlate visual and quantitative data by generating their own standard curve against which they compare unknown solutions. This experiment was found to decisively increase students’ understanding of concepts related to standard curves and spectrophotometry, and it also demonstrated gains in student understanding of solubility and its impact on colorimetry. Opportunities within this experimental framework exist for additional conceptual development related to the chemical, technological, and social aspects of the assay. Combined with its pedagogical effectiveness, this represents an excellent resource for diverse chemistry classrooms.