Elucidating the Priming Mechanism of ClpXP Protease by Single-Domain Response Regulator CpdR in Caulobacter crescentus

In Caulobacter crescentus, progression through the cell cycle is regulated by the AAA+ protease ClpXP, and there are several classes of cell-cycle substrates that require adaptors in order to be degraded. CpdR, a single domain-response regulator, binds the N-terminal domain of ClpXP and primes the protease for degradation of downstream factors (Lau et al., 2015). The ability of CpdR to bind ClpX is regulated by its phosphorylation state. In the unphosphorylated state, CpdR binds ClpXP and guides its localization to the cell pole during the swarmer to stalked transition, where CpdR is mediates degradation of substrates such as PdeA. Phosphorylation of response regulator receiver domains requires magnesium as a cofactor to stabilize the phosphorylated aspartate and reciprocally, phosphorylated receiver domains bind magnesium more effectively. While it is understood that CpdR primers ClpX for substrate degradation, the mechanism by which it does so has remained unclear. Using CollabFold, we identified putative residues involved in CpdR-ClpX binding and validated them using a BACTH screening. In vitro, we characterized the role that magnesium plays in regulating CpdR binding to ClpX. In this work, we directly test the role of magnesium in CpdR priming of ClpXP to show that magnesium may play a regulatory role in CpdR-mediated degradation, and thus binding to ClpX. We identify residues in ClpX that seem to be important for CpdR binding, which prior to this work was not clear

Integrating Campus Partners Towards Equitable Open Educational Resources Adoption: A Case Study of a California State University Affordable Learning Solutions Program at Humboldt State University

The Humboldt State University (HSU) Library and HSU Center for Teaching and Learning (CTL) collaborated with the campus’ own Accessibility Resource Center, Student Disability Resources Center, and Office of Diversity, Equity, and Inclusion through five training sessions, to facilitate the integration of open educational resources (OER) into the classroom. Faculty and students, from disparate disciplines, participated as equal stakeholders in the discovery and implementation of OER into a course, throughout a year-long immersive program. This summary covers the history of the California State University (CSU) Affordable Learning Solutions (AL$) OER program at Humboldt State University; the structure of the 2019-2020 implementation of the program; and the results and conclusion to gauge the efficacy of our collective goal to reduce the material costs for students and expand open, equitable, and accessible practices into the classroom. This summary will specifically address the process of integrating inclusion, diversity, equity, and accessibility into OER adoption and curriculum design, for the purpose of advancing student success. The trainings for the CSU/AL$/OER program are publicly available through our OER Canvas Commons course and our collected OER resources, organized by discipline, are available through our OER Research Guid

The establishment and function of lung resident memory B cells after bacterial respiratory infection

Streptococcus pneumoniae, or pneumococcus, remains the most prevalent cause of bacterial pneumonia worldwide. The burden of pneumococcal disease peaks among children and the elderly, while young adults are well protected against disease from all pneumococcal serotypes. Whether memory B cells play a role in this naturally developing serotype-independent immunity has not been determined. Additionally, lung resident memory B cells (BRM cells) are elicited after influenza infections of mice, but their relevance to bacterial pathogens and to humans remains unknown, as do the signals required for their establishment. We sought to address these knowledge gaps. We found that respiratory pneumococcal exposures in mice elicited lung BRM cells without concurrent tertiary lymphoid structure formation. Additionally, normal human lung tissue is enriched for B cells bearing a resident memory phenotype. Mice exposed to a low virulence pneumococcal strain were protected from a subsequent serotype-mismatched pneumococcal challenge. To address the role of B cells in this lung defense, we used a genetically engineered mouse strain allowing effective depletion of lung B cells bearing programmed death-ligand 2 (PD-L2, a memory B cell marker). When pneumococcus-experienced mice were depleted of PD-L2+ B cells just before the challenge infection, they experienced substantial defects in bacterial clearance compared to mice with lung B cells intact. These results provide the first evidence of a role for lung BRM cells in anti-bacterial immunity. Notably, this defense was pneumococcal serotype-independent, distinguishing it from the serotype-specific immunity elicited by current pneumococcal vaccines. Finally, we found that the establishment of lung BRM cells in mice requires CD4+ T cells and multiple respiratory pneumococcal exposures. A second pneumococcal infection, but not the first, induces lung chemokine (C-X-C motif) ligand 13 (CXCL13) production, establishment of local germinal center reactions, and accumulation of class-switched BRM cells in the lung. In conclusion, herein we show that lung BRM cells are a common feature of antigen-experienced lungs and describe the kinetics of lung BRM cell establishment and the role these cells play in serotype-independent lung immunity against pneumococcal pneumonia

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress

Genetically encoded, fluorescent biosensors have been developed to probe the activities of various signaling molecules inside cells ranging from changes in intracellular ion concentrations to dynamics of lipid second messengers. HyPer is a member of this class of biosensors and is the first to dynamically respond to hydrogen peroxide (H[subscript 2]O[subscript 2]), a reactive oxygen species that functions as a signaling molecule. However, detailed characterization of HyPer's signal is not currently available within the context of bacteria exposed to external oxidative stress, which occurs in the immunological response of higher organisms against invasive pathogenic bacteria. Here, we performed this characterization, specifically in Escherichia coli exposed to external H[subscript 2]O[subscript 2]. We found that the temporal behavior of the signal does not correspond exactly to peroxide concentration in the system as a function of time and expression of the sensor decreases the peroxide scavenging activity of the cell. We also determined the effects of cell number, both before and after normalization of externally added H[subscript 2]O[subscript 2] to the number of cells. Finally, we report quantitative characteristics of HyPer's signal in this context, including the dynamic range of the signal, the signal-to-noise ratio, and the half saturation constant. These parameters show statistically meaningful differences in signal between two commonly used strains of E. coli, demonstrating how signal can vary with strain. Taken together, our results establish a systematic, quantitative framework for researchers seeking to better understand the role of H[subscript 2]O[subscript 2] in the immunological response against bacteria, and for understanding potential differences in the details of HyPer's quantitative performance across studies.Massachusetts Institute of Technology. James H. Ferry Fund for Innovation in Research EducationNational Science Foundation (U.S.) (NSF Graduate Research Fellowship)Massachusetts Institute of Technology (Joseph R. Mares endowed chair in chemical engineering)Burroughs Wellcome Fund (Career Award at the Scientific Interface

Testing self-report time-use diaries against objective instruments in real time

This study provides a new test of time-use diary methodology, comparing diaries with a pair of objective criterion measures: wearable cameras and accelerometers. A volunteer sample of respondents (n = 148) completed conventional self-report paper time-use diaries using the standard UK Harmonised European Time Use Study (HETUS) instrument. On the diary day, respondents wore a camera that continuously recorded images of their activities during waking hours (approximately 1,500–2,000 images/day) and also an accelerometer that tracked their physical activity continuously throughout the 24-hour period covered by the diary. Of the initial 148 participants recruited, 131 returned usable diary and camera records, of whom 124 also provided a usable whole-day accelerometer record. The comparison of the diary data with the camera and accelerometer records strongly supports the use of diary methodology at both the aggregate (sample) and individual levels. It provides evidence that time-use data could be used to complement physical activity questionnaires for providing population-level estimates of physical activity. It also implies new opportunities for investigating techniques for calibrating metabolic equivalent of task (MET) attributions to daily activities using large-scale, population-representative time-use diary studies

Individually addressable double quantum dots formed with nanowire polytypes and identified by epitaxial markers

Double quantum dots (DQDs) hold great promise as building blocks for quantum technology as they allow for two electronic states to coherently couple. Defining QDs with materials rather than using electrostatic gating allows for QDs with a hard-wall confinement potential and more robust charge and spin states. An unresolved problem is how to individually address these quantum dots, which is necessary for controlling quantum states. We here report the fabrication of double quantum dot devices defined by the conduction band edge offset at the interface of the wurtzite and zinc blende crystal phases of InAs in nanowires. By using sacrifical epitaxial GaSb markers selectively forming on one crystal phase, we are able to precisely align gate electrodes allowing us to probe and control each QD independently. We hence observe textbook-like charge stability diagrams, a discrete energy spectrum and electron numbers consistent with theoretical estimates and investigate the tunability of the devices, finding that changing the electron number can be used to tune the tunnel barrier as expected by simple band diagram arguments.Comment: 5 pages, 3 figure

Show Me My Health Plans: a study protocol of a randomized trial testing a decision support tool for the federal health insurance marketplace in Missouri

BACKGROUND: The implementation of the ACA has improved access to quality health insurance, a necessary first step to improving health outcomes. However, access must be supplemented by education to help individuals make informed choices for plans that meet their individual financial and health needs. METHODS/DESIGN: Drawing on a model of information processing and on prior research, we developed a health insurance decision support tool called Show Me My Health Plans. Developed with extensive stakeholder input, the current tool (1) simplifies information through plain language and graphics in an educational component; (2) assesses and reviews knowledge interactively to ensure comprehension of key material; (3) incorporates individual and/or family health status to personalize out-of-pocket cost estimates; (4) assesses preferences for plan features; and (5) helps individuals weigh information appropriate to their interests and needs through a summary page with “good fit” plans generated from a tailored algorithm. The current study will evaluate whether the online decision support tool improves health insurance decisions compared to a usual care condition (the healthcare.gov marketplace website). The trial will include 362 individuals (181 in each group) from rural, suburban, and urban settings within a 90 mile radius around St. Louis. Eligibility criteria includes English-speaking individuals 18–64 years old who are eligible for the ACA marketplace plans. They will be computer randomized to view the intervention or usual care condition. DISCUSSION: Presenting individuals with options that they can understand tailored to their needs and preferences could help improve decision quality. By helping individuals narrow down the complexity of health insurance plan options, decision support tools such as this one could prepare individuals to better navigate enrollment in a plan that meets their individual needs. The randomized trial was registered in clinicaltrials.gov (NCT02522624) on August 6, 2015

Delivering More Than Food: Understanding and Operationalizing Racial Equity in Food Hubs

This report is a look at a how U.S.-based food hubs understand engagement in racial equity work. The sample of food hubs interviewed for this report are diverse in their structures, leadership, and missions. Through interviews with food hub managers and other roles, we identify common facilitators and inhibitors to food hubs engaging in racial equity work. After presenting the major themes of our findings, we provide an analysis of those findings through multiple frames. We offer takeaways in the form of identifying deeper questions for food hubs, funders, and researchers about how to meaningfully support racial equity within the food system. We also offer specifics of how to operationalize some of our findings by providing a few examples of food hubs/food system organizations that have taken clear action toward achieving racial equity goals.

Host-parasite interactions between Lernaeocera branchialis (Copepoda: Pennellidae) and its host Gadus morhua (Teleosti: Gadidae)

Lernaeocera branchialis (Linnaeus, 1767) is a parasitic copepod possessing a complex dual-host lifecycle. The “definitive” gadoid hosts, including Gadus morhua (Atlantic cod), Melanogrammus aeglefinus (haddock) and Merlangius merlangus (whiting), are infected by the fertilised female, which penetrates the host’s ventral aorta or bulbus arteriosus whilst undertaking extensive metamorphosis and a haematophagous lifestyle. The pathogenic effects of this activity upon the host have been well documented and mortality may occur, especially when multiple parasites are present. These negative impacts on cod, particularly juveniles, by L. branchialis have the potential to adversely affect cod aquaculture in the future, and already vulnerable wild cod stocks. This PhD project therefore, investigated the immune response of wild haddock and cultured-cod post-infection by L. branchialis, and the possible mechanisms by which the parasite modulates/evades the host’s immune response. The systemic immune response of both wild haddock and cultured-cod post-infection by L. branchialis depended on the maturation stage of the parasite, and in the former host species, upon the infection intensity. Wild haddock harbouring fully metamorphosed females showed an increase in circulating thrombocytes and a decrease in serum protein levels however; if multiple mature L. branchialis were present the haddock possessed reduced circulating monocytes, and increased circulating thrombocytes and serum anti-trypsin activity. Infection by L. branchialis was also associated with a suppressive effect on haddock serum spontaneous haemolytic activity. These responses were thought to be due to the host trying to counteract the increased damage caused by the massive increase in size and the feeding of the mature parasite, which is more pronounced when multiple parasites are present, resulting in the increase in some parameters and the ‘consumption’ of others. However, the effect of parasite-derived secretions and other pathogens due to observations on wild fish could not be discounted. The laboratory-infection of cultured-cod from two different sources was also performed in order to study the immune response over time. The two groups of cod showed differences in their immune response to L. branchialis. The first group showed suppressed respiratory burst activity of phagocytes, as the parasite reached the early penella sub-stage, whilst no suppression in phagocyte respiratory burst activity was found in the second group. The parasite was found to migrate along the afferent branchial artery of the cod where a thrombus formed and was present throughout its migration into the ventral aorta. At 14 d post-infection, leukocytes expressing Interleukin 8 mRNA were observed within the free-flowing blood at the periphery of the organising thrombus within the lumen of the ventral aorta. This was speculated to aid the recruitment and activation of leukocytes to the site, and the maturation and neovascularisation of granulation tissue. The infection of the second group subsided with the death of the parasite, and none of the parasites metamorphosed past the early penella sub-stage. The live parasites infecting the first group of cod did not possess IgM or complement component C3 binding on their cuticle, however, both IgM and C3 binding occurred on the dead parasites in the second infection trial. This may highlight the importance of these opsonins and the cytotoxic effect of phagocytes in the elimination of L. branchialis by some cod. However, the first infection was terminated as the parasite reached the early penella sub-stage due to a loss of stock cod prior to the study, so the long-term success of the infection can not be concluded. Therefore, the immune response to infection needs to be determined over the entire metamorphosis of L. branchialis to determine whether the infection was successful or not, and preferably in populations with varying susceptibility to L. branchialis. This will not be possible without further studies into the resistance of different stocks of cultured-cod. Many arthropod parasites, such as ticks and salmon lice, have been previously documented to produce pharmacologically active secretions, aiding host invasion and parasite feeding, preventing the host immune response from working effectively against the parasite, all aimed at improving survival of the parasite. Therefore, the effects of the secretory/excretory products (SEPs) produced during the initial infective stage and by the mature, fully metamorphosed female on the immune response of cultured-cod in vitro, and the location of exocrine glands associated with the oral region of the parasite were investigated. The SEPs from the infective stage of the parasite were found not to affect the intracellular hydrogen peroxide (H2O2) production of phagocytes. The practical difficulties in collecting large quantities of the SEPs from the infective stage meant that their effects could not be tested on the other host immune parameters studied. The SEPs from fully metamorphosed female L. branchialis, however, had a number of suppressive effects on the host immune response in vitro including: 1) suppression of the intracellular production of cytotoxic H2O2 during the respiratory burst of phagocytic leukocytes post-PMA stimulation, 2) suppression of the production of macrophage activating factor by leukocytes with a priming effect on naïve phagocyte function, and 3) suppression of the chemo-attraction ‘power’ of zymosan activated cod serum, i.e. anaphylatoxin activity, on head kidney-derived leukocytes. These effects were dose-dependent, and highlight the capacity of L. branchialis to suppress its host’s innate immune response at the local feeding area. Further work is required to establish the mechanisms by which the parasite-derived SEPs suppress these host immune parameters, and to identify which molecules produced by the parasite are responsible. The correlation between these in vitro results, and systemic immune parameters measured from laboratory-infected Atlantic cod and wild infected haddock are discussed. Host immuno-modulation by other arthropod parasites is mediated by pharmacologically active secretions produced by exocrine glands. Therefore, the exocrine glands of the infective and fully metamorphosed female L. branchialis were also investigated in order to identify those that might be responsible for the secretion of host-modifying products. Adult female exocrine glands were mapped using diaminobenzidine (DAB), most commonly known to stain peroxidases and catalases. These compounds are known to be involved in the neutralisation of harmful free radicals which are released during the respiratory burst and tissue damage. Such products may therefore be important protective secretory components at the site of feeding / infection. Exocrine glands were located in the infective stage associated with the oral region, one pair termed the anterior gland complex (AGC), and the other pair extending either side of the oral cone termed the circum-oral glands (CG). These were further investigated using light microscopy and transmission electron microcopy. The AGC and CGs possessed multi-component secretions and they possessed secretory vesicles, abundant and highly active rough endoplasmic reticulum and Golgi apparatus suggesting that protein is an important component of the secretory products. These glands were also observed in the fully metamorphosed females where they had increased in size within the cephalothorax post-metamorphosis. It is hoped that the identification of these glandular structures, which are thought to secrete within the local vicinity of the oral cone, will aid future studies regarding the identification and secretion kinetics of parasite-derived molecules during the infection and feeding process.EThOS - Electronic Theses Online ServiceFisheries Society of the British IslesGBUnited Kingdo