OBJECTIVES/GOALS: Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). In this study, we perform an epigenetic-focused functional genomics screen in GBM organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. METHODS/STUDY POPULATION: Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche. Our niche-specific screens identified WDR5, an H3K4 histone methyltransferase responsible for activating specific gene expression, as indispensable for GBM CSC growth and survival. RESULTS/ANTICIPATED RESULTS: In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, required for stem cell maintenance and including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment resulted in dose-dependent silencing of stem cell reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. DISCUSSION/SIGNIFICANCE: Our results unveiled the role of WDR5 in maintaining the CSC state in GBM and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. This conceptual and experimental framework can be applied to many cancers, and can unmask unique microenvironmental biology and rationally designed combination therapies.

The ancient city of Gabii—an Italian polity of the first millennium BC and a peer to early Rome—has often been presented as an example of urban decline, a counterpoint to Rome's rise from a collection of hilltop huts to a Mediterranean hegemon. Here the authors draw on the results from recent excavations at Gabii that challenge such simplistic models of urban history. Diachronic evidence documenting activity at the site over the course of 1400 years highlights shifting values and rhythms materialised in the maintenance, transformation and abandonment of different urban components. This complex picture of adaptation and resilience provides a model of ancient urbanism that calls into question outdated narratives of urban success and failure.

Previously, it was suggested that haemadipsid leeches represent an important vector of trypanosomes amongst native animals in Australia. Consequently, Chtonobdella bilineata leeches were investigated for the presence of trypanosome species by polymerase chain reaction (PCR), DNA sequencing and in vitro isolation. Phylogenetic analysis ensued to further define the populations present. PCR targeting the 28S rDNA demonstrated that over 95% of C. bilineata contained trypanosomes; diversity profiling by deep amplicon sequencing of 18S rDNA indicated the presence of four different clusters related to the Trypanosoma (Megatrypanum) theileri. Novy–MacNeal–Nicolle slopes with liquid overlay were used to isolate trypanosomes into culture that proved similar in morphology to Trypanosoma cyclops in that they contained a large numbers of acidocalcisomes. Phylogeny of 18S rDNA/GAPDH/ND5 DNA sequences from primary cultures and subclones showed the trypanosomes were monophyletic, with T. cyclops as a sister group. Blood-meal analysis of leeches showed that leeches primarily contained blood from swamp wallaby (Wallabia bicolour), human (Homo sapiens) or horse (Equus sp.). The leech C. bilineata is a host for at least five lineages of Trypanosoma sp. and these are monophyletic with T. cyclops; we propose Trypanosoma cyclops australiensis as a subspecies of T. cyclops based on genetic similarity and biogeography considerations.

Background: Antibiotic stewardship programs (ASPs) have traditionally focused on inpatient prescribing, but they are now mandated to involve ambulatory settings. We developed and tested an educational tool in resident physicians to empower outpatient providers to perform self-reflection stewardship (SRS) to improve their antibiotic use. Results of the first SRS workshop are reported. Methods: A 90-minute SRS workshop focusing on the evaluation and management of sinusitis in ambulatory care was developed for PGY 2-3 internal medicine residents. Participants received a 15-minute didactic on the evaluation and management of adults with sinusitis, including typical microbiology, differentiation of bacterial sinusitis, and guideline recommendations on antibiotic treatment. In a computer lab, participants were instructed how to review charts of patients they had treated with antibiotics for sinusitis during the past year using the SlicerDicer application in Epic. Over 1 hour, they worked in pairs to complete and discuss a self-reflection inventory for 5 patients from each of their respective reviews. They evaluated pertinent history, comorbidities, presenting symptoms and signs, diagnostic testing performed, and a self-assessment of the subsequent antibiotic prescribing, including appropriateness of using an antibiotic, antibiotic choice and duration. In addition, they reflected on potential patient and prescriber challenges. Residents then identified common themes and developed a personal improvement plan for antibiotic prescribing for sinusitis. The last 15 minutes were spent debriefing with ASP faculty on reasons for overprescription of antibiotics for URIs and individual improvement plans. Residents completed workshop evaluations using a Likert scale and open-ended comments. Results: In total, 26 residents participated. All (100%) agreed or strongly agreed that the SRS workshop improved their understanding of how to obtain data on their own practice habits. Moreover, 23 (88%) agreed or strongly agreed that the workshop improved their understanding of when to prescribe antibiotics and how to practice antibiotic stewardship in the outpatient setting. Also, 20 participants (77%) agreed or strongly agreed that the SRS workshop helped them gain insight into reasons why they might overprescribe antibiotics in the outpatient setting. Furthermore, 25 (96%) agreed or strongly agreed that the SRS workshop helped them identify at least 1 way they could improve their antibiotic prescribing in the outpatient setting. Conclusions: The SRS workshop was well received by residents and offers a tool to empower primary care resident physicians to access their own antibiotic prescribing data, perform a structured self-reflection, and enhance their understanding of antibiotic stewardship in the ambulatory setting. SRS is a potential tool to improve ambulatory antibiotic use.

Disclosures: None

Funding: None

The last three decades have seen the biotherapeutic drug market evolve from promising concept to market dominance in a range of clinical indications. This growth has been spurred by the success of established drug classes like monoclonal antibodies, but also by the introduction of biosimilars, and more recently, multiple novel cell and gene therapies. Biotherapeutic drug development presents many unique challenges, but unintended immune responses are among the most common reasons for program attrition. Anti-drug antibodies can impact the safety and efficacy of drug products, and related immune responses, like the cytokine release syndrome that occurred in the infamous TGN-1412 clinical trial, can be challenging to predict with nonclinical models. For this reason, it is important that development programs proceed with a scientifically grounded and measured approach to these responses. This process begins at the discovery stage with the application of “quality by design,” continues into the clinic with the development of quality assays and management strategies, and culminates in the effective presentation of this information in regulatory documents. This review provides an overview of some of the key strategic and regulatory considerations for biotherapeutics as they pertain to immunogenicity and related responses.

Americans are engaged in a heated, sometimes violent, debate over the fate of Confederate monuments. As communities decide whether to remove these monuments, elected and appointed officials typically have had the final say. What if instead of allowing elected officials to make such decisions, voters had the power? Would this affect how the public feels about the outcome, win or lose? We used a survey experiment to examine whether the mode of decision making affects public attitudes, testing the effects of a decision made by public referendum versus by a city council. We found that respondents view decisions made by referendum to be fairer and more legitimate and allow multiple perspectives to be heard. These results hold even for respondents who oppose the referendum’s outcome. Our results speak to the potential of direct democracy to enhance public acceptance of decisions, particularly when the public is divided.

The public opinion literature stresses the importance of source cues in determining which types of messages affect attitudes and which types do not. Building upon such research, we seek to determine if messenger ethnicity influences how individuals evaluate candidates speaking on immigration in the context of a campaign. Do Americans (and Anglo Americans in particular) view Latino candidates as more experienced, stronger leaders, more trustworthy, and more qualified on immigration than Anglo candidates? Moreover, do such relationships hold regardless of the valence of the message itself? Through an original survey experiment presenting subjects with immigration talk on the campaign trail, we find Latino candidates are reviewed more positively than Anglo candidates when it comes to the immigration messages they speak (especially when it comes to pro-immigration messages). Such findings give us insight into whether or not Latino candidates have the potential to “own” the issue of immigration, as well as offering another path by which Latino candidates can gain a strong foothold with the public in the context of a campaign.

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.

We studied 7 patients with nosocomial Legionnaires' disease to determine the relationship between isolates of Legionella pneumophila recovered from potable water and those recovered from patients. Potable water was cultured from all rooms in which patients had stayed prior to the diagnosis of Legionnaires' disease. The 38 isolates of L. pneumophila (31 environmental, 7 patient) were resolved into 9 distinct patterns by pulse-field gel electrophoresis (PFGE), 3 by plasmid content and 2 each with monoclonal antibodies and conventional agarose gel electrophoresis of small fragments of DNA.

Using PFGE it was determined that 4 of the 7 patients were infected with L. pneumophila identical to an isolate recovered from the potable water supply in one of the rooms each had occupied prior to the diagnosis of Legionnaires' disease. Patients had resided in a mean of 3·57 rooms before a diagnosis of nosocomial Legionnaires' disease. We conclude that in the setting of contaminated potable water and nosocomial Legionnaires' disease water from all the rooms which the patient has occupied prior to this diagnosis should be cultured. PFGE of large DNA fragments discriminated best among the isolates of L. pneumophila.

Lobaria pulmonaria is an epiphytic lichen that, in south-eastern Canada, inhabits deciduous forests where it must acclimate to large seasonal changes in temperature and in light caused by closing and opening of the leaf canopy. On a seasonal timescale, this acclimation occurs via large shifts in the macromolecular complexes of the photosynthetic system, within a photobiont population that shows no seasonal change in cell numbers. In this study, samples of L. pulmonaria were harvested in February and in May from a natural population near Sackville, New Brunswick, and subjected to two simulated intense seasonal changes: (1) early spring warming, simulated by a shift from high light at 5°C to high light at 16°C (February shift), and (2) late spring canopy closure, simulated by a shift from high light at 16°C to low light at 16°C (May shift). Thallus samples were collected daily throughout each week-long shift. There were no significant changes in photobiont cell population size or in the fraction of cells dividing during either shift. During the first day of the February temperature shift, there were, however, large changes in the pools of chlorophyll, the major light capture molecule in the photobionts, the PsbA (D1) core protein of photosystem II whose turnover is highly responsive to changing light and temperature, and the RbcL major subunit of the carbon-fixing RUBISCO enzyme whose levels correlate strongly with achieved photosynthesis in lichens. A static population of photobionts was therefore able to perform large and rapid macromolecular reallocations to cope with rapid environmental change. No significant changes were seen in the chlorophyll, photosystem II or RUBISCO pools across the May light shift, although seasonal-scale macromolecular reallocation does occur in response to decreased light in the summer.

Thirty-seven Vibrio cholerae and four non-cholera Vibrio isolates from Ukraine, including strains from the epidemic of 1994–5, were analysed by molecular methods. Results from PFGE and ribotyping indicated that all Ukrainian toxigenic V. cholerae were closely related to each other and to an isolate from a patient from Pakistan. A non-toxigenic river water strain obtained during the height of the epidemic was more distantly related to these V. cholerae strains, while the Vibrio parahaemolyticus isolates and Vibrio alginolyticus isolate were not closely related to V. cholerae or each other. ERIC- and REP-PCR allowed the differentiation of strains identical by other methods. The results obtained confirm that the epidemic Ukrainian strains are most closely related to seventh pandemic strains from Asia and support a hypothesis that the Ukrainian epidemic of 1994–5 was caused by toxigenic environmental strains surviving since the time of the 1991 Ukrainian epidemic or before.

Control programmes for contagious mastitis pathogens, primarily Streptococcus agalactiae and Staphylococcus aureus, consisting of milking hygiene, treatment or isolation of infected cows and post-milking teat disinfection are relatively effective (McDonald, 1970; Natzke, 1977; Fox & Gay, 1993). Similar control programmes are often ineffective in the prevention of intramammary infections caused by environmental bacteria (Eberhart, 1977; Smith et al. 1985; Erskine et al. 1991; Smith & Hogan, 1993). This disparity of success relates to the fact that the principal source of environmental mastitis pathogens is the cow's environment, rather than another cow with an intramammary infection (Eberhart, 1977; Smith et al. 1985). Infections of environmental origin are particularly noteworthy because they may remain common in well managed herds, and these infections often cause severe clinical signs and high case fatality rates (Eberhart, 1977; Smith et al. 1985; Erskine et al. 1991; Smith & Hogan, 1993).

Control programmes for environmental mastitis usually include premilking teat disinfection, decreased use of water in udder preparation, increased concentrations of dietary vitamin E and selenium, improved sanitation of the environment and modification of cow behaviour in the post-milking period (Eberhart, 1977; Smith et al. 1984, 1985; Weiss et al. 1990; Erskine et al. 1991; Smith & Hogan, 1993). Environmental infections are thought to result when bacteria lacking specific virulence factors penetrate the teat sphincter (Eberhart et al. 1979; Smith et al. 1985). The bulk of these exposures are thought to occur between milkings. Control of these infections is problematic because the cow's teat sphincter remains open after mechanical milking (McDonald, 1975a). Presumably, cows are predisposed to intramammary infections when the open teat sphincter is exposed when cows lie down. Hence, dairy managers are advised to offer fresh feed to cows immediately after milking to increase the likelihood that the teat sphincter will close at least partly before cows become recumbent.

The purpose of this study was to determine the effect of feed availability on post-milking behaviour in dairy cows. Specifically, the measured dependent variable was the length of time that cows remained standing after leaving the milking parlour.