bluuebunny/crossref_metadata_2025 huggingface dataset:
The Motor Neuron-Like Cell Line NSC-34 and Its Parent Cell Line N18TG2 Have Glycogen that is Degraded Under Cellular Stress
Authors: Pfeiffer-Guglielmi Brigitte , Jansen Ralf-Peter
Published: March 2021

Brain glycogen has a long and versatile history: Primarily regarded as an evolutionary remnant, it was then thought of as an unspecific emergency fuel store. A dynamic role for glycogen in normal brain function has been proposed later but exclusively attributed to astrocytes, its main storage site. Neuronal glycogen had long been neglected, but came into focus when sensitive technical methods allowed quantification of glycogen at low concentration range and the detection of glycogen metabolizing enzymes in cells and cell lysates. Recently, an active role of neuronal glycogen and even its contribution to neuronal survival could be demonstrated. We used the neuronal cell lines NSC-34 and N18TG2 and could demonstrate that they express the key-enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase and contain glycogen which is mobilized on glucose deprivation and elevated potassium concentrations, but not by hormones stimulating cAMP formation. Conditions of metabolic stress, namely hypoxia, oxidative stress and pH lowering, induce glycogen degradation. Our studies revealed that glycogen can contribute to the energy supply of neuronal cell lines in situations of metabolic stress. These findings shed new light on the so far neglected role of neuronal glycogen. The key-enzyme in glycogen degradation is glycogen phosphorylase. Neurons express only the brain isoform of the enzyme that is supposed to be activated primarily by the allosteric activator AMP and less by covalent phosphorylation via the cAMP cascade. Our results indicate that neuronal glycogen is not degraded upon hormone action but by factors lowering the energy charge of the cells directly.
Mental Imagery in Patients with Prolonged Grief Disorder: a Comparison with Matched Bereaved Healthy Controls
Authors: Harrison Octavia , Wiedenmann Claudio , Rosner Rita , Steil Regina
Published: March 2021

Mental imagery is a transdiagnostic feature that has been increasingly researched in mental disorders in the past years. This study is the first to investigate mental imagery in individuals suffering from Prolonged Grief Disorder (PGD), a new disorder which will be included into the new edition of the International Classification of Diseases and Related Health Problems (ICD-11).

Our objective was to find out to what extent patients suffering from PGD differ from healthy, but equally bereaved, controls in terms of mental imagery, and how mental imagery is related to psychopathology. Patients with PGD and matched bereaved healthy controls (n = 54) completed a mental imagery questionnaire specifically designed for the study, and other established measures of psychopathology. Patients suffering from PGD reported mental images more frequently, had less control over them, and described negative images as more vivid than did healthy controls. Also, in reaction to mental images, patients less frequently experienced joy, but more often grief, anger and guilt. Besides these group differences, significant correlations between mental imagery other psychopathological measures could be found. Mental imagery is clearly related to PGD. The underlying mechanisms on whether it is a developing or maintaining factor need to be addressed in future studies. Future research should also investigate in what way mental imagery might be used in therapeutic approaches.
Threats to epistemic agency in young people with unusual experiences and beliefs
Authors: Houlders Joseph W. , Bortolotti Lisa , Broome Matthew R.
Published: March 2021

A good therapeutic relationship in mental health services is a predictor of positive clinical outcomes for people who seek help for distressing experiences, such as voice hearing and paranoia. One factor that may affect the quality of the therapeutic relationship and raises further ethical issues is the impact of the clinical encounter on users’ sense of self, and in particular on their sense of agency. In the paper, we discuss some of the reasons why the sense of epistemic agency may be especially fragile in young people with unusual experiences and beliefs. We argue that it is important to identify and avoid behaviours that can undermine young people’s contributions as epistemic agents in the clinical encounter.
Investigating the Effects of Surface Adsorbates on Gold and Palladium Deposition on Carbon
Authors: Bowden Bethany , Davies-Jones Josh A. , Davies Matthew , Davies Philip R. , Morgan David J. , Sainna Mala A. , Willock David J.
Published: March 2021

Surface functional groups have a strong influence on the deposition and final state of nanoparticles adsorbed on to the surface, a role discussed by Professor Spencer in his work. This tribute to Spencer explores the formation of hydroxyls, thiosulfates, sulfites and sulfur atoms on carbon (HOPG) surfaces and their effect on the deposition of gold and palladium from aqueous solutions. Hydroxyls formed from ammonium hydroxide treatment have identical behaviour to those formed by acid treatment, and gold adsorption from Au3+ solutions gives Au0 initially, with Au3+ formed at higher concentrations on these surfaces. In contrast, palladium adsorption is hindered by the presence of the hydroxyls and there is no indication of any reduction to the metallic state. Ammonium thiosulfate adsorbs dissociatively from aqueous solutions on HOPG if the surface is pre-activated by the presence of surface hydroxyls. At low concentrations of ammonium thiosulfate, adsorbed sulfite and sulfur are formed in equimolar concentrations whereas adsorption of high concentrations of ammonium thiosulfate gives some degree of molecular adsorption, with evidence in XP spectra for an ammonium ion and a sulfur 2p peak at 282.9 eV attributed to the undissociated thiosulfate ion. Both sulfur and the sulfite are stable at the surface in neutral solutions but the sulfite desorbs when treated with acidified solutions (~ pH ≤ 6). These two groups are also stable at 373 K but begin to desorb by 473 K. Exposure to a weak chloroauric acid solution causes the desorption of the sulfite and formation of a gold species with an XP binding energy of 84.6 eV; we cannot determine from the present data whether this peak is due to a Au(I) state or very small nanoparticles of Au(0).

Graphic Abstract
Postprandial Normoglycemic Hypokalemia—an Overlooked Complication to Gastric Bypass Surgery?
Authors: Abrahamsson Niclas
Published: March 2021

Obesity is one of the major health problems of the world, and one of the most common surgical treatments is the Roux-en-Y gastric bypass surgery. This can however lead to problems with postprandial hypoglycemia, but sometimes, the meal test does not render any signs of hypoglycemia. Here, 3 cases are presented with postprandial normoglycemic hypokalemia.

Graphical abstract
The Regulation of Farmer’s Privilege Under Vietnamese IP Law and the Law of the European Union
Authors: Nguyen Ho Bich Hang , Weckström Lindroos Katja
Published: March 2021

The International Union for the Protection of New Varieties of Plants Convention 1991 (UPOV Convention) recognizes the need to protect plant breeders’ legitimate interests. Without breeders’ innovations, new high-productivity plant varieties can neither be created nor contribute to society as a whole. Without these new varieties, it is impossible to create new high-yield generations of plants for the benefit of society. The absolute protection of breeders’ rights, however, would create many negative impacts for society. In particular, farmers would have to pay higher prices for seeds if breeders’ exclusive rights were to be overprotected, which would also mean consumers having to pay more for basic foodstuffs. This would lead to food insecurity nationally or even globally, in contravention of the right to food as recognized by Art. 25 of the United Nations Universal Declaration of Human Rights in 1948. These situations represent the dilemmas that governments have to cope with in developing national economies. The concept of farmer’s privilege is an exemption that the UPOV Convention recognizes in order to balance benefits between breeders and farmers. Under the optional exception set out in Art. 15(2) of the UPOV Convention, Contracting Parties may adopt the farmer’s privilege exemption in national law. Vietnam’s Law on Intellectual Property contains the farmer’s privilege exemption, yet there are many loopholes regarding this provision, and in reality, its application is limited. This article analyzes European Union regulation on farmer’s privilege and the way in which this exception has been interpreted, and compares and applies it to the Vietnamese law.
African American Unemployment and the Disparity in Periviable Births
Authors: Catalano Ralph , Karasek Deborah , Bruckner Tim , Casey Joan A. , Saxton Katherine , Ncube Collette N. , Shaw Gary M. , Elser Holly , Gemmill Alison
Published: March 2021

Periviable infants (i.e., born before 26 complete weeks of gestation) represent fewer than .5% of births in the US but account for 40% of infant mortality and 20% of billed hospital obstetric costs. African American women contribute about 14% of live births in the US, but these include nearly a third of the country’s periviable births. Consistent with theory and with periviable births among other race/ethnicity groups, males predominate among African American periviable births in stressed populations. We test the hypothesis that the disparity in periviable male births among African American and non-Hispanic white populations responds to the African American unemployment rate because that indicator not only traces, but also contributes to, the prevalence of stress in the population. We use time-series methods that control for autocorrelation including secular trends, seasonality, and the tendency to remain elevated or depressed after high or low values. The racial disparity in male periviable birth increases by 4.45% for each percentage point increase in the unemployment rate of African Americans above its expected value. We infer that unemployment—a population stressor over which our institutions exercise considerable control—affects the disparity between African American and non-Hispanic white periviable births in the US.
3D-semantic segmentation and classification of stomach infections using uncertainty aware deep neural networks
Authors: Amin Javaria , Sharif Muhammad , Gul Eman , Nayak Ramesh Sunder
Published: March 2021

Wireless capsule endoscopy (WCE) might move through human body and captures the small bowel and captures the video and require the analysis of all frames of video due to which the diagnosis of gastrointestinal infections by the physician is a tedious task. This tiresome assignment has fuelled the researcher’s efforts to present an automated technique for gastrointestinal infections detection. The segmentation of stomach infections is a challenging task because the lesion region having low contrast and irregular shape and size. To handle this challenging task, in this research work a new deep semantic segmentation model is suggested for 3D-segmentation of the different types of stomach infections. In the segmentation model, deep labv3 is employed as a backbone of the ResNet-50 model. The model is trained with ground-masks and accurately performs pixel-wise classification in the testing phase. Similarity among the different types of stomach lesions accurate classification is a difficult task, which is addressed in this reported research by extracting deep features from global input images using a pre-trained ResNet-50 model. Furthermore, the latest advances in the estimation of uncertainty and model interpretability in the classification of different types of stomach infections is presented. The classification results estimate uncertainty related to the vital features in input and show how uncertainty and interpretability might be modeled in ResNet-50 for the classification of the different types of stomach infections. The proposed model achieved up to 90% prediction scores to authenticate the method performance.
Exploring the disparity of influence between users in the discussion of Brexit on Twitter
Authors: Rajabi Amirarsalan , Mantzaris Alexander V. , Atwal Kuldip Singh , Garibay Ivan
Published: March 2021

The topic of political polarization has received increased attention for valid reasons. Given that an increased amount of the social exchange for opinions happens online, social media platforms provide a good source of information to investigate various aspects of the phenomena. In this work, data collected from Twitter are used to examine polarization surrounding the topic of the Brexit referendum on the membership of the European Union. The analysis specifically focuses on the question of how different tiers of users in terms of influence can project their opinions and if the polarized conditions affect the relative balance in the broadcast capabilities of the tiers. The results show that during polarization periods, users of the higher tier have increased capabilities to broadcast their information in relation to the lower tiers thereby further dominating the discussion. This validates previous modeling investigations and the hypothesis that polarization provides an opportunity for influencers to increase their relative social capital.
Documenting Cultures of Harassment in Archaeology: A Review and Analysis of Quantitative and Qualitative Research Studies
Authors: Voss Barbara L.
Published: March 2021

This article is the first of a two-part series to analyze current research on harassment in archaeology. Harassment has shaped the discipline of archaeology since at least the late 1800s. Since the 1970s, harassment has been recognized as a significant factor impacting gender equity in archaeology. Recent qualitative and quantitative research has verified that harassment occurs at epidemic rates in archaeology. Archaeologists are primarily harassed by other archaeologists, and harassment occurs not only in field research settings but also in classrooms, laboratories, museums, office workplaces, and conferences. Although women in archaeology experience a higher frequency of harassment, both men and women report harassment at disturbingly high rates. Archaeologists of color, LGBTQIA+ archaeologists, nonbinary archaeologists, and archaeologists with disabilities are also disproportionately harassed. As reflected in the author's own career experiences, harassment creates a cognitive burden for survivors and reduces access to professional opportunities, directly impacting diversity within archaeology. Fortunately, there are evidence-based interventions and policies that can reduce harassment and support survivors. These are discussed in the second article, “Disrupting Cultures of Harassment in Archaeology.”
Disrupting Cultures of Harassment in Archaeology: Social-Environmental and Trauma-Informed Approaches to Disciplinary Transformation
Authors: Voss Barbara L.
Published: March 2021

This article is the second in a two-part series that analyzes current research on harassment in archaeology. Both qualitative and quantitative studies, along with activist narratives and survivor testimonials, have established that harassment is occurring in archaeology at epidemic rates. These studies have also identified key patterns in harassment in archaeology that point to potential interventions that may prevent harassment, support survivors, and hold perpetrators accountable. This article reviews five key obstacles to change in the disciplinary culture of archaeology: normalization, exclusionary practices, fraternization, gatekeeping, and obstacles to reporting. Two public health paradigms—the social-environmental model and trauma-informed approaches—are used to identify interventions that can be taken at all levels of archaeological practice: individual, relational, organizational, community, and societal.
22511 Glycolipid-loaded nanoparticles harness iNKT cells for tumor immunotherapy
Authors: Shute Travis , Dudley Elizabeth , Lai Andrew , Salas Briana , Vincent Brandy , Angel Daniel , Nash Kelly , Leadbetter Elizabeth
Published: March 2021

ABSTRACT IMPACT: My work is on the development of a novel tumor immunotherapy to treat various types of cancer OBJECTIVES/GOALS: As iNKT cells can have direct and indirect killing effects on tumor cells, we propose a novel strategy for activating iNKT cells, via a PLGA nanoparticle delivery platform, to promote anti-tumor immune responses. METHODS/STUDY POPULATION: Poly-lactic-co-glycolic acid (PLGA) nanoparticles can be reproducibly loaded with an iNKT cell glycolipid agonist, alpha-galactosylceramide (αGalCer), and a tumor associated antigen, ovalbumin (OVA). We then test our nanoP prophylactically and therapeutically against a murine model of melanoma, B16F10-OVA. RESULTS/ANTICIPATED RESULTS: These dual-loaded PLGA nanoparticles rapidly activate iNKT cells in vivo to produce IFNgamma. Furthermore, in an in vivo model of melanoma, using B16F10-OVA cells, both prophylactic and therapeutic administration of nanoparticles containing αGalCer and OVA led to decreased tumor cell growth and increased survival. We also show our nanoparticle therapy has synergistic potential with clinically used immune checkpoint blockade (ICB) therapies, anti-PD-1 and anti-CTLA-4, indicated by the significance increase in survival and lower tumor growth rate of ICB +nanoP treated mice compared to either ICB or nanoP alone. DISCUSSION/SIGNIFICANCE OF FINDINGS: This novel delivery system provides a platform with tremendous potential to harness iNKT cells for cancer immunotherapy purposes against many cancer types.
31547 Regulation and function of the i6A37 tRNA modification
Authors: Aubee Joseph I , Williams Kinlyn , Adigun Alexandria , Olusanya Olufolakemi , Thompson Karl M
Published: March 2021

ABSTRACT IMPACT: MiaA has a human homolog known as TRIT1. Mutations in TRIT1 have been associated with rare diseases such as MELAS and MERRF syndromes. These diseases are associated with mitochondrial disfunction.Understanding the mechanisms of bacterial sRNAs, and the miRNAs associated with these diseases could potentially afford the insight into effective cures. OBJECTIVES/GOALS: The aim is to investigate the regulation and function of tRNA isopentyladenine transferase enzyme in Escherichia coli. We aimed to execute screens for the identification of small RNA regulators of MiaA. The study will also investigate if i6A tRNA modification is necessary for the expression of major heat shock and mitochondrial proteins. METHODS/STUDY POPULATION: We constructed a chromosomal miaA-lacZ translational fusion driven by the arabinose responsive PBAD promoter and used it to screen against an Escherichia coli small RNA library. Using CsrB, one of our candidate sRNA regulators from our genetic screen, we measured the steady state levels of MiaA by Northern Blot in a PBAD-miaA2(P2HS)-lacZ translational fusion strain whereby pBR-pLac-csrB, pBR-pLac-csrA and the pBR-pLac vector are over-expressed, and under the control of an IPTG inducible promoter. Additionally, and in the same PBAD-miaA2(P2HS)-lacZ translational fusion strain background, we measured the steady state levels of MiaA in the wild type, csrA:zeo mutant strain, and csrA:zeo pBR-pLac-csrA complementation strain to determine if a combination of the pair would restore the wild-type genotype. RESULTS/ANTICIPATED RESULTS: Upon measuring the effect of small RNAs on miaA expression using quantitative b-galactosidase assays, we saw a 5-fold decrease in the expression of MiaA in the miaA-lacZ translational fusion containing sRNA CsrB, suggesting that this sRNA may play a role in the regulation of post-transcriptional expression of MiaA.From our northern blotting analysis, we observed a 6-fold decrease in MiaA expression in the absence of csrA, suggesting that csrA is essential for MiaA expression. DISCUSSION/SIGNIFICANCE OF FINDINGS: Identifying, mapping and characterizing how MiaA is regulated post-transcriptionally will give us an increased understanding in the maintenance and regulation of the normal function of E.coli to conserve homeostasis and translation fidelity.
60404 HIV Tat Induced Neuroinflammation
Authors: Carey Sean , Maguire-Zeiss Kathleen
Published: March 2021

ABSTRACT IMPACT: Demonstrate the role of astrocyte released MMPs in response to pathogenic HIV protein Tat. OBJECTIVES/GOALS: In the presence of the pathogenic HIV protein Tat, astrocytes have been demonstrated to adopt an inflammatory phenotype as well as release extracellular matrix degrading enzymes, MMPs. Our work aims to identify whether MMPs alter perineuronal net integrity and working memory in a mouse model of Tat-induced neuroinflammation. METHODS/STUDY POPULATION: Stereotaxic Injection: C57BL6/J mice were injected bilaterally with HIV-1 IIIB Tat 5ug in 5uL or Vehicle (0.2M KCl, 5mM DTT, 50mM Tris, pH 8.0), into the hippocampus (CA1; -1.9mm AP, ±1.6mm ML, -1.5mm DV from pial surface). All outcome measurements were performed 14-days post injection. Behavior: T-maze was used to assess working memory following Tat exposure. qRT-PCR: TaqMan probes were used according to manufacturer on extracted whole hippocampus mRNA. IF: GFAP and CD68 immunofluorescence was used to determine inflammation post injection. Inhibitory interneurons (parvalbumin positive) and peri-neuronal nets (WFA positive) were quantified. WB: Synaptosomes from whole hippocampi (Syn-PER) were isolated and synaptic excitatory markers were quantified (PSD-95, synaptophysin, GluR2a). RESULTS/ANTICIPATED RESULTS: Tat exposure resulted in impairments in working memory as measured by T-maze alternations and an increase in hippocampal mRNA expression of MMP-13 and IL-1β, indicative of neuroinflammation. We also noted an increase in GFAP+ injection site width 14 days post-Tat injection, suggesting robust gliosis. While there were no changes in the excitatory pre and post synaptic markers we found a significant decrease in the percent of PV+ interneurons with peri-neuronal nets (PNNs) following Tat exposure. Taken together, this preliminary data supports a role for inflammation and PNN integrity in Tat-induced alterations in working memory. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings suggest that Tat contributes to cognitive impairment and that astrogliosis with elevated MMP-13 facilitates the degradation of peri-neuronal nets (PNNs) within the hippocampus. Since PNN degradation can alter neuronal circuitry future studies will focus on Tat-induced changes in hippocampal signaling.
23255 Devices Engineered to Collect Exhaled Breath Condensate (EBC) and their Applications
Authors: Schmidt Alexander J. , Borras Eva , Nguyen Anh , Kenyon Nicholas J. , Davis Cristina E.
Published: March 2021

ABSTRACT IMPACT: Human exhaled breath is rich in metabolomic content that represents pulmonary function and gas exchange with blood, which can provide insights into an individual’s state of health. OBJECTIVES/GOALS: Human exhaled breath is rich in metabolomic content that represents pulmonary function and gas exchange with blood. It contains a mixture of compounds that offer insight into an individual’s state of health. Here, we present two novel non-invasive breath sampling devices for use in basic medical practice. METHODS/STUDY POPULATION: The two breath samplers have a disposable mouthpiece, a set of inhale and exhale one-way flap valves to allow condensation of exhaled breath only, and a saliva filter. The housing is constructed out of Teflon®, a chemically inert material to reduce chemical absorbance. The first device condenses exhaled breath into a frozen condensate using dry ice pellets and the other is a miniaturized design that liquifies exhaled breath on a condenser surface with micropatterned features on a cooling plate. Both designs have individual strategic and analytical advantages: frozen exhaled breath condensate (EBC) has high retention of analytes and sample volume; EBC collected in liquid phase offers facilitated sample collection and device portability. RESULTS/ANTICIPATED RESULTS: We investigated if breath aerosol size distribution affects the types or abundances of metabolites. We modified the geometry of the first device to redirect aerosol trajectories based on size. The trapping of larger aerosols increases with filter length, thus altering the aerosol size distribution although no significant changes in the metabolite profiles were found. With the miniaturized device, metabolite abundances were measured in a small cohort of healthy control and mild asthmatic subjects. Differences among subjects were found, as well as main differences between control and asthmatic groups. All analyses of EBC were performed with liquid chromatography - mass spectrometry. Inflammatory suppression found in asthmatic subjects can be explained by prescribed daily use of inhaled corticosteroids. DISCUSSION/SIGNIFICANCE OF FINDINGS: Breath collection devices can be used in intensive care units, outpatient clinics, workplaces, and at home. EBC analysis has been used to monitor asthma and chronic obstructive pulmonary disease. It can be applied to infectious respiratory diseases (e.g. influenza, COVID-19) and for monitoring environmental and occupational chemical exposures.
90449 Can Ultrasound detect changes to spinal cord blood flow before and after injury?
Authors: Manbachi Amir , Theodore Nicholas
Published: March 2021

ABSTRACT IMPACT: To track recovery and mitigate additional spinal cord injury (avoiding further paralysis), we are assessing the applicability of an implantable ultrasound device that can monitor the tissue health postoperatively. OBJECTIVES/GOALS: To date, no method has been developed that monitors spinal cord perfusion rate (mL/min/g) or pressure (mmHg) successfully after the surgery. Our goal is to design, construct, and validate (in animal models) a novel sensor that quantifies postoperative tissue perfusion in patients with SCI at the site of and downstream from the injury. METHODS/STUDY POPULATION: A sample size of 10 animals will allow us to test our hypothesis to track tissue perfusion before and after the SCI using ultrasound. After prepping and scrubbing the animal, the skin will be incised with a blade, bony structures will be removed and the spinal cord will be revealed. A 25-g weight will then be dropped from a height of 15 cm, and the animal will be observed for contraction of the lower extremities, a sign that the cord was damaged. Using Doppler ultrasound settings available on commercial transducers, we will investigate the acceptable frequency, as well as proper Doppler mode with and without contrast agents, and with and without elastography (stiffness mapping of the tissue). A range of frequencies will be tested (5 -25 MHz). RESULTS/ANTICIPATED RESULTS: It is expected that at frequencies 12 MHz and above, our radiologist collaborators would be able to easily detect the blood flow. It is also expected that the injury will have a noticeable effect on the changes of this detected blood flow. We aim to present figures demonstrating ultrasound image qualities obtained at various frequencies. We expect three such figures: one for gray scale ultrasound imaging, one for color Doppler and finally, one for spectral Doppler, which is the one mostly used to quantify blood flow. DISCUSSION/SIGNIFICANCE OF FINDINGS: To monitor recovery and mitigate secondary injury in patients with traumatic SCI, there is a need to monitor tissue perfusion intra-operatively. To address this need, we will design, construct, and validate a novel sensor that will postoperatively quantify tissue perfusion for the SCI patients at the site of the injury.
32460 The Bench Tutorials Program: An Essential Educational Pivot in response to COVID-19
Authors: Singleton Chantele , Croisant Sharon A. , Hallberg Lance , Prochaska John , Bohn Krista , Puig Michelle , Elferink Cornelis
Published: March 2021

ABSTRACT IMPACT: The Bench Tutorials Program is an independent study course in biomedical research in which high school students are paired with graduate and post-doctoral students during the academic year. The purpose is to enhance the rigor of high school science education and build the pipeline of tomorrow’s researchers. OBJECTIVES/GOALS: The Bench Tutorials Program: ο Proficiency in research design, implementation, and presentation; ο Acquisition of hands-on laboratory skills; ο Increase in scientific literacy; ο Increase in analytical skills and critical thinking; ο Career in science; ο Build the pipeline of tomorrow’s biomedical researchers METHODS/STUDY POPULATION: High School seniors are paired with graduate and postdoc mentors through a matching process. Students spend approximately four hours/week in supervised instruction and research from a participating laboratory in addition to classroom experience at their High School. Mentors design research projects relating to the larger research framework of their laboratories. In light of COVID-19, approaches have been adjusted to maintain the program safely through a hybrid method of using the high school lab for hands-on learning and through the use of Go-Pros ’s to enable our mentors to video and narrate as they conduct experiments in their own labs to teach their mentees scientific methods and processes. RESULTS/ANTICIPATED RESULTS: Since inception, more than 400 students and mentors have participated in the Bench Tutorial’s program. This year we found a way to continue the program under COVID-19 restraints without putting anyone in harms way. Go-Pros have been essential for our program to maintain continuity for high school students who receive academic credit for this course. This program is also one of few in which our graduate students have the opportunity to serve as mentors in the scientific setting. Using Go-Pro’s will also enable us to provide teaching videos online for other academic institutions, so even in the absence of COVID-19 in the future, the continued use of these devices will still be of great value. DISCUSSION/SIGNIFICANCE OF FINDINGS: High school students are afforded the ability to work on cutting edge research projects alongside graduate students and postdocs, who are afforded the chance to mentor and teach. Due to the COVID-19 pandemic, we have successfully adjusted our methods for teaching through the use of Go-Pro technology.
10399 DNA-PK(cs) Regulates Stability of Egr1 During T Cell Activation
Authors: Waldrip Zachary , Harrison David , Burdine Marie , Burdine Lyle
Published: March 2021

ABSTRACT IMPACT: This work provides supporting evidence for the development of a novel immunosuppression therapy for transplant patients. OBJECTIVES/GOALS: Our laboratory reported that inhibition of the kinase DNA-PK(cs) in mice delays allogeneic graft rejection in part by mitigating the induction of certain cytokines. We hypothesized that this was due to an inhibition of intracellular signaling programs in T cells and designed studies to identify the mechanism(s) by which this occurs. METHODS/STUDY POPULATION: The immortalized Jurkat T cell line was used to evaluate the effect of the DNA-PK(cs) inhibitor NU7441 on T cell activation by PMA/Ionomycin or PMA/PHA. Mouse primary splenocytes also were used to demonstrate the universality and reproducibility of our observations. Initially, protein mass spectrometry of lysates from untreated and NU7441-treated Jurkat cells identified proteins of interest regulated by DNA-PK(cs) that play a role in T cell activation and cytokine production. CRISPR genome editing was used to validate a potential downstream target of DNA-PK(cs). Western blot, ELISA, and flow cytometry were used to document changes in protein levels with respect to treatments. RESULTS/ANTICIPATED RESULTS: We observed that expression of the transcription factor Egr1 was highly induced after activation but attenuated after treatment with NU7441 in both Jurkat T cells and mouse splenocytes. Phosphorylated serine 301 of Egr1 was identified by mass spectrometry in stimulated cells and fits the kinase consensus sequence for DNA-PK(cs). Both an endogenous CRISPR-generated serine 301 to alanine mutant and expression of a plasmid-based S301A mutant resulted in an unstable form of Egr1 that was barely detectable. In contrast, expression of either a S301 to D or E phospho-mimetic mutant resulted in a stable form of the protein detectable by Western blot. Further evaluation of these mutants and Egr1 phosphorylation is underway to determine the mechanism by which DNA-PK(cs) kinase regulates protein stability. DISCUSSION/SIGNIFICANCE OF FINDINGS: We previously reported a role for DNA-PK(cs) in immunomodulation. We now have evidence that this occurs in part through stabilization of Egr1. We believe this novel finding will lead to uncovering a broader role for DNA-PK(cs) as a mediator of protein stability in T cells and provide support for targeting DNA-PK(cs) in immunosuppression therapy.
15727 KATP channel prodrugs as therapeutics for chronic pain and substance abuse disorders
Authors: Doucette Alexis , Johnson Kayla , Dosa Peter I. , Klein Amanda H
Published: March 2021

ABSTRACT IMPACT: Pharmacological activation of KATP channels may provide analgesia and attenuate opioid tolerance and withdrawal OBJECTIVES/GOALS: Our long term goal is to develop therapeutics for the treatment of the overuse of opioids. The objective of this application is to test novel KATP channel-targeting prodrugs in rodent models of neuropathic and inflammatory pain in addition to opioid tolerance after chronic morphine administration. METHODS/STUDY POPULATION: In one study, two different measures for chronic pain were implemented in mice. Male and female mice (n=10) were subjected to spinal nerve ligation (SNL) or intraplantar injection of Complete Freund’s Adjuvant (CFA) to induce neuropathic and inflammatory pain, respectively. Administration of KATP channel prodrugs (60ug, it) attenuated mechanical hypersensitivity after SNL or CFA compared to vehicle (saline). In a separate study, changes in mechanical hypersensitivity were tested while mice undergo chronic morphine treatment (15mg/kg, 2x, 5 days) with administration of the prodrugs. Tolerance was measured as the loss of antinociception, and withdrawal is measured ˜24 hours after the final morphine injection. RESULTS/ANTICIPATED RESULTS: Intrathecal administration of either KATP channel prodrugs significantly attenuated mechanical hypersensitivity after SNL and significantly attenuated mechanical hypersensitivity after CFA in mice. We predict that intrathecal administration of these prodrugs will also attenuate morphine tolerance and withdrawal in mice. This hypothesis is based off our previous data indicating non-water soluble KATP channel agonists produce analgesia and attenuate morphine tolerance in mice. DISCUSSION/SIGNIFICANCE OF FINDINGS: Pharmaceutical strategies to utilize KATP channels for therapeutics have been hindered due to the low solubility and low ability to cross the neurovascular unit. Newly developed, water-soluble KATP channel openers could be useful pharmaceutical strategy to reduce chronic pain, opioid tolerance, and withdrawal in human populations.
19233 Basis profile curve identification to understand electrical stimulation effects in human brain networks
Authors: Miller Kai J. , Muller Klaus-Robert , Hermes Dora
Published: March 2021

ABSTRACT IMPACT: Brain networks can be explored by delivering brief pulses of electrical current in one area while measuring responses in other areas, and this describes an open-source novel algorithm to carry out this exploration. OBJECTIVES/GOALS: If we focus on a single brain site and observe the average effect of stimulating each of many other brain sites, visually-apparent motifs in the temporal response shape emerge from adjacent stimulation sites. There are no existing approaches to identify and quantify the spatiotemporal structure of these motifs. METHODS/STUDY POPULATION: Individual stimulation trials are correlated with one another, then a correlation-significance matrix quantifying similarity between stimulation sites is decomposed with non-negative matrix factorization, in which the inner dimension is iteratively reduced. The dimensionality reduction identifies stimulation sites that produce a common elicited temporal response, and linear kernel PCA is applied to obtain the robust profile of this response cluster. RESULTS/ANTICIPATED RESULTS: We describe and illustrate a data-driven approach to determine characteristic spatiotemporal structure in these response shapes, summarized by a set of unique ‘basis profile curves’ (BPCs). Each BPC may be mapped back to underlying anatomy in a natural way, quantifying projection strength from each stimulation site using simple metrics. Our technique is demonstrated for an array of implanted brain surface electrodes in a human patient, and our code is shared at https://purl.stanford.edu/rc201dv0636. DISCUSSION/SIGNIFICANCE OF FINDINGS: This framework enables straightforward interpretation of single-pulse brain stimulation data, and can be applied generically to explore the diverse milieu of interactions that comprise the connectome.
21813 Changes in Electrophysiologic Activity in the Rat Visual Cortex following Traumatic Brain Injury (TBI)
Authors: Germi James , Fruchet Oceane , Wolf John , Chen Isaac
Published: March 2021

ABSTRACT IMPACT: This research aims to identify changes in visual network function after TBI as a way to define potential therapeutic targets for neuromodulation or neural tissue substrates. OBJECTIVES/GOALS: The objectives of this study are to compare neural activity in the visual cortex following TBI with cortical activity in the uninjured brain. This study aims to characterize functional changes in single neuron activity, spike-field relationships and oscillatory activity. METHODS/STUDY POPULATION: The effects of TBI will be studied by comparing electrophysiologic recordings from Long-Evans rats with a fluid percussion injury (FPI) to rats with a sham injury. Four days after the injury or sham procedure, a laminar probe with multiple electrode contacts will be chronically implanted in the ipsilesional primary visual cortex (V1). Afterwards, rats will be anesthetized weekly for 3 weeks (up to 4 weeks post-injury) to assess visual processing in response to drifting grating visual stimulation. To assess behavioral correlates, neural activity will also be recorded while rats perform a visual discrimination task in an operant, touchscreen chamber twice weekly. Recordings will be analyzed for visually evoked units, unit entrainment to local field potentials (LFPs) and evoked oscillatory activity. RESULTS/ANTICIPATED RESULTS: Consistent with other studies, our preliminary evidence from V1 recordings in naive rats has shown that individual neurons are responsive to visual stimuli, visual stimuli are associated with evoked oscillations and unit activity is correlated with LFPs. While activity of individual V1 neurons in injured animals is expected to recover to resemble activity in uninjured animals over time, patterns of functional organization in the two groups are expected to diverge over time. We anticipate that TBI-associated axonal damage, neuronal loss and changes in synaptic weights will lead to disruptions in the timing of neural activity in V1. These perturbations of neural communication within the visual system are expected to be associated with behavioral deficits in the awake, visual discrimination task. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study helps define how cortical network disruption after TBI. These changes are potential targets for novel TBI therapeutics, including neuromodulation and neural tissue transplantation. Thus, this work lays the groundwork for future studies aimed at mitigating the effects of TBI with rationally designed experimental therapeutics.
32219 Differences in cell death in methionine versus cysteine depletion
Authors: Wallis Katherine F. , Miousse Isabelle R.
Published: March 2021

ABSTRACT IMPACT: Reducing methionine levels has repeatedly been shown to reduce cancer growth in vivo, while at the same time increasing lifespan in healthy animals. However, the mechanisms behind the beneficial effects of methionine restriction are currently unknown. OBJECTIVES/GOALS: We hypothesized that comparing the response of a cancer cell line to depletion of the amino acids methionine and cysteine would give us insight into the critical role of these two closely related amino acids in cancer, and help advance methionine restriction on the translational science spectrum. METHODS/STUDY POPULATION: We used the human melanoma cell line A101D to analyze the response to three conditions: methionine depletion, methionine replacement with homocysteine, and cysteine depletion in vitro. We measured proliferation/viability, gene expression patterns, and glutathione levels. We also assessed ferroptotic versus apoptotic cell death. We then used a normal human fibroblast cell line to compare responses. RESULTS/ANTICIPATED RESULTS: The transcription factors ATF4 and NRF2 were activated by all three tested conditions in melanoma cells. Glutathione levels were decreased by ˜40% in cells grown without methionine, and by 95% in cells grown without cysteine. Lipid peroxidation was increased in cells grown without cysteine, but not in cells grown without methionine. Inhibiting ferroptotic cell death partially rescued proliferation in cysteine-depleted but not in methionine-depleted cells. Almost 70% of cells grown in methionine-depleted media stained positive for Annexin V, an indicator of apoptosis, compared to only 20% of cells grown in cysteine-depleted media. In normal cells, ferrostatin recued proliferation/viability to 86% of control levels in cysteine-depleted cells. Ferrostatin did not affect methionine-depleted normal cells. DISCUSSION/SIGNIFICANCE OF FINDINGS: These results indicate that methionine depletion leads to apoptosis, while cysteine depletion leads to ferroptosis. We found overlapping pathways activated by methionine and cysteine depletion at the gene expression levels, but divergences in cell death pathways ultimately activated.
41224 REDUCED FRONTOSTRIATAL FUNCTIONAL CONNECTIVITY IN 41- TO 70-YEAR-OLD ADULTS WITH HIV
Authors: Hassanzadeh-Behbahani Shiva , Yang Fan Nils , Bronshteyn Margarita , Dawson Matthew , Kumar Princy , VanMeter John , Moore David J. , Ellis Ronald J. , Jiang Xiong
Published: March 2021

ABSTRACT IMPACT: The knowledge acquired from my research can inform the development of early diagnostic methods for HIV-associated neurocognitive disorders. OBJECTIVES/GOALS: In the era of combination antiretroviral therapy (cART), the prevalence of HIV-associated neurocognitive disorders (HAND) remains high but the neural mechanisms are unclear. We examined whether older people with HIV (PWH) with minimal cognitive impairment have reduced functional connectivity in frontostriatal circuits compared to controls. METHODS/STUDY POPULATION: 99 PWH (mean age 56.6 years, 75% male, 62% Black, mean duration of HIV-infection 26.2 years ±9.3, 90% viral load <50 copies, 98% on stable cART) and 38 demographically-comparable controls (mean age 54.5 years, 71% male, 58% Black) participated in a cross-sectional study. A 7-domain neuropsychological battery and an Activities of Daily Living index were used to determine HAND diagnoses: 32 PWH met criteria for asymptomatic to mild HAND. Motor skill was assessed using the Grooved Pegboard Test by measuring performance speed. Structural MRI and resting-state functional MRI were collected. Seed-to-voxel analyses were conducted using 4 distinct regions in the striatum as seed regions. We used a voxel threshold of p<0.001 and cluster threshold of p<0.05 (FDR-corrected) after controlling for demographic variables. RESULTS/ANTICIPATED RESULTS: Compared to controls, PWH had lower resting state functional connectivity between the default mode region of the striatum (i.e., medial caudate) and bilateral superior frontal gyrus, supplementary motor cortex and paracingulate gyrus (p<0.05; cluster size: 567 voxels). Also, compared to controls, PWH had reduced resting state functional connectivity between the motor division of the striatum (i.e., posterior putamen) and anterior cingulate cortex and left supplementary motor cortex (p<0.05, cluster size: 405 voxels). Performance speed on the Grooved Pegboard motor test negatively correlated with functional connectivity between the motor region of the striatum and supplementary motor frontal regions in all participants (Spearman’s rho=-0.18, p=0.04). DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results support the hypothesis that frontostriatal abnormalities are widely present in PWH and might play a key role in HAND development. Our data suggest that dysfunction within the frontostriatal circuits may be involved in motor impairment in PWH, and ongoing inflammation may contribute to motor impairment and frontostriatal injury.
47461 Regulation of the immune response in the tumor microenvironment of lung adenocarcinoma
Authors: Simmons Glenn
Published: March 2021

ABSTRACT IMPACT: This work will provide a rational approach to improve the efficacy of current immunotherapy approaches in patients that have historically responded poorly to immune checkpoint inhibitors. OBJECTIVES/GOALS: Recent evidence of immunogenic cell death as a predictor of response to therapy has increased the interest in monitoring the presence of damage-associated molecular pattern protein (DAMPs). By regulating DAMP expression, our lab is interested in discovering new ways to improve the patient response rate to immune checkpoint inhibition. METHODS/STUDY POPULATION: Using cultured cell, and a limited number of patient tumors and serum (n=4), we measured intracellular and extracellular levels of DAMP molecule, high mobility group box 1 (HMGB1) using enzyme-linked immunosorbent assays and immunoblots. Immunological assayed were compared to the expression of immune checkpoint molecules PD-1/PDL1 on patient tumors as presented in pathology reports. RESULTS/ANTICIPATED RESULTS: HMGB1 release was associated with increased levels of PD-L1 on tumor cells. Targeted inhibition of HMGB1 altered the expression of programmed death-ligand 1 (PD-L1), a target for immune checkpoint inhibition therapy. Patients with higher levels of PD-L1 possessed increased levels of HMGB1 in serum. DISCUSSION/SIGNIFICANCE OF FINDINGS: This implies that regulating the expression of HMGB1 could have an effect on the response of patients to immunotherapy. The main objective of the work is to determine the potential benefit of targeting HMGB1 to improve the efficacy of current therapeutic approaches to treating lung cancer.