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Since the school's inception in 1979, students in the Morningside Graduate School of Biomedical Sciences at UMass Chan Medical School have contributed thousands of research publications to the field of biomedical sciences. This collection makes this body of work accessible to our students, faculty, potential recruits, the citizens of Massachusetts, and the world.

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Recently Published

  • Feasibility of At-Home Serial Testing Using Over-the-Counter SARS-CoV-2 Tests With a Digital Smartphone App for Assistance: Longitudinal Cohort Study

    Herbert, Carly; Broach, John; Heetderks, William; Qashu, Felicia; Gibson, Laura; Pretz, Caitlin; Woods, Kelsey; Kheterpal, Vik; Suvarna, Thejas; Nowak, Christopher; et al. (2022-10-18)
    Background: The ongoing SARS-CoV-2 pandemic necessitates the development of accurate, rapid, and affordable diagnostics to help curb disease transmission, morbidity, and mortality. Rapid antigen tests are important tools for scaling up testing for SARS-CoV-2; however, little is known about individuals' use of rapid antigen tests at home and how to facilitate the user experience. Objective: This study aimed to describe the feasibility and acceptability of serial self-testing with rapid antigen tests for SARS-CoV-2, including need for assistance and the reliability of self-interpretation. Methods: A total of 206 adults in the United States with smartphones were enrolled in this single-arm feasibility study in February and March 2021. All participants were asked to self-test for COVID-19 at home using rapid antigen tests daily for 14 days and use a smartphone app for testing assistance and to report their results. The main outcomes were adherence to the testing schedule, the acceptability of testing and smartphone app experiences, and the reliability of participants versus study team's interpretation of test results. Descriptive statistics were used to report the acceptability, adherence, overall rating, and experience of using the at-home test and MyDataHelps app. The usability, acceptability, adherence, and quality of at-home testing were analyzed across different sociodemographic, age, and educational attainment groups. Results: Of the 206 enrolled participants, 189 (91.7%) and 159 (77.2%) completed testing and follow-up surveys, respectively. In total, 51.3% (97/189) of study participants were women, the average age was 40.7 years, 34.4% (65/189) were non-White, and 82% (155/189) had a bachelor's degree or higher. Most (n=133/206, 64.6%) participants showed high testing adherence, meaning they completed over 75% of the assigned tests. Participants' interpretations of test results demonstrated high agreement (2106/2130, 98.9%) with the study verified results, with a κ score of 0.29 (P<.001). Participants reported high satisfaction with self-testing and the smartphone app, with 98.7% (157/159) reporting that they would recommend the self-test and smartphone app to others. These results were consistent across age, race/ethnicity, and gender. Conclusions: Participants' high adherence to the recommended testing schedule, significant reliability between participants and study staff's test interpretation, and the acceptability of the smartphone app and self-test indicate that self-tests for SARS-CoV-2 with a smartphone app for assistance and reporting is a highly feasible testing modality among a diverse population of adults in the United States.
  • Building integrative functional maps of gene regulation

    Xu, Jinrui; Pratt, Henry; Moore, Jill E.; Gerstein, Mark B.; Weng, Zhiping (2022-09-09)
    Every cell in the human body inherits a copy of the same genetic information. The three billion base pairs of DNA in the human genome, and the roughly 50 000 coding and non-coding genes they contain, must thus encode all the complexity of human development and cell and tissue type diversity. Differences in gene regulation, or the modulation of gene expression, enable individual cells to interpret the genome differently to carry out their specific functions. Here we discuss recent and ongoing efforts to build gene regulatory maps, which aim to characterize all sequences in a genome for their roles in regulating gene expression. Many researchers and consortia have identified such regulatory elements using functional assays and evolutionary analyses; we discuss the results, strengths, and shortcomings of their approaches. We also discuss new techniques the field can leverage and emerging challenges it will face while striving to build gene regulatory maps of ever-increasing resolution and comprehensiveness.
  • Shear stress activates nociceptors to drive Drosophila mechanical nociception

    Gong, Jiaxin; Chen, Jiazhang; Gu, Pengyu; Shang, Ye; Ruppell, Kendra Takle; Yang, Ying; Wang, Fei; Wen, Qi; Xiang, Yang (2022-09-02)
    Mechanical nociception is essential for animal survival. However, the forces involved in nociceptor activation and the underlying mechanotransduction mechanisms remain elusive. Here, we address these problems by investigating nocifensive behavior in Drosophila larvae. We show that strong poking stimulates nociceptors with a mixture of forces including shear stress and stretch. Unexpectedly, nociceptors are selectively activated by shear stress, but not stretch. Both the shear stress responses of nociceptors and nocifensive behavior require transient receptor potential A1 (TrpA1), which is specifically expressed in nociceptors. We further demonstrate that expression of mammalian or Drosophila TrpA1 in heterologous cells confers responses to shear stress but not stretch. Finally, shear stress activates TrpA1 in a membrane-delimited manner, through modulation of membrane fluidity. Together, our study reveals TrpA1 as an evolutionarily conserved mechanosensitive channel specifically activated by shear stress and suggests a critical role of shear stress in activating nociceptors to drive mechanical nociception.
  • Validation of DREADD agonists and administration route in a murine model of sleep enhancement

    Ferrari, Loris L; Ogbeide-Latario, Oghomwen E; Gompf, Heinrich S; Anaclet, Christelle (2022-07-30)
    Chemogenetics is a powerful tool to study the role of specific neuronal populations in physiology and diseases. Of particular interest, in mice, acute and specific activation of parafacial zone (PZ) GABAergic neurons expressing the Designer Receptors Activated by Designer Drugs (DREADD) hM3Dq (PZGABA-hM3Dq) enhances slow-wave-sleep (SWS), and this effect lasts for up to 6 h, allowing prolonged and detailed study of SWS. However, the most widely used DREADDs ligand, clozapine N-oxide (CNO), is metabolized into clozapine which has the potential of inducing non-specific effects. In addition, CNO is usually injected intraperitoneally (IP) in mice, limiting the number and frequency of repeated administration.
  • Healthcare use in commercially insured youth with mental health disorders

    Hugunin, Julie; Davis, Maryann; Larkin, Celine; Baek, Jonggyu; Skehan, Brian; Lapane, Kate L (2022-07-26)
    Background: The objective of this study is to describe age-related patterns of outpatient healthcare utilization in youth and young adults with mental health disorders. Method: We used the IBM® MarketScan® Commercial Database to identify 359,413 youth and young adults (12-27 years) with a mental health disorder continuously enrolled in private health insurance in 2018. Exploratory analysis was used to describe patterns of outpatient healthcare use (e.g., primary, reproductive, mental health care) and therapeutic management (e.g., medication prescriptions, psychotherapy) by age. Period prevalence and median number of visits are reported. Additional analysis explored utilization patterns by mental health disorder. Results: The prevalence of outpatient mental health care and primary care decreased with age, with a larger drop in primary care utilization. While 74.0-78.4% of those aged 12-17 years used both outpatient mental health care and primary care, 53.1-59.7% of those aged 18-27 years did. Most 18-19-year-olds had a visit with an internal medicine or family medicine specialist, a minority had a pediatrician visit. The prevalence of medication management increased with age, while the prevalence of psychotherapy decreased. Conclusions: Taken together, this descriptive study illustrates age-related differences in outpatient healthcare utilization among those with mental health disorders. Additionally, those with the most severe mental health disorders seem to be least connected to outpatient care. This knowledge can inform efforts to improve utilization of healthcare across the transition to adulthood.
  • Development and test-retest reliability of a screening tool for axial spondyloarthritis

    Shridharmurthy, Divya; Lapane, Kate L.; Khan, Sara; Yi, Esther; Baek, Jonggyu; Kay, Jonathan; Liu, Shao-Hsien (2022-07-08)
    Background: People with axial Spondyloarthritis (axSpA) suffer from lengthy diagnostic delays of ~7 years. The usage of screening tools to identify axSpA patients in primary care can reduce diagnostic delays by facilitating early referral to rheumatologic care. The purpose of this study was to examine the psychometric properties of a potential screening tool for patients with axSpA. Method: Content validity was evaluated by soliciting feedback from 7 rheumatologists regarding the relevance and content representativeness of the proposed screening questions. For the test-retest study, participants ≥18 years of age with chronic back pain (≥3 months) without a diagnosis of mechanical or inflammatory back pain (n = 91) were e-recruited through ResearchMatch. Participation included completing identical baseline and follow-up questionnaires ~14 days apart. Weighted quadratic kappa was used to measure test-retest reliability between the two ratings of the ordinal scales. Construct validity was examined using exploratory factor analysis (EFA) and items with factor loadings ≥0.6 were extracted. Scale dimensionality and simplified factorial solutions were measured using Kaiser's criteria (Eigenvalue >1). Cronbach's alpha was used to measure internal consistency. Results: Most participants were women, non-Hispanic white, and had at least some college education, with a mean age of 45 years. On average, the age at onset of back pain was 31 years. Eleven questions yielded test-retest reliabilities ranging from 0.6 to 0.76. Results from EFA extracted two factors relating to: 1) how pain affects daily life functioning and 2) whether pain improves with movement. Internal consistency was high for questions evaluating how pain affects life, with a Cronbach's alpha of 0.81. Following assessment for validity and reliability, the questionnaire was revised to create the 6-item screening tool. Conclusions: The 6-item SpA-SED screening tool designed to identify potential cases of axSpA was found to have good test-retest reliability and high internal consistency.
  • Elevated TNF-α Leads to Neural Circuit Instability in the Absence of Interferon Regulatory Factor 8

    Feinberg, Philip A; Becker, Shannon C; Chung, Leeyup; Ferrari, Loris; Stellwagen, David; Anaclet, Christelle; Durán-Laforet, Violeta; Faust, Travis E; Sumbria, Rachita K; Schafer, Dorothy P. (2022-07-05)
    Interferon regulatory factor 8 (IRF8) is a transcription factor necessary for the maturation of microglia, as well as other peripheral immune cells. It also regulates the transition of microglia and other immune cells to a pro-inflammatory phenotype. Irf8 is also a known risk gene for multiple sclerosis and lupus, and it has recently been shown to be downregulated in schizophrenia. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. Here, we show by RNAseq from Irf8 -/- male and female mouse brains that several genes involved in regulation of neural activity are dysregulated. We then show that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to lethal seizures in male and female Irf8 -/- mice. Finally, we identify that TNF-α is elevated specifically in microglia in the CNS, and genetic or acute pharmacological blockade of TNF-α in the Irf8 -/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. Our data further suggest that neuronal function is impacted by loss of IRF8, a factor involved in neuropsychiatric and neurodegenerative diseases.SIGNIFICANCE STATEMENT Here, we identify a previously unknown and key role for interferon regulator factor 8 (IRF8) in regulating neural excitability and seizures. We further determine that these effects on neural circuits are through elevated TNF-α in the CNS. As IRF8 has most widely been studied in the context of regulating the development and inflammatory signaling in microglia and other immune cells, we have uncovered a novel function. Further, IRF8 is a risk gene for multiple sclerosis and lupus, IRF8 is dysregulated in schizophrenia, and elevated TNF-α has been identified in a multitude of neurologic conditions. Thus, elucidating these IRF8 and TNF-α-dependent effects on brain circuit function has profound implications for understanding underlying, therapeutically relevant mechanisms of disease.
  • Comprehensive fitness landscape of SARS-CoV-2 M(pro) reveals insights into viral resistance mechanisms

    Flynn, Julia; Samant, Neha; Nachum, Gily S.; Bakan, David T.; Yilmaz, Nese Kurt; Schiffer, Celia A.; Moquin, Stephanie A.; Dovala, Dustin; Bolon, Daniel N. (2022-06-20)
    With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs SARS-CoV-2 main protease (M(pro)) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting M(pro) appear promising but will elicit selection pressure for resistance. To understand resistance potential in M(pro), we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of M(pro) function in yeast, based on either the ability of M(pro) variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to M(pro) function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the M(pro) dimer. The clinical variants of M(pro) were predominantly functional in our screens, indicating that M(pro) is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to M(pro) evolution and that are likely to contribute to drug resistance. This complete mutational guide of M(pro) can be used in the design of inhibitors with reduced potential of evolving viral resistance.
  • Perspectives on addressing bipolar disorder in the obstetric setting

    Masters, Grace A; Xu, Lulu; Cooper, Katherine M; Moore Simas, Tiffany A; Brenckle, Linda; Mackie, Thomas I; Schaefer, Ana J; Straus, John; Byatt, Nancy (2022-05-25)
    Objective: Perinatal Psychiatry Access Programs have emerged to help obstetric professionals meet the needs of perinatal individuals with mental health conditions, including bipolar disorder (BD). We elucidate obstetric professionals' perspectives on barriers and facilitators to managing BD in perinatal patients, and how Access Programs may affect these processes. Methods: We conducted three focus groups with obstetric professionals, two with- and one without-exposure to an Access Program, the Massachusetts Child Psychiatry Access Program (MCPAP) for Moms. Focus groups discussed experiences, barriers, facilitators, and solutions to caring for perinatal individuals with BD. Qualitative data were coded and analyzed by two independent coders; emergent themes were examined across exposure groups. Results: Thirty-one obstetric professionals (7 without-exposure, 24 with-exposure) participated. Identified themes included: (1) gaps in perinatal BD education; (2) challenges in patient assessment; (3) MCPAP for Moms as a facilitator for addressing BD; and (4) importance of continued outreach and destigmaization to increase care collaboration. Conclusions: Barriers to obstetric professionals accessing adequate mental healthcare for their patients with BD abound. With psychiatric supports in place, it is possible to build obstetric professionals' capacity to address BD. Perinatal Psychiatry Access Programs can facilitate obstetric professionals bridging these gaps in mental health care.
  • Co-transmission of neuropeptides and monoamines choreograph the C. elegans escape response

    Florman, Jeremy T.; Alkema, Mark J. (2022-03-03)
    Co-localization and co-transmission of neurotransmitters and neuropeptides is a core property of neural signaling across species. While co-transmission can increase the flexibility of cellular communication, understanding the functional impact on neural dynamics and behavior remains a major challenge. Here we examine the role of neuropeptide/monoamine co-transmission in the orchestration of the C. elegans escape response. The tyraminergic RIM neurons, which coordinate distinct motor programs of the escape response, also co-express the neuropeptide encoding gene flp-18. We find that in response to a mechanical stimulus, flp-18 mutants have defects in locomotory arousal and head bending that facilitate the omega turn. We show that the induction of the escape response leads to the release of FLP-18 neuropeptides. FLP-18 modulates the escape response through the activation of the G-protein coupled receptor NPR-5. FLP-18 increases intracellular calcium levels in neck and body wall muscles to promote body bending. Our results show that FLP-18 and tyramine act in different tissues in both a complementary and antagonistic manner to control distinct motor programs during different phases of the C. elegans flight response. Our study reveals basic principles by which co-transmission of monoamines and neuropeptides orchestrate in arousal and behavior in response to stress.
  • Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice

    Smith, Ciearra B.; van der Vinne, Vincent; McCartney, Eleanor; Stowie, Adam C.; Leise, Tanya L.; Martin-Burgos, Blanca; Molyneux, Penny C.; Garbutt, Lauren A.; Brodsky, Michael H.; Davidson, Alec J.; et al. (2022-02-01)
    Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.
  • A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior

    Ramachandran, Shankar; Banerjee, Navonil; Bhattacharya, Raja; Lemons, Michele L.; Florman, Jeremy; Lambert, Christopher M.; Touroutine, Denis; Alexander, Kellianne; Schoofs, Liliane; Alkema, Mark J.; et al. (2021-11-12)
    Neuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here, we show that the Caenorhabditis elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits.
  • A phase transition enhances the catalytic activity of SARM1, an NAD(+) glycohydrolase involved in neurodegeneration

    Loring, Heather S.; Czech, Victoria L.; Icso, Janneke D.; O'Connor, Lauren C.; Parelkar, Sangram; Byrne, Alexandra B.; Thompson, Paul R. (2021-06-29)
    Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is a neuronally expressed NAD(+) glycohydrolase whose activity is increased in response to stress. NAD(+) depletion triggers axonal degeneration, which is a characteristic feature of neurological diseases. Notably, loss of SARM1 is protective in murine models of peripheral neuropathy and traumatic brain injury. Herein, we report that citrate induces a phase transition that enhances SARM1 activity by ~2000-fold. This phase transition can be disrupted by mutating a residue involved in multimerization, G601P. This mutation also disrupts puncta formation in cells. We further show that citrate induces axonal degeneration in C. elegans that is dependent on the C. elegans orthologue of SARM1 (TIR-1). Notably, citrate induces the formation of larger puncta indicating that TIR-1/SARM1 multimerization is essential for degeneration in vivo. These findings provide critical insights into SARM1 biology with important implications for the discovery of novel SARM1-targeted therapeutics.
  • PAPPA-mediated adipose tissue remodeling mitigates insulin resistance and protects against gestational diabetes in mice and humans

    Rojas-Rodriguez, Raziel; Ziegler, Rachel; DeSouza, Tiffany; Majid, Sana; Madore, Aylin S.; Amir, Nili S.; Pace, Veronica A.; Nachreiner, Daniel; Alfego, David; Mathew, Jomol; et al. (2020-11-25)
    Pregnancy is a physiological state of continuous adaptation to changing maternal and fetal nutritional needs, including a reduction of maternal insulin sensitivity allowing for appropriately enhanced glucose availability to the fetus. However, excessive insulin resistance in conjunction with insufficient insulin secretion results in gestational diabetes mellitus (GDM), greatly increasing the risk for pregnancy complications and predisposing both mothers and offspring to future metabolic disease. Here, we report a signaling pathway connecting pregnancy-associated plasma protein A (PAPPA) with adipose tissue expansion in pregnancy. Adipose tissue plays a central role in the regulation of insulin sensitivity, and we show that, in both mice and humans, pregnancy caused remodeling of adipose tissue evidenced by altered adipocyte size, vascularization, and in vitro expansion capacity. PAPPA is known to be a metalloprotease secreted by human placenta that modulates insulin-like growth factor (IGF) bioavailability through prolteolysis of IGF binding proteins (IGFBPs) 2, 4, and 5. We demonstrate that recombinant PAPPA can stimulate ex vivo human adipose tissue expansion in an IGFBP-5- and IGF-1-dependent manner. Moreover, mice lacking PAPPA displayed impaired adipose tissue remodeling, pregnancy-induced insulin resistance, and hepatic steatosis, recapitulating multiple aspects of human GDM. In a cohort of 6361 pregnant women, concentrations of circulating PAPPA are inversely correlated with glycemia and odds of developing GDM. These data identify PAPPA and the IGF signaling pathway as necessary for the regulation of maternal adipose tissue physiology and systemic glucose homeostasis, with consequences for long-term metabolic risk and potential for therapeutic use.
  • Flexible motor sequence generation during stereotyped escape responses

    Wang, Yuan; Zhang, Xiaoqian; Xin, Qi; Hung, Wesley; Florman, Jeremy; Huo, Jing; Xu, Tianqi; Xie, Yu; Alkema, Mark J.; Zhen, Mei; et al. (2020-06-05)
    Complex animal behaviors arise from a flexible combination of stereotyped motor primitives. Here we use the escape responses of the nematode Caenorhabditis elegans to study how a nervous system dynamically explores the action space. The initiation of the escape responses is predictable: the animal moves away from a potential threat, a mechanical or thermal stimulus. But the motor sequence and the timing that follow are variable. We report that a feedforward excitation between neurons encoding distinct motor states underlies robust motor sequence generation, while mutual inhibition between these neurons controls the flexibility of timing in a motor sequence. Electrical synapses contribute to feedforward coupling whereas glutamatergic synapses contribute to inhibition. We conclude that C. elegans generates robust and flexible motor sequences by combining an excitatory coupling and a winner-take-all operation via mutual inhibition between motor modules.
  • The 'Take a Break' game: Randomized trial protocol for a technology-assisted brief abstinence experience designed to engage lower-motivated smokers.

    Amante, Daniel J.; Blok, Amanda C.; Nagawa, Catherine S.; Wijesundara, Jessica G.; Allison, Jeroan J.; Person, Sharina D.; Morley, Jeanne; Conigliaro, Joseph; Mattocks, Kristin M.; Garber, Lawrence D.; et al. (2020-06-01)
    BACKGROUND: While smoking continues to be the most preventable cause of mortality in the United States, most current smokers remain not ready to quit at any given time. Engaging these 'motivation phase' smokers with brief experiences to build confidence and practice skills related to cessation could lead to sooner and more successful quit attempts. Increasingly available mobile technology and gamification can be used to provide smokers with accessible and engaging support. METHODS: We describe our protocol for conducting a randomized controlled trial evaluating Take a Break, an mHealth-based smoking pre-cessation challenge designed for smokers not ready to quit. Participants in the intervention receive 1) Motivational Messages, 2) text message Challenge Quizzes, 3) Goal-setting with tobacco treatment specialist, 4) Coping Mini-Games apps, and 5) Recognition and Rewards for participation during a 3-week challenge. Access to coping mini-games and motivational messaging continues for 6-months. Both intervention and comparison group participants receive brief Nicotine Replacement Therapy (NRT) sampling and daily smoking assessment text messages for three weeks. Primary outcomes include number of days abstinent during the challenge, change in patient-reported self-efficacy after the challenge, time to first quit attempt following the challenge, and 7-day point prevalent smoking cessation at six months. CONCLUSION: Take a Break is an innovative approach to engage those not prepared for a quit attempt. Take a Break provides motivation phase smokers with tools and a brief experience to prepare them for a quit attempt, filling a gap in tobacco cessation support and current research.
  • A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture

    Keener, David G.; Cheung, Amy; Futai, Kensuke (2020-05-01)
    BACKGROUND: Exogenous gene introduction by transfection is one of the most important approaches for understanding the function of specific genes at the cellular level. Electroporation has a long-standing history as a versatile gene delivery technique in vitro and in vivo. However, it has been underutilized in vitro because of technical difficulty and insufficient transfection efficiency. NEW METHOD: We have developed an electroporation technique that combines the use of large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses. Here, we describe the technique and compare it with existing methods. RESULTS: Our method achieves a high transfection efficiency ( approximately 80 %) in both excitatory and inhibitory neurons with no detectable side effects on their function. We demonstrate this method is capable of transferring at least three different genes into a single neuron. In addition, we demonstrate the ability to transfect different genes into neighboring cells. COMPARISON WITH EXISTING METHODS: The majority of existing methods use fine-tipped glass electrodes (i.e. > 10MOmega) and apply high voltage (10V) pulses with high frequency (100Hz) for 1s. These parameters contribute to practical difficulties thus lowering the transfection efficiency. Our unique method minimizes electrode clogging and therefore procedure duration, increasing transfection efficiency and cellular viability. CONCLUSIONS: Our modifications, relative to current methods, optimize electroporation efficiency and cell survival. Our approach offers distinct research strategies not only in elucidating cell-autonomous functions of genes but also for assessing genes contributing to intercellular functions, such as trans-synaptic interactions.
  • In Situ Regulated Dopamine Transporter Trafficking: There's No Place Like Home

    Fagan, Rita R.; Kearney, Patrick J.; Melikian, Haley E. (2020-03-07)
    Dopamine (DA) is critical for motivation, reward, movement initiation, and learning. Mechanisms that control DA signaling have a profound impact on these important behaviors, and additionally play a role in DA-related neuropathologies. The presynaptic SLC6 DA transporter (DAT) limits extracellular DA levels by clearing released DA, and is potently inhibited by addictive and therapeutic psychostimulants. Decades of evidence support that the DAT is subject to acute regulation by a number of signaling pathways, and that endocytic trafficking strongly regulates DAT availability and function. DAT trafficking studies have been performed in a variety of model systems, including both in vitro and ex vivo preparations. In this review, we focus on the breadth of DAT trafficking studies, with specific attention to, and comparison of, how context may influence DAT's response to different stimuli. In particular, this overview highlights that stimulated DAT trafficking not only differs between in vitro and ex vivo environments, but also is influenced by both sex and anatomical subregions.
  • Drosophila Cryptochrome: Variations in Blue

    Foley, Lauren E.; Emery, Patrick (2020-02-01)
    CRYPTOCHROMES (CRYs) are structurally related to ultraviolet (UV)/blue-sensitive DNA repair enzymes called photolyases but lack the ability to repair pyrimidine dimers generated by UV exposure. First identified in plants, CRYs have proven to be involved in light detection and various light-dependent processes in a broad range of organisms. In Drosophila, CRY's best understood role is the cell-autonomous synchronization of circadian clocks. However, CRY also contributes to the amplitude of circadian oscillations in a light-independent manner, controls arousal and UV avoidance, influences visual photoreception, and plays a key role in magnetic field detection. Here, we review our current understanding of the mechanisms underlying CRY's various circadian and noncircadian functions in fruit flies.
  • Role of Interferon-γ-Producing Th1 Cells in a Murine Model of Type I Interferon-Independent Autoinflammation Resulting From DNase II Deficiency

    Pawaria, Sudesh; Nündel, Kerstin; Gao, Kevin M; Moses, Stephanie; Busto, Patricia; Holt, Kevin; Sharma, Rohit B; Brehm, Michael A; Gravallese, Ellen M; Socolovsky, Merav; et al. (2019-12-29)
    Objective: Patients with hypomorphic mutations in DNase II develop a severe and debilitating autoinflammatory disease. This study was undertaken to compare the disease parameters in these patients to those in a murine model of DNase II deficiency, and to evaluate the role of specific nucleic acid sensors and identify the cell types responsible for driving the autoinflammatory response. Methods: To avoid embryonic death, Dnase2-/- mice were intercrossed with mice that lacked the type I interferon (IFN) receptor (Ifnar-/- ). The hematologic changes and immune status of these mice were evaluated using complete blood cell counts, flow cytometry, serum cytokine enzyme-linked immunosorbent assays, and liver histology. Effector cell activity was determined by transferring T cells from Dnase2-/- × Ifnar-/- double-knockout (DKO) mice into Rag1-/- mice, and 4 weeks after cell transfer, induced changes were assessed in the recipient mice. Results: In Dnase2-/- × Ifnar-/- DKO mice, many of the disease features found in DNase II-deficient patients were recapitulated, including cytopenia, extramedullary hematopoiesis, and liver fibrosis. Dnase2+/+ × Rag1-/- mice (n > 22) developed a hematologic disorder that was attributed to the transfer of an unusual IFNγ-producing T cell subset from the spleens of donor Dnase2-/- × Ifnar-/- DKO mice. Autoinflammation in this murine model did not depend on the stimulator of IFN genes (STING) pathway but was highly dependent on the chaperone protein Unc93B1. Conclusion: Dnase2-/- × Ifnar-/- DKO mice may be a valid model for exploring the innate and adaptive immune mechanisms responsible for the autoinflammation similar to that seen in DNASE2-hypomorphic patients. In this murine model, IFNγ is required for T cell activation and the development of clinical manifestations. The role of IFNγ in DNASE2-deficient patient populations remains to be determined, but the ability of Dnase2-/- mouse T cells to transfer disease to Rag1-/- mice suggests that T cells may be a relevant therapeutic target in patients with IFN-related systemic autoinflammatory diseases.

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