32 results on '"Holt LM"'
Search Results
2. Risk factors for perioperative blood transfusions after urogenital fistula repair in Uganda: a retrospective cohort study.
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Potluri, TK, Holt, LM, Tanner, JP, Wasingya, L, Duffy, S, and Greene, KA
- Subjects
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BLOOD transfusion , *FISTULA , *COHORT analysis , *RETROSPECTIVE studies ,DEVELOPED countries - Abstract
Objective: To determine the incidence of and risk factors for perioperative blood transfusions after urogenital fistula repairs in Uganda. Design: A retrospective cohort study. Setting: A community hospital in Masaka, Uganda. Population: Women who underwent fistula repair at the Kitovu Hospital between 2013 and 2019. Methods: Retrospective review of demographics and clinical perioperative characteristics of patients surgically treated for urogenital fistula. Patient characteristics were compared between those who did and those who did not require a blood transfusion. Main outcome measures: Need for perioperative blood transfusion and risk factors. Results: A total of 546 patients treated for urogenital fistulas were included in this study. The median age was 31.1 ± 13.2 years. A vaginal surgical approach was used in the majority of patients (84.6%). Complications occurred in 3.5% of surgical repairs, and the incidence of blood transfusions was 6.2%. In multivariable analyses, for each gram per deciliter (g/dl) increase in preoperative haemoglobin, the odds of blood transfusion decreased by approximately 28% (adjusted OR 0.72, 95% CI 0.59–0.86). Women who had their fistula repaired abdominally were 3.4 times more likely to require transfusions (95% CI 1.40–8.08). Conclusions: The incidence of blood transfusions among urogenital fistula repairs in our population is twice that of developed nations. An abdominal surgical approach to urogenital fistula is a significant risk factor for perioperative blood transfusions. The timing of the repair may warrant further study. One of the first studies to look at blood transfusion risk factors after fistula repair in a low‐resource setting. One of the first studies to look at blood transfusion risk factors after fistula repair in a low‐resource setting. [ABSTRACT FROM AUTHOR]
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- 2022
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3. Cell-Type-Specific Regulation of Cocaine Reward by the E2F3a Transcription Factor in Nucleus Accumbens.
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Martínez-Rivera FJ, Yim YY, Godino A, Minier-Toribio A, Tofani S, Holt LM, Torres-Berrío A, Futamura R, Browne CJ, Markovic T, Hamilton PJ, Neve RL, and Nestler EJ
- Abstract
The development of drug addiction is characterized by molecular changes in brain reward regions that lead to the transition from recreational to compulsive drug use. These neurobiological processes in brain reward regions, such as the nucleus accumbens (NAc), are orchestrated in large part by transcriptional regulation. Our group recently identified the transcription factor E2F3a as a novel regulator of cocaine's rewarding effects and gene expression regulation in the NAc of male mice. Despite this progress, no information is available about the role of E2F3a in regulating cocaine reward at the sex- and cell-specific levels. Here, we used male and female mice expressing Cre-recombinase in either D1- or D2-type medium spiny neurons (MSNs) combined with viral-mediated gene transfer to bidirectionally control levels of E2F3a in a cell-type-specific manner in the NAc during conditioned place preference (CPP) to cocaine. Our findings show that selective overexpression of E2F3a in D1-MSNs increased cocaine CPP in both male and female mice, whereas opposite effects were observed under knockdown conditions. In contrast, equivalent E2F3a manipulations in D2-MSNs had no significant effects. To further explore the role of E2F3a in sophisticated operant and motivated behaviors, we performed viral manipulations of all NAc neurons in combination with cocaine self-administration and behavioral economics procedures in rats and demonstrated that E2F3a regulates sensitivity aspects of cocaine seeking and taking. These results confirm E2F3a as a central substrate of cocaine reward and demonstrate that this effect is mediated in D1-MSNs, thereby providing increased knowledge of cocaine action at the transcriptional level.
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- 2024
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4. Circuit-Wide Gene Network Analysis Reveals Sex-Specific Roles for Phosphodiesterase 1b in Cocaine Addiction.
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Teague CD, Markovic T, Zhou X, Martinez-Rivera FJ, Minier-Toribio A, Zinsmaier A, Pulido NV, Schmidt KH, Lucerne KE, Godino A, van der Zee YY, Ramakrishnan A, Futamura R, Browne CJ, Holt LM, Yim YY, Azizian CH, Walker DM, Shen L, Dong Y, Zhang B, and Nestler EJ
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- Animals, Male, Female, Mice, Rats, Cocaine pharmacology, Reward, Cyclic Nucleotide Phosphodiesterases, Type 1 genetics, Cyclic Nucleotide Phosphodiesterases, Type 1 metabolism, Cocaine-Related Disorders genetics, Cocaine-Related Disorders metabolism, Gene Regulatory Networks drug effects, Gene Regulatory Networks genetics, Nucleus Accumbens drug effects, Nucleus Accumbens metabolism, Sex Characteristics, Mice, Inbred C57BL
- Abstract
Cocaine use disorder is a significant public health issue without an effective pharmacological treatment. Successful treatments are hindered in part by an incomplete understanding of the molecular mechanisms that underlie long-lasting maladaptive plasticity and addiction-like behaviors. Here, we leverage a large RNA sequencing dataset to generate gene coexpression networks across six interconnected regions of the brain's reward circuitry from mice that underwent saline or cocaine self-administration. We identify phosphodiesterase 1b ( Pde1b ), a Ca
2+ /calmodulin-dependent enzyme that increases cAMP and cGMP hydrolysis, as a central hub gene within a nucleus accumbens (NAc) gene module that was bioinformatically associated with addiction-like behavior. Chronic cocaine exposure increases Pde1b expression in NAc D2 medium spiny neurons (MSNs) in male but not female mice. Viral-mediated Pde1b overexpression in NAc reduces cocaine self-administration in female rats but increases seeking in both sexes. In female mice, overexpressing Pde1b in D1 MSNs attenuates the locomotor response to cocaine, with the opposite effect in D2 MSNs. Overexpressing Pde1b in D1/D2 MSNs had no effect on the locomotor response to cocaine in male mice. At the electrophysiological level, Pde1b overexpression reduces sEPSC frequency in D1 MSNs and regulates the excitability of NAc MSNs. Lastly, Pde1b overexpression significantly reduced the number of differentially expressed genes (DEGs) in NAc following chronic cocaine, with discordant effects on gene transcription between sexes. Together, we identify novel gene modules across the brain's reward circuitry associated with addiction-like behavior and explore the role of Pde1b in regulating the molecular, cellular, and behavioral responses to cocaine., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)- Published
- 2024
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5. Astrocytic transcriptional and epigenetic mechanisms of drug addiction.
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Holt LM and Nestler EJ
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- Humans, Animals, Transcription, Genetic physiology, Epigenesis, Genetic physiology, Astrocytes metabolism, Astrocytes drug effects, Substance-Related Disorders genetics
- Abstract
Addiction is a leading cause of disease burden worldwide and remains a challenge in current neuroscience research. Drug-induced lasting changes in gene expression are mediated by transcriptional and epigenetic regulation in the brain and are thought to underlie behavioral adaptations. Emerging evidence implicates astrocytes in regulating drug-seeking behaviors and demonstrates robust transcriptional response to several substances of abuse. This review focuses on the astrocytic transcriptional and epigenetic mechanisms of drug action., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)
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- 2024
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6. Bictegravir Use During Pregnancy: A Multi-Center Retrospective Analysis Evaluating HIV Viral Suppression and Perinatal Outcomes.
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Holt LM, Short WR, Momplaisir F, Hyun E, McKinney J, Lugo Morales A, Duque A, Druyan B, Ndubizu C, Duthely L, Joseph N, Sheth A, and Badell ML
- Abstract
This study describes the largest cohort to date (n=147) of pregnant patients living with HIV on bictegravir (BIC). BIC in pregnancy was associated with high levels of viral suppression and similar perinatal outcomes to published literature. These findings support consideration for use of BIC in management of HIV during pregnancy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)
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- 2024
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7. Transcriptional characterization of cocaine withdrawal versus extinction within nucleus accumbens.
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Martínez-Rivera FJ, Holt LM, Minier-Toribio A, Estill M, Yeh SY, Tofani S, Futamura R, Browne CJ, Mews P, Shen L, and Nestler EJ
- Abstract
Substance use disorder is characterized by a maladaptive imbalance wherein drug seeking persists despite negative consequences or drug unavailability. This imbalance correlates with neurobiological alterations some of which are amplified during forced abstinence, thereby compromising the capacity of extinction-based approaches to prevent relapse. Cocaine use disorder (CUD) exemplifies this phenomenon in which neurobiological modifications hijack brain reward regions such as the nucleus accumbens (NAc) to manifest craving and withdrawal-like symptoms. While increasing evidence links transcriptional changes in the NAc to specific phases of addiction, genome-wide changes in gene expression during withdrawal vs. extinction (WD/Ext) have not been examined in a context- and NAc-subregion-specific manner. Here, we used cocaine self-administration (SA) in rats combined with RNA-sequencing (RNA-seq) of NAc subregions (core and shell) to transcriptionally profile the impact of experiencing withdrawal in the home cage or in the previous drug context or experiencing extinction training. As expected, home-cage withdrawal maintained drug seeking in the previous drug context, whereas extinction training reduced it. By contrast, withdrawal involving repetitive exposure to the previous drug context increased drug-seeking behavior. Bioinformatic analyses of RNA-seq data revealed gene expression patterns, networks, motifs, and biological functions specific to these behavioral conditions and NAc subregions. Comparing transcriptomic analysis of the NAc of patients with CUD highlighted conserved gene signatures, especially with rats that were repetitively exposed to the previous drug context. Collectively, these behavioral and transcriptional correlates of several withdrawal-extinction settings reveal fundamental and translational information about potential molecular mechanisms to attenuate drug-associated memories.
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- 2024
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8. Astrocytic CREB in nucleus accumbens promotes susceptibility to chronic stress.
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Holt LM, Gyles TM, Parise EM, Minier-Toribio A, Markovic T, Rivera M, Yeh SY, and Nestler EJ
- Abstract
Background: Increasing evidence implicates astrocytes in stress and depression in both rodent models and human Major Depressive Disorder (MDD). Despite this, little is known about the transcriptional responses to stress of astrocytes within the nucleus accumbens (NAc), a key brain reward region, and their influence on behavioral outcomes., Methods: We used whole cell sorting, RNA-sequencing, and bioinformatic analyses to investigate the NAc astrocyte transcriptome in male mice in response to chronic social defeat stress (CSDS). Immunohistochemistry was used to determine stress-induced changes in astrocytic CREB within the NAc. Finally, astrocytic regulation of depression-like behavior was investigated using viral-mediated manipulation of CREB in combination with CSDS., Results: We found a robust transcriptional response in NAc astrocytes to CSDS in stressed mice, with changes seen in both stress-susceptible and stress-resilient animals. Bioinformatic analysis revealed CREB, a transcription factor widely studied in neurons, as one of the top-predicted upstream regulators of the NAc astrocyte transcriptome, with opposite activation states seen in resilient versus susceptible mice. This bioinformatic result was confirmed at the protein level with immunohistochemistry. Viral overexpression of CREB selectively in NAc astrocytes promoted susceptibility to chronic stress., Conclusions: Together, our data demonstrate that the astrocyte transcriptome responds robustly to CSDS and, for the first time, that transcriptional regulation in astrocytes contributes to depressive-like behaviors. A better understanding of transcriptional regulation in astrocytes may reveal unknown molecular mechanisms underlying neuropsychiatric disorders., Competing Interests: Disclosures The authors declare no competing financial interests.
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- 2024
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9. Transcriptional correlates of cocaine-associated learning in striatal ARC ensembles.
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Salery M, Godino A, Xu YQ, Fullard JF, Durand-de Cuttoli R, LaBanca AR, Holt LM, Russo SJ, Roussos P, and Nestler EJ
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Learned associations between the rewarding effects of drugs and the context in which they are experienced underlie context-induced relapse. Previous work demonstrates the importance of sparse neuronal populations - called neuronal ensembles - in associative learning and cocaine seeking, but it remains unknown whether the encoding vs. retrieval of cocaine-associated memories involves similar or distinct mechanisms of ensemble activation and reactivation in nucleus accumbens (NAc). We use ArcCreER
T2 mice to establish that mostly distinct NAc ensembles are recruited by initial vs. repeated exposures to cocaine, which are then differentially reactivated and exert distinct effects during cocaine-related memory retrieval. Single-nuclei RNA-sequencing of these ensembles demonstrates predominant recruitment of D1 medium spiny neurons and identifies transcriptional properties that are selective to cocaine-recruited NAc neurons and could explain distinct excitability features. These findings fundamentally advance our understanding of how cocaine drives pathological memory formation during repeated exposures.- Published
- 2023
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10. Shared and divergent transcriptomic regulation in nucleus accumbens D1 and D2 medium spiny neurons by cocaine and morphine.
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Browne CJ, Mews P, Zhou X, Holt LM, Estill M, Futamura R, Schaefer A, Kenny PJ, Hurd YL, Shen L, Zhang B, and Nestler EJ
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Substance use disorders (SUDs) induce widespread molecular dysregulation in the nucleus accumbens (NAc), a brain region pivotal for coordinating motivation and reward. These molecular changes are thought to support lasting neural and behavioral disturbances that promote drug-seeking in addiction. However, different drug classes exert unique influences on neural circuits, cell types, physiology, and gene expression despite the overlapping symptomatology of SUDs. To better understand common and divergent molecular mechanisms governing SUD pathology, our goal was to survey cell-type-specific restructuring of the NAc transcriptional landscape in after psychostimulant or opioid exposure. We combined fluorescence-activated nuclei sorting and RNA sequencing to profile NAc D1 and D2 medium spiny neurons (MSNs) across cocaine and morphine exposure paradigms, including initial exposure, prolonged withdrawal after repeated exposure, and re-exposure post-withdrawal. Our analyses reveal that D1 MSNs display many convergent transcriptional responses across drug classes during exposure, whereas D2 MSNs manifest mostly divergent responses between cocaine and morphine, with morphine causing more adaptations in this cell type. Utilizing multiscale embedded gene co-expression network analysis (MEGENA), we discerned transcriptional regulatory networks subserving biological functions shared between cocaine and morphine. We observed largely integrative engagement of overlapping gene networks across drug classes in D1 MSNs, but opposite regulation of key D2 networks, highlighting potential therapeutic gene network targets within MSNs. These studies establish a landmark, cell-type-specific atlas of transcriptional regulation induced by cocaine and by morphine that can serve as a foundation for future studies towards mechanistic understanding of SUDs. Our findings, and future work leveraging this dataset, will pave the way for the development of targeted therapeutic interventions, addressing the urgent need for more effective treatments for cocaine use disorder and enhancing the existing strategies for opioid use disorder., Competing Interests: Competing Interests The authors declare no competing financial interests.
- Published
- 2023
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11. Transcriptional control of nucleus accumbens neuronal excitability by retinoid X receptor alpha tunes sensitivity to drug rewards.
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Godino A, Salery M, Durand-de Cuttoli R, Estill MS, Holt LM, Futamura R, Browne CJ, Mews P, Hamilton PJ, Neve RL, Shen L, Russo SJ, and Nestler EJ
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- Mice, Male, Female, Animals, Nucleus Accumbens metabolism, Retinoid X Receptor alpha genetics, Retinoid X Receptor alpha metabolism, Neurons physiology, Receptors, Dopamine D1 metabolism, Reward, Mice, Inbred C57BL, Cocaine pharmacology, Mental Disorders metabolism
- Abstract
The complex nature of the transcriptional networks underlying addictive behaviors suggests intricate cooperation between diverse gene regulation mechanisms that go beyond canonical activity-dependent pathways. Here, we implicate in this process a nuclear receptor transcription factor, retinoid X receptor alpha (RXRα), which we initially identified bioinformatically as associated with addiction-like behaviors. In the nucleus accumbens (NAc) of male and female mice, we show that although its own expression remains unaltered after cocaine exposure, RXRα controls plasticity- and addiction-relevant transcriptional programs in both dopamine receptor D1- and D2-expressing medium spiny neurons, which in turn modulate intrinsic excitability and synaptic activity of these NAc cell types. Behaviorally, bidirectional viral and pharmacological manipulation of RXRα regulates drug reward sensitivity in both non-operant and operant paradigms. Together, this study demonstrates a key role for NAc RXRα in promoting drug addiction and paves the way for future studies of rexinoid signaling in psychiatric disease states., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)
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- 2023
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12. Distinct forms of regret linked to resilience versus susceptibility to stress are regulated by region-specific CREB function in mice.
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Durand-de Cuttoli R, Martínez-Rivera FJ, Li L, Minier-Toribio A, Holt LM, Cathomas F, Yasmin F, Elhassa SO, Shaikh JF, Ahmed S, Russo SJ, Nestler EJ, and Sweis BM
- Abstract
Regret describes recognizing alternative actions could have led to better outcomes. It remains unclear whether regret derives from generalized mistake appraisal or instead comprises dissociable, action-specific processes. Using a neuroeconomic task, we found that mice were sensitive to fundamentally distinct types of regret following exposure to chronic social defeat stress or manipulations of CREB, a transcription factor implicated in stress action. Bias to make compensatory decisions after rejecting high-value offers (regret type I) was unique to stress-susceptible mice. Bias following the converse operation, accepting low-value offers (regret type II), was enhanced in stress-resilient mice and absent in stress-susceptible mice. CREB function in either the prefrontal cortex or nucleus accumbens was required to suppress regret type I but bidirectionally regulated regret type II. We provide insight into how maladaptive stress response traits relate to distinct forms of counterfactual thinking, which could steer therapy for mood disorders, such as depression, toward circuit-specific computations through a careful description of decision narrative.
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- 2022
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13. Toward a Next-Generation Digital Chest Tube.
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DeArmond DT, Holt LM, Wang AP, Errico KN, and Das NA
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- Swine, Animals, Pneumonectomy methods, Suction methods, Drainage methods, Chest Tubes, Pneumothorax prevention & control, Pneumothorax diagnosis
- Abstract
Chest tubes in patients who have undergone pulmonary resection with pleural air leak are painful, impair ventilatory mechanics, and increase hospital length of stay and costs. Despite these well-documented concerns, current protocols for chest tube management in this setting are not well supported by evidence. Excessive suction applied to chest tubes has been associated with prolonged air leak due to alveolar over-distension, and most practitioners intuit that suction should be minimized to the lowest level needed to maintain desired pleural apposition. Unfortunately, there is no evidence-based protocol for the establishment of minimal adequate suction. Digital suction devices in current clinical use can identify air leak resolution preventing the delay of chest tube removal but cannot guide suction minimization while an air leak persists. We recently described a monitor of lung expansion in a porcine model of pleural air leak that could detect loss of pleural apposition continuously in real-time based on electrical impedance readings obtained directly from the surface of the lung via chest tube-embedded electrodes. The value of the impedance signal was "in-range" when pleural apposition was present but became abruptly "out-of-range" when pneumothorax due to inadequate suction developed. These findings suggested that a digitally controlled suction pump system could be programmed to recognize the development of pneumothorax and automatically identify and set the minimum level of suction required to maintain pleural apposition. We present here preliminary proof of concept for this system.
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- 2022
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14. Beyond the neuron: Role of non-neuronal cells in stress disorders.
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Cathomas F, Holt LM, Parise EM, Liu J, Murrough JW, Casaccia P, Nestler EJ, and Russo SJ
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- Brain, Humans, Mental Disorders, Neurons
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Stress disorders are leading causes of disease burden in the U.S. and worldwide, yet available therapies are fully effective in less than half of all individuals with these disorders. Although to date, much of the focus has been on neuron-intrinsic mechanisms, emerging evidence suggests that chronic stress can affect a wide range of cell types in the brain and periphery, which are linked to maladaptive behavioral outcomes. Here, we synthesize emerging literature and discuss mechanisms of how non-neuronal cells in limbic regions of brain interface at synapses, the neurovascular unit, and other sites of intercellular communication to mediate the deleterious, or adaptive (i.e., pro-resilient), effects of chronic stress in rodent models and in human stress-related disorders. We believe that such an approach may one day allow us to adopt a holistic "whole body" approach to stress disorder research, which could lead to more precise diagnostic tests and personalized treatment strategies. Stress is a major risk factor for many psychiatric disorders. Cathomas et al. review new insight into how non-neuronal cells mediate the deleterious effects, as well as the adaptive, protective effects, of stress in rodent models and human stress-related disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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15. Astrocytes in cocaine addiction and beyond.
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Wang J, Holt LM, Huang HH, Sesack SR, Nestler EJ, and Dong Y
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- Humans, Neuronal Plasticity physiology, Synapses physiology, Synaptic Transmission physiology, Astrocytes metabolism, Cocaine-Related Disorders metabolism
- Abstract
Drug addiction remains a key biomedical challenge facing current neuroscience research. In addition to neural mechanisms, the focus of the vast majority of studies to date, astrocytes have been increasingly recognized as an "accomplice." According to the tripartite synapse model, astrocytes critically regulate nearby pre- and postsynaptic neuronal substrates to craft experience-dependent synaptic plasticity, including synapse formation and elimination. Astrocytes within brain regions that are implicated in drug addiction exhibit dynamic changes in activity upon exposure to cocaine and subsequently undergo adaptive changes themselves during chronic drug exposure. Recent results have identified several key astrocytic signaling pathways that are involved in cocaine-induced synaptic and circuit adaptations. In this review, we provide a brief overview of the role of astrocytes in regulating synaptic transmission and neuronal function, and discuss how cocaine influences these astrocyte-mediated mechanisms to induce persistent synaptic and circuit alterations that promote cocaine seeking and relapse. We also consider the therapeutic potential of targeting astrocytic substrates to ameliorate drug-induced neuroplasticity for behavioral benefits. While primarily focusing on cocaine-induced astrocytic responses, we also include brief discussion of other drugs of abuse where data are available., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2022
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16. Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1.
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Holt LM, Hernandez RD, Pacheco NL, Torres Ceja B, Hossain M, and Olsen ML
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- Animals, Cells, Cultured, Membrane Glycoproteins deficiency, Mice, Mice, Knockout, Protein Isoforms metabolism, Protein-Tyrosine Kinases deficiency, Astrocytes cytology, Brain-Derived Neurotrophic Factor metabolism, Cell Differentiation, Membrane Glycoproteins metabolism, Morphogenesis, Protein-Tyrosine Kinases metabolism, Signal Transduction
- Abstract
Brain-derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of the CNS, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF's receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1, which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1, globally and astrocyte-specifically, in mice revealed morphologically immature astrocytes with significantly reduced volume, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte function. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development., Competing Interests: LH, RH, NP, BT, MH, MO No competing interests declared, (© 2019, Holt et al.)
- Published
- 2019
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17. Magnetic Cell Sorting for In Vivo and In Vitro Astrocyte, Neuron, and Microglia Analysis.
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Holt LM, Stoyanof ST, and Olsen ML
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- Animals, Brain physiology, Female, Male, Mice, Mice, Inbred C57BL, Astrocytes physiology, Brain cytology, Cell Separation methods, Magnetic Phenomena, Microglia physiology, Neurons physiology
- Abstract
Interest in evaluating individual cellular populations in the central nervous system has prompted the development of several techniques enabling the enrichment of single-cell populations. Herein we detail a relatively inexpensive method to specifically isolate neurons, astrocytes, and microglia from a mixed homogenate utilizing magnetic beads conjugated to cell-type specific antibodies. We have used this technique to isolate astrocytes across development and into late adulthood. Finally, we detail the utilization of this technique in novel astrocyte and astrocyte/neuron co-culture paradigms. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)
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- 2019
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18. A Real-Time Hemodynamic ICD Measurement: Evaluation in Chronically Implanted Canines With Pacing-Induced Dilated Cardiomyopathy.
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Holt LM, Oglesby ML, Wang AP, Valvano JW, and Feldman MD
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- Algorithms, Animals, Cardiac Pacing, Artificial adverse effects, Cardiomyopathy, Dilated etiology, Dogs, Hemodynamics, Tachycardia, Ventricular physiopathology, Cardiac Output, Cardiomyopathy, Dilated physiopathology, Defibrillators, Implantable, Electric Countershock methods, Tachycardia, Ventricular diagnosis
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- 2019
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19. MeCP2 Deficiency Leads to Loss of Glial Kir4.1.
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Kahanovitch U, Cuddapah VA, Pacheco NL, Holt LM, Mulkey DK, Percy AK, and Olsen ML
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- Animals, Gene Expression Regulation, Male, Methyl-CpG-Binding Protein 2 genetics, Mice, Transgenic, Rett Syndrome metabolism, Astrocytes metabolism, Methyl-CpG-Binding Protein 2 metabolism, Potassium Channels, Inwardly Rectifying metabolism, Rett Syndrome genetics
- Abstract
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder usually caused by mutations in methyl-CpG-binding protein 2 (MeCP2). RTT is typified by apparently normal development until 6-18 mo of age, when motor and communicative skills regress and hand stereotypies, autonomic symptoms, and seizures present. Restoration of MeCP2 function selectively to astrocytes reversed several deficits in a murine model of RTT, but the mechanism of this rescue is unknown. Astrocytes carry out many essential functions required for normal brain functioning, including extracellular K
+ buffering. Kir4.1, an inwardly rectifying K+ channel, is largely responsible for the channel-mediated K+ regulation by astrocytes. Loss-of-function mutations in Kir4.1 in human patients result in a severe neurodevelopmental disorder termed EAST or SESAME syndrome. Here, we evaluated astrocytic Kir4.1 expression in a murine model of Rett syndrome. We demonstrate by chromatin immunoprecipitation analysis that Kir4.1 is a direct molecular target of MeCP2. Astrocytes from Mecp2 -deficient mice express significantly less Kir4.1 mRNA and protein, which translates into a >50% deficiency in Ba2+ -sensitive Kir4.1-mediated currents, and impaired extracellular potassium dynamics. By examining astrocytes in isolation, we demonstrate that loss of Kir4.1 is cell autonomous. Assessment through postnatal development revealed that Kir4.1 expression in Mecp2 -deficient animals never reaches adult, wild-type levels, consistent with a neurodevelopmental disorder. These are the first data implicating a direct MeCP2 molecular target in astrocytes and provide novel mechanistic insight explaining a potential mechanism by which astrocytic dysfunction may contribute to RTT.- Published
- 2018
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20. The α2β2 isoform combination dominates the astrocytic Na + /K + -ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation.
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Stoica A, Larsen BR, Assentoft M, Holm R, Holt LM, Vilhardt F, Vilsen B, Lykke-Hartmann K, Olsen ML, and MacAulay N
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- Adenosine Triphosphatases genetics, Animals, Animals, Newborn, Arginine genetics, Astrocytes drug effects, Astrocytes physiology, CD11b Antigen metabolism, Cation Transport Proteins genetics, Cell Adhesion Molecules, Neuronal genetics, Cells, Cultured, Excitatory Amino Acids pharmacology, Female, Glycine genetics, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neurons physiology, Oocytes physiology, Protein Isoforms metabolism, Rats, Rats, Sprague-Dawley, Xenopus laevis, Adenosine Triphosphatases metabolism, Cation Transport Proteins metabolism, Cell Adhesion Molecules, Neuronal metabolism, Mutation genetics, Sodium-Potassium-Exchanging ATPase genetics, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Synaptic activity results in transient elevations in extracellular K
+ , clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+ /K+ -ATPase. The Na+ /K+ -ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na+ /K+ -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na+ /K+ -ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics., (© 2017 Wiley Periodicals, Inc.)- Published
- 2017
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21. RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2 -deficient mice, a model for Rett syndrome.
- Author
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Pacheco NL, Heaven MR, Holt LM, Crossman DK, Boggio KJ, Shaffer SA, Flint DL, and Olsen ML
- Subjects
- Animals, Chromatography, High Pressure Liquid, Disease Models, Animal, Female, Genotype, Male, Methyl-CpG-Binding Protein 2 deficiency, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurons metabolism, Phenotype, Proteome analysis, RNA chemistry, RNA isolation & purification, Rett Syndrome pathology, Sequence Analysis, RNA, Tandem Mass Spectrometry, Transcriptome, Cerebral Cortex metabolism, Methyl-CpG-Binding Protein 2 genetics, Proteome metabolism, Proteomics, RNA metabolism, Rett Syndrome genetics
- Abstract
Background: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation., Methods: We therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates ( n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity® Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets., Results: Our results indicate these two "omics" data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2 -deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits., Conclusions: This comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT.
- Published
- 2017
- Full Text
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22. Novel Applications of Magnetic Cell Sorting to Analyze Cell-Type Specific Gene and Protein Expression in the Central Nervous System.
- Author
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Holt LM and Olsen ML
- Subjects
- Animals, Astrocytes metabolism, Biomarkers, Cerebral Cortex growth & development, Cerebral Cortex metabolism, Excitatory Amino Acid Transporter 2 analysis, Excitatory Amino Acid Transporter 2 immunology, Green Fluorescent Proteins, Mice, Mice, Inbred C57BL, Microglia metabolism, Neurons metabolism, RNA, Messenger genetics, Rats, Real-Time Polymerase Chain Reaction, Cerebral Cortex cytology, Gene Expression, Immunomagnetic Separation, Nerve Tissue Proteins analysis
- Abstract
The isolation and study of cell-specific populations in the central nervous system (CNS) has gained significant interest in the neuroscience community. The ability to examine cell-specific gene and protein expression patterns in healthy and pathological tissue is critical for our understanding of CNS function. Several techniques currently exist to isolate cell-specific populations, each having their own inherent advantages and shortcomings. Isolation of distinct cell populations using magnetic sorting is a technique which has been available for nearly 3 decades, although rarely used in adult whole CNS tissue homogenate. In the current study we demonstrate that distinct cell populations can be isolated in rodents from early postnatal development through adulthood. We found this technique to be amendable to customization using commercially available membrane-targeted antibodies, allowing for cell-specific isolation across development and animal species. This technique yields RNA which can be utilized for downstream applications-including quantitative PCR and RNA sequencing-at relatively low cost and without the need for specialized equipment or fluorescently labeled cells. Adding to its utility, we demonstrate that cells can be isolated largely intact, retaining their processes, enabling analysis of extrasomatic proteins. We propose that magnetic cell sorting will prove to be a highly useful technique for the examination of cell specific CNS populations.
- Published
- 2016
- Full Text
- View/download PDF
23. Field characterization of external grease abatement devices.
- Author
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Aziz TN, Holt LM, Keener KM, Groninger JW, and Ducoste JJ
- Subjects
- Fats analysis, Food Services, Hydrogen-Ion Concentration, Oils analysis, Oils isolation & purification, Temperature, Fats isolation & purification, Sewage analysis, Waste Disposal, Fluid instrumentation
- Abstract
This study characterized some of the physical and chemical features of large outside field grease abatement devices (GADs). 24-hour measurements of several food service establishments' (FSEs') influent GAD flowrates indicated highly intermittent conditions with hydraulic retention times (HRTs) that exceeded the common recommendation (30 minutes) by two to five times. Investigation into the chemical characteristics of GADs indicated highly variable influent and effluent fat, oil, and grease (FOG) concentrations. Low pH and dissolved oxygen values were measured throughout the GAD, indicating the likely occurrence of anaerobic microbial processes. Detailed spatial and temporal observations of the accumulation of FOG and food solids were also discussed. Though the FOG layer remained relatively constant for all GAD configurations investigated, results indicated that commonly-used GAD configurations with a straight submerged inlet tee or no-inlet tee configuration may result in the transport of food solids into the second compartment. The present research showed increased accumulation of food solids in the first compartment with a retro-fit flow distributive inlet. This retro-fit displays promise for potentially improving the separation characteristics of existing GADs.
- Published
- 2012
- Full Text
- View/download PDF
24. Properties influencing fat, oil, and grease deposit formation.
- Author
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Keener KM, Ducoste JJ, and Holt LM
- Subjects
- Water Purification, Fats chemistry, Sewage chemistry, Waste Disposal, Fluid
- Abstract
Fat, oil, and grease (FOG) deposits are the reported cause of 50 to 75% of sanitary sewer overflows in the United States, resulting in 1.8 X 10(6) m3 (500 mil. gal) of raw wastewater released into the environment annually. The objective of this research was to characterize the chemical and physical properties of FOG deposits. Twenty-three cities from around the United States contributed FOG samples for the study. The FOG deposits showed a wide range in yield strength (4 to 34 kPa), porosity (10 to 24%), and moisture content (10 to 60%), suggesting uncontrolled formation processes. A majority of these deposits display hard, sandstonelike texture, with distinct layering effects, suggesting a discontinuous formation process. The results found that 84% of FOG deposits contained high concentrations of saturated fatty acids and calcium, suggesting preferential accumulation.
- Published
- 2008
- Full Text
- View/download PDF
25. Development and characterisation of a line of bread wheat, Triticum aestivum, which lacks the short-arm satellite of chromosome 1B and the Gli-B1 locus.
- Author
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Payne PI, Holt LM, Hutchinson J, and Bennett MD
- Abstract
About 360 offspring of a tri-parental cross were screened by gel electrophoresis and unexpectedly one of them did not contain chromosome 1B ω-gliadins derived from either of the primary parents. A line disomic for the ω-gliadin null was developed from the surviving embryo half of the unique grain. Two dimensional electrophoresis revealed that all the storage protein genes at Gli-B1, coding for γ-gliadins, β-gliadins and low-molecular-weight subunits of glutenin as well as the ω-gliadin, were not expressed. The nuclei of dividing root-tip cells were shown by light microscopy to lack the normal short-arm satellites of chromosome 1B, indicating that the genes for the missing storage proteins had been lost through a terminal deletion. Using a radioactive ribosomal RNA probe, the deficient 1B chromosomes were shown to contain ribosomal RNA genes demonstrating that at least two-thirds of the short arm was still present. Examination of serial sections of chromosome 1B at metaphase by low-power electron microscopy showed that the point of scission of this chromosome was within the secondary constriction where the ribosomal RNA genes are located. The Gli-B1 locus must therefore be carried on the short-arm satellite. Transmission of the deficient chromosome from female gametes to progeny was normal (i.e., about 50%) but from pollen it was poor (8.8%). Recombination mapping indicated that the distance from the ribosomal RNA genes (Nor1) to Glu-B1 was 22 cM, equivalent to 13 cM from Nor1 to the centromere.
- Published
- 1984
- Full Text
- View/download PDF
26. Chromosomal location of genes coding for endosperm proteins of Hordeum chilense, determined by two-dimensional electrophoresis of wheat-H. chilense chromosome addition lines.
- Author
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Payne PI, Holt LM, Reader SM, and Miller TE
- Subjects
- Chromosome Mapping, Electrophoresis, Polyacrylamide Gel, Isoelectric Point, Molecular Weight, Edible Grain genetics, Hordeum genetics, Plant Proteins genetics, Triticum genetics
- Abstract
The proteins of Hordeum chilense grain were resolved into 25 major components by two-dimensional electrophoresis. Their solubilities in aqueous alcohol solutions were determined to distinguish prolamin storage proteins from metabolic and structural proteins. The prolamins were divided into two groups, based on the presence or absence of intermolecular disulfide bonds determined by gel-filtration chromatography. Using an incomplete set of Chinese Spring wheat-H. chilense disomic addition lines, the structural genes of 21 of the 26 most dominant seed proteins were assigned to chromosomes. The great majority of the prolamin genes, including those coding for a high molecular weight (HMW) prolamin subunit, was present on chromosome 1 Hch. However, a small number of prolamin genes also occurred on chromosomes 5 Hch and 7 Hch. A minor protein, probably belonging to the nonstorage group of proteins, is coded by genes on 5 Hch. Various ditelosomic addition lines and ditelosomic and disomic substitution lines for chromosome 7 Hch were also analyzed by electrophoresis. This technique revealed that the genes for three major prolamins occur on the beta arm of chromosome 7 Hch and that a gene for a minor protein, also thought to be a prolamin, occurs on the alpha arm. These results are discussed in relation to the evolution of prolamin genes in the Triticeae.
- Published
- 1987
- Full Text
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27. Genetic linkage between endosperm storage protein genes on each of the short arms of chromosomes 1A and 1B in wheat.
- Author
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Payne PI, Jackson EA, Holt LM, and Law CN
- Abstract
The storage proteins of the endosperm of wheat grain which are known to be controlled by genes on the short arms of the homoeologous group 1 chromosomes are (1) the ω-gliadins, (2) most of the γ-gliadins, (3) a few β-gliadins and (4) the major lowmolecular-weight subunits of glutenin. Several crosses were made between varieties or genetic lines which had contrasting allelic variants for some of these proteins and which were coded by genes on chromosomes 1A or 1B. The progeny were analysed by one or more of several electrophoretic procedures. The results of all the analyses are consistent with the hypothesis that chromosomes 1A and 1B each contain just one, complex locus, named Gli-A 1 and Gli-B 1 respectively, which contain the genes for the ω-, γ- and β-gliadins and the low-molecular-weight subunits of glutenin.
- Published
- 1984
- Full Text
- View/download PDF
28. Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin : Part 3. Telocentric mapping of the subunit genes on the long arms of the homoeologous group 1 chromosomes.
- Author
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Payne PI, Holt LM, Worland AJ, and Law CN
- Abstract
The genes controlling the synthesis of the high-molecular-weight subunits of glutenin on the long arms of chromosomes 1A and IB were mapped to the ω-gliadin genes on the short arms by analysing the progeny of three test crosses by sodium dodecyl sulphate, polyacrylamide-gel electrophoresis. Only very weak linkages were detected: the percentage recombination ranged from 39% to 47% and as the values did not significantly differ from each other, the data was pooled. A mean recombination of 43% was obtained and the map distance between glutenin and gliadin genes was calculated to be 66 cM. The analysis of three crosses involving telocentric lines revealed that the glutenin subunit genes on chromosomes 1A, IB and ID are tightly linked to the centromere, the mean map distance being 9.0 cM.
- Published
- 1982
- Full Text
- View/download PDF
29. Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin : Part 1: Allelic variation in subunits amongst varieties of wheat (Triticum aestivum).
- Author
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Payne PI, Holt LM, and Law CN
- Abstract
The high-molecular-weight (HMW) subunits of glutenin from about 185 varieties were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). About 20 different, major subunits were distinguished by this technique although each variety contained, with only a few exceptions, between 3 and 5 subunits. Further inter-varietal substitution lines to those already described (Payne et al. 1980) were analysed and the results indicate that all the HMW subunits are controlled by the homoeologous group 1 chromosomes. All hexaploid varieties studied except 'NapHal' contained two major subunits controlled by chromosome 1D. Their genes were shown to be tightly linked genetically for only four different types of banding patterns were observed. The nominal molecular weights determined after fractionation in 10% polyacrylamide gels were between 110,000 and 115,000 for the larger of the two subunits and between 82,000 and 84,000 for the smaller. One quarter of the varieties contained only one major HMW subunit controlled by chromosome 1B whereas the rest had two. The chromosome 1B subunits were the most varied and nine different banding patterns were detected. All the subunits had mobilities which were intermediate between those of the two chromosome 1D-controlled subunits. Only two types of HMW subunit controlled by chromosome 1A were detected in all the varieties examined; a single variety never contained both of these subunits and 40% of varieties contained neither. The chromosome 1A-controlled subunits had slightly slower mobilities in 10% gels than the largest HMW subunit controlled by chromosome 1D. About 100 single grains were analysed from each of five different crosses of the type (F1 of variety A × variety B) × variety C. The results indicate that the genes on chromosome 1B which control the synthesis of subunits 6, 7, 13, 14 and 17 are allelic, as are the genes of the chromosome 1A-controlled subunits, 1 and 2.
- Published
- 1981
- Full Text
- View/download PDF
30. Characterisation of high molecular weight gliadin and low-molecular-weight glutenin subunits of wheat endosperm by two-dimensional electrophoresis and the chromosomal localisation of their controlling genes.
- Author
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Jackson EA, Holt LM, and Payne PI
- Abstract
Gliadins, here defined as those proteins of defatted wheat endosperm which dissolve in 70% (v/v) ethanol at room temperature, were fractionated by gel filtration using Sephadex G-100. The protein which eluted with the void volume of the column, often described as high-molecular-weight (HMW) gliadin, was fractionated by the two different, two dimensional gel electrophoresis procedures of O'Farrell (1975) and O'Farrell et al. (1977). The next two fractions to elute from the gel column, ω-gliadin and α-, β-, γ-gliadin, were analysed similarly. The subunits of HMW gliadin and the classical (i.e. non-aggregated) gliadins map at distinctive positions on the electrophoregrams, the majority of the HMW gliadin subunits being more basic and having a slightly slower electrophoretic mobility than the α-, β-, γ-gliadins. These experiments demonstrate that those gliadins which aggregate to form HMW gliadin are distinct molecular entities and thus coded by different genes to those gliadins which do not aggregate. Glutenin, here prepared by a modification of the pH 6.4 precipitation procedure of Orth and Bushuk (1973), was also analysed by two-dimensional electrophoresis. The low-molecular-weight subunits were found to correspond exactly with the HMW gliadin subunits. Using the nullisomic-tetrasomic lines and the ditelocentric lines of 'Chinese Spring', the genes controlling the synthesis of all the major HMW gliadin subunits were shown to be located on the short arms of chromosomes 1A, 1B and 1D, as are the genes coding for the ω-gliadins and the majority of the γ-gliadins.
- Published
- 1983
- Full Text
- View/download PDF
31. Celiac disease--what is it?
- Author
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HOLT LM Jr
- Subjects
- Child, Humans, Infant, Celiac Disease, Diet
- Published
- 1955
- Full Text
- View/download PDF
32. Effects of oestrogen, progesterone and betamethasone on the guinea-pig uterus.
- Author
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Atkinson G, Holt LM, and Knifton A
- Subjects
- Acetylcholine pharmacology, Animals, Betamethasone administration & dosage, Calcium, Electric Stimulation, Estrus, Female, Guinea Pigs, In Vitro Techniques, Pregnancy, Betamethasone pharmacology, Estrogens pharmacology, Progesterone pharmacology, Uterus drug effects
- Published
- 1972
- Full Text
- View/download PDF
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