bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hyperspectral Imaging Approaches for Measuring Three‐Dimensional FRET
Authors: Annamdevula Naga S , Sweat Rachel , Britain Andrea , Rich Thomas C , Leavesley Silas J 
Published: November 2023

Förster Resonance energy transfer (FRET) imaging has become a standard method for assessing protein‐protein interactions as well as changes in the concentration of signaling molecules, such as cAMP. However, many FRET probes or pairs have low signal‐to‐noise ratios and limited dynamic range. Our previous work suggested that hyperspectral imaging and analysis allows quantitative measurement of FRET efficiency in 3D (x, y, and t) within single cell. Here, we present data demonstrating that hyperspectral imaging approaches allow 4D (x, y, z, and t) assessment of FRET efficiency in living cells. In these studies, we have utilized a FRET biosensor consisting of a cAMP binding domain from Epac sandwiched between donor and acceptor fluorophores, Turquoise and Venus. Binding of cAMP induces a conformational change that reduces FRET efficiency. The FRET probe was transfected into HEK293 or pulmonary microvascular endothelial cells. To further demonstrate the potential of 4D hyperspectral approaches to simultaneously assess multiple fluorescence signals in single cells, we co‐transfected the calcium probe RCaMP into cells. 4D hyperspectral images were acquired using a Nikon A1R confocal microscope as follows: Excitation was provided using 405 and 561 nm laser lines. Spectral emission was detected from 424 to 724 nm in increments of 10 nm using a 32‐wavelength spectral detector on a Nikon A1R confocal microscope. A 3D (x, y, z) hyperspectral image was acquired every 15 seconds for 15 minutes. Following one minute measurement of baseline signals, we treated the cells with either 50 μM forskolin (an adenylyl cyclase activator), 0.1 μM or 1 μM isoproterenol (a beta adrenergic agonist), 10 μM rolipram (a PDE4 inhibitor), 1 μM prostaglandin, and/or calcium modulating agents such as 0.1 or 1 μM thapsigargin. Spectral images were processed using custom MATLAB scripts. Briefly, spectral images were unmixed to individual endmembers including Turquoise, Venus, DRAQ5 (nuclear label), WGA‐TRITC (plasma membrane label), and RCaMP. 4D FRET images were calculated from unmixed images. Data were re‐sliced to visualize fluorescence signals in different orientations/projections through each cell. This overall approach allowed assessment of changes in FRET signals in 4D. We observed little or no gradients in the lateral direction with real time 2D (x, y, and t) imaging. However, with 3D imaging (x, y, and z), we found axial gradients (from apical to basolateral) in the cell. With 4D (x, y, z, and t) imaging, we observed that cAMP gradients move from apical to basolateral side over time. Our data suggest that 3D (x, y, and t) imaging approaches may provide only a limited perspective of cAMP gradients and that 4D (x, y, z and t) studies are required to study cAMP gradients and signaling mechanisms in a cell.Support or Funding InformationThis work is supported by P01HL066299, S10RR027535, S10OD020149, R01HL058506, and the Abraham Mitchell Cancer Research Fund.
Contribution of Oligomeric Forms of Tripartite Motif E3 Ubiquitin Ligase Proteins to Plasma Membrane Repair in Striated Muscle
Authors: Gushchina Liubov V. , Alloush Jenna , Choi Jin Hyuk , Bhattacharya Sayak , Beck Eric , Weisleder Noah 
Published: November 2023

Defects in the intrinsic plasma membrane repair function underlie the progression of many human diseases associated with membrane repair disruptions, such as ischemia/reperfusion damage and similar injuries to the cardiovascular system. Our previous experimental results showed that mitsugumin 53 (MG53), a novel muscle enriched protein from the tripartite motif (TRIM) family (known as TRIM72), is an essential component of the cell membrane repair machinery that protects injury to skeletal muscle and the myocardium. We recently determined that recombinant human MG53 (rhMG53) protein can be applied directly to cells or animal models to increase membrane repair in muscle cells, showing the potential of rhMG53 as a protein therapeutic agent. At the molecular level, MG53 can form dimers to allow movement of intracellular vesicles to patch membrane injury sites. Dimerization through their central coiled‐coil domain is a common property of TRIM proteins, which open the possibility that other TRIM proteins could form heterodimers with MG53. We screened for novel TRIM proteins that can increase membrane repair capacity and found that one such TRIM, TRIM27, can interact with MG53 to form heterodimers. The role of these homo‐ and heterodimers in the cardiac membrane repair is unclear and has to be elucidated in future studies. To study whether selected TRIM family proteins mediate membrane repair in the heart and to understand the molecular mechanism of this process, we generated several versions of human MG53 and TRIM27 cDNAs, which were optimized for expression in different host organisms for protein production, including E. coli and CHO cells. To create MG53 and TRIM27 and their homo‐ and heterodimers, we performed structural analysis of all TRIM family proteins and their separate domains that are available in the RCSB Protein Data Bank (PDB). Our analysis showed that PRY‐SPRY domains of protein are within several angstroms spatially. This allowed us to propose that the distance between domains can be covered by a linker to generate proteins without losing their activity. Thus, oligomeric forms of TRIM family proteins were created by fusing their human monomeric variants in anti‐parallel orientation (tail‐to‐tail). The therapeutic effects of TRIM proteins and their oligomeric forms on cardiac function and vulnerability to ischemia‐reperfusion injury will be further defined using in vitro and in vivo models.Support or Funding InformationThis work was supported by grant AR063084 from the National Institutes of Health and the Muscular Dystrophy Association (N.W.), Internal Muscle Health and Neuromuscular Disorders Program (L.G.) and the American Heart Association (AHA) program (14POST19990020, L.G.).
Dysregulation of ZnT2‐Mediated Zn Homeostasis Impairs Epithelial Polarity And Breast Differentiation
Authors: Lee Sooyeon , Kelleher Shannon L 
Published: November 2023

Epithelial polarity is essential for the architecture and function of the mammary gland. We previously found that the loss of the vesicular zinc (Zn) transporter ZnT2 in ZnT2‐null mice compromises polarity, reduces STAT5 activation and impairs mammary gland differentiation. Here, we aimed to determine how the loss of ZnT2 impairs polarity and mammary gland differentiation. We used ZnT2‐attenuated mammary epithelial cells (MECs) in vitro and hypothesized that the loss of ZnT2‐mediated Zn transport dysregulates (PTEN), an epithelial polarity factor that disrupts spatial orientation of polarity, which subsequently impairs prolactin‐mediated Jak2/STAT5 signaling. First, we found that without the ability to sequester Zn into vesicles via ZnT2, Zn accumulated in the cytoplasm and significantly reduced PTEN expression. Moreover, ZnT2‐attenuated MECs had reduced membrane expression of, Annexin A2, a protein involved in apical membrane transport, and disrupted localization of ZO‐1, a protein critical for tight junction assembly, indicating impaired spatial orientation. Consequently, ZnT2‐attenuated MECs failed to differentiate into 3D mammospheres with expanded alveolar lumen. Importantly, we showed that the loss polarity disrupted cell surface localization of the principal lactogenic hormone receptor, prolactin receptor, and reduced downstream Jak2/STAT5 signaling, collectively indicating that cytoplasmic Zn accumulation dysregulates epithelial polarity and mammary differentiation. Our recent studies in breastfeeding women identified several non‐synonymous genetic variants in ZnT2 that result in “loss‐of‐function”. To investigate effects of these variants (ZnT2K66N and ZnT2Q71H) on MEC polarity, we transfected MECs to express these ZnT2 variants and found that expression increased cytoplasmic Zn and reduced PTEN expression in MECs, implicating defects of ZnT2 function in breast differentiation in women. Taken together, these results establish that ZnT2‐mediated Zn homeostasis is critical for maintaining epithelial polarity and regulating prolactin signaling for proper mammary gland differentiation and this suggests that “loss‐of‐function” variants of ZnT2 may be associated with impaired breast differentiation and lactation insufficiency in women.Support or Funding InformationIntramural funds to SLK
Unbuckling the Buccal oscillator in the American Bullfrog
Authors: Baghdadwala Mufaddal I , Trask William M , Wilson Richard JA 
Published: November 2023

The American Bullfrog is a remarkable model organism to study rhythm generation underlying breathing. It exhibits a breathing pattern that can be divided into four distinct ventilatory phases: lung‐priming, lung‐powerstroke, buccal‐dilation, and buccal‐constriction. We recently established that lung‐priming and lung‐powerstroke are generated by two separate burst generating substrates in the brainstem—the Priming area and Powerstroke area—located in rhombomere 4–5. The neural origin of the two phases of buccal, on the other hand, remains elusive. Our previous experiments suggest that a Buccal area located in rhombomere 7 is responsible, at least in part, for generating the biphasic buccal rhythm; however, whether any additional burst generating substrates are involved is yet unknown.The objective of this study was to characterize areas in the frog brainstem that generate the biphasic buccal rhythm. We utilized the isolated frog brainstem preparation and recorded fictive respiratory motor output from the trigeminal and facial cranial nerves using extracellular suction electrodes. We progressively transected the brainstem in both directions (caudal‐to‐rostral, and rostral‐to‐caudal) to identify regions where transection eliminated one or both buccal burst phases.Our preliminary results suggest that both phases of the buccal rhythm are generated by substrates located at the same level as the previously identified Buccal area. Nevertheless, using our current methodology, we are unable to discern whether multiple substrates or a single substrate is generating the buccal rhythm. To address this question, we developed a ‘buccal slice’ preparation at the level of the Buccal area which, when exposed to 100nM AMPA, produces buccal‐like bursts. We continue to develop this preparation as it holds promise for further dissecting the properties of this system and exploring it at the cellular and molecular levels.Our analysis suggests that the Buccal area is an evolutionary homolog of the mammalian preBötzinger complex. Both are located in rhombomere 7, are resilient oscillators, being staunchly insensitive to CO2, and co‐opted for sniffing. We anticipate that having two comparable preparations, i.e. ‘buccal slice’ and ‘preBötC slice’, will allow for more direct comparisons between these oscillators, further testing their homology and thus take us closer towards understanding the evolution of breathing.Support or Funding InformationNational Science and Engineering Council of Canada (NSERC) and Alberta Innovates Health Solutions (AIHS) provided funding for this study.
Disturbances in central respiratory rhythm generation may contribute to breathing disturbances in prematurely born mice
Authors: Ramirez Sanja Carina , Koschnitzky Jenna E. , Baertsch Nathan Andrew , Anderson Tatiana M , Smith Charles V , Ramirez Jan‐Marino 
Published: November 2023

Nearly one million premature infants die annually, due to respiratory distress. While immaturity of the lungs is a major contributor to morbidity and mortality, much less is known about the potential contribution of disturbances in central neuronal networks. Here we tested the hypothesis that central neuronal mechanisms contribute to neonatal apneas and breathing disturbances occurring within the first few hours of life. We used an animal model of prematurity, in which we administered lipopolysaccharide (LPS) into pregnant dams eliciting a maternal inflammatory response that resulted in mouse pups being born prematurely. This experimental model parallels the human condition because a large fraction of human premature births are associated with Chorioamnionitis and other forms of maternal infections. Administration of 5 micrograms of LPS to C57BL/6 mice at G17.5 resulted in delivery of pups at G18.5, about half of which were viable for at least 2 h post delivery. In a flow, whole‐body plethysmograph, premature mice from LPS‐treated dams showed abnormally large breathing patterns occurring at low frequency, which in some pups transitioned to more mature eupneic respiratory activity. Transverse, rhythmically active slices containing the pre‐Bötzinger complex, the putative respiratory rhythm generator, obtained within 30 minutes after birth from LPS pups, showed large amplitude bursts occurring at a slow and irregular frequency. Conversely term and C‐sectioned pups exhibited respiratory rhythmic activity with a smaller and more regular frequency and amplitude. Bath applied caffeine effectively stimulated and regularized burst activity. The in vitro preparations will enable us to compare the cellular mechanisms that underlie the abnormally large breathing patterns seen primarily in premature mouse pups, with the respiratory patterns generated in term and c‐sectioned pups. Understanding the central mechanisms that underlie disordered breathing in prematurity may lead to novel treatment strategies for neonatal apneas and breathing failures in human infants.Support or Funding InformationInvestigators Inter‐center seed funds
Mapping projections of excitatory and inhibitory preBötzinger Complex neurons
Authors: Yang Cindy F , Feldman Jack L 
Published: November 2023

Understanding the neural control of breathing requires a detailed neuroanatomical and functional map of the breathing central pattern generator (CPG). The logical entry point in constructing such a map is the “engine” of respiratory rhythm, the preBötzinger Complex (preBötC), a bilateral microcircuit of ~3000 interconnected neurons in the ventrolateral medulla. The preBötC generates respiratory rhythm in rodents (and presumably all mammals, including humans); rhythmic activity in the preBötC is required to produce inspiratory movements. To delineate the breathing CPG circuit in mice, we used a viral strategy to identify projections from molecularly‐defined populations of preBötC neurons. Using stereotaxic delivery of Cre‐dependent AAV expressing EGFP (AAV‐flex‐EGFP) into the preBötC of somatostatin‐Cre or GlyT2‐Cre mice, we located efferent projections emanating from two broad, functionally distinct classes of neurons: excitatory neurons that express somatostatin (SST) and inhibitory neurons that express the glycine transporter GlyT2. Critically, we optimized the viral delivery method to restrict viral transfections within the preBötC, while sufficiently labeling neurons to visualize long distance projections, enabling reliable identification of preBötC projections. Glutamatergic SST+ preBötC neurons project to brainstem nuclei implicated in the control of breathing including the contralateral preBötC, parahypo‐glossal/nucleus of the solitary tract, ventral respiratory group, and parabrachial/Kölliker‐Fuse nuclei, similar to efferent projections in rats; these neurons also had suprapontine projections to multiple hypothalamic nuclei, periaquaductal grey, as well as sparse projections to amygdala, suggesting that the preBötC relays respiratory information to higher brain regions that may modulate physiological or behavioral processes. Strikingly, glycinergic preBötC neurons send projections in parallel with glutamatergic SST+ neurons to the same brainstem and suprapontine targets.Support or Funding InformationA.P. Giannini Foundation, NIH T32 NS007101, NIH F32 HL126522‐01, NIH RO1 HL070029‐13
MCT Expression in the Ventromedial Hypothalamus is Not Increased by Recurrent Hypoglycemia
Authors: Oberlin Douglas J , Fecych Mariel , Murray Coleman , Morrison Ron , Wideman Laurie , Beverly Lee 
Published: November 2023

Recurrent hypoglycemia (RH) has been shown to lead to deficits in the counter‐regulatory response to subsequent hypoglycemia; however, the exact mechanisms of this effect are unknown. A proposed explanation has included increased lactate shuttling from astrocytes to neurons, which would blunt the change in glucose availability in the ventromedial hypothalamus (VMH). The objective of the current study was to measure changes in the lactate shuttling monocarboxylate transporter (MCT) proteins in the VMH after RH or control. Male Sprague‐Dawley rats (~250 g) received a subcutaneous injection of 0.9% saline or insulin (3–5 U/kg) on 3 consecutive days. Blood glucose was measured 30 minutes post‐injection via a pedal vein stick and glucometer. Animals were sacrificed 24 hr after the last episode of hypoglycemia, and the VMH was rapidly extracted, homogenized, and frozen at −80 c. MCT1, MCT2, MCT4, and GAPDH (as a loading control) were measured by western blot analysis. Quality One software was used to determine band density and the ratio of each MCT:GAPDH was calculated for each MCT in the VMH. MCT differences between groups were assessed using ANOVA (SAS 9.3).ResultsThere was a significant decrease in average 30 minute post‐injection glucose in the RH group (f=402.237, p<0.001). The results showed no significant differences between the RH and control group for MCT proteins (λ=0.867, p=0.404; MCT1 F=2.87, p=0.104; MCT2 F=2.14, p=0.157; MCT4 F=0.07, p=0.796). In conclusion, these findings suggest that RH does not increase MCT expression in the VMH and that increased lactate shuttling is not likely to mediate the impaired counter‐regulatory response after recurrent hypoglycemia.Support or Funding InformationSupported by NIH Grant DK082609
The 3D organization of the Mammalian Genome
Authors: Ren Bing 
Published: November 2023

The 3D genome organization plays an essential role in orchestrating the many essential nuclear processes including gene regulation. Great strides have been made recently to characterize the 3D chromatin architecture in mammalian cells, with the development of both technologies for genome‐wide study of chromatin interactions at increasing precision and concepts such as “chromosome territories”, “topologically associating domains” and “chromatin loops”. I will discuss how these new concepts are transforming the study of gene regulatory mechanisms in mammalian cells, by giving specific examples that illustrate the role of 3D chromatin organization in developing revolutionary tools in sequencing diploid genomes, understanding molecular basis of genetic disorders caused by genome structural variations, and predicting target genes of enhancers.Support or Funding InformationLICR, U54 HG006997, U54 DK107977, U01 CA200147
Complex Multi‐Enhancer Contacts Captured By Genome Architecture Mapping, A Novel Ligation‐Free Approach
Authors: Pombo Ana , Beagrie Robert A , Scialdone Antonio , Schueler Markus , Chotalia Mita , Xie Sheila Q , Kraemer Dorothee , de Santiago Ines , Fraser James , Dostie Josee , Game Laurence , Dillon Niall , Edwards Paul AW , Nicodemi Mario 
Published: November 2023

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key elements of transcriptional control and their disruption causes disease. Technologies based on chromosome conformation capture (3C) have profoundly expanded our understanding of the role of genome architecture in gene regulation. However, 3C‐based techniques have important limitations, many of which arise from their reliance on digestion and ligation of the interacting DNA segments. We present a new genome‐wide method, Genome Architecture Mapping (GAM) for measuring three‐dimensional chromatin topology without ligation.We use this new method to generate a genome‐wide dataset of chromatin interactions in mouse ES cells, and compare to published Hi‐C data. GAM identifies specific chromatin contacts enriched for interactions between active genes and enhancers across very large genomic distances. GAM also reveals abundant three‐way contacts genome‐wide, especially between the enhancers most highly occupied by pluripotency transcription factors and highly transcribed genomic regions. These contacts are most prominent for sequences further away from the nuclear lamina. Our results shed light on a previously inaccessible complexity in genome architecture and a major role for gene‐expression specific contacts in organizing genome architecture of mammalian nuclei.Support or Funding InformationThe work was supported by the Medical Research Council, UK (AP, RAB, MC, SQX, IdS, LG, ND), by the MRC‐Technology (AP, MC), by the Helmholtz Foundation (AP, MS, MB, DK), by Breast Cancer Campaign (PAWE) and by Cancer Research UK (PAWE). Work by JD and JF was supported by grants from the Canadian Institutes of Health Research (CIHR) [MOP‐86716, CAP‐120350].
Chronic Binge Alcohol Exacerbates Apoptotic Signaling in Simian Immunodeficiency Virus‐infected Macaques
Authors: Maxi John K. , Bagby Gregory J. , Nelson Steve , Winsauer Peter J. , Molina Patricia E. 
Published: November 2023

HIV‐associated neurocognitive disorder (HAND), characterized by altered pre‐frontal cortex (PFC) mediated behaviors, affects ≈50% of persons living with HIV/AIDS. Alcohol use accelerates the development of HAND. Our studies have shown that chronic binge alcohol administration (CBA) unmasks behavioral deficits in simian immunodeficiency virus (SIV)‐infected macaques. However, the mechanisms underlying these alcohol‐induced behavioral deficits remain unknown. Alcohol administration and HIV/SIV neuronal damage result, at least in part, from excitotoxic NMDA receptor signaling stimulating mitochondrial pro‐apoptotic mechanisms. Recently, it has been recognized that loss of pro‐survival factors, specifically Brain‐Derived Neurotrophic Factor (BDNF), also contributes to alcohol and HIV/SIV‐induced neuronal damage. Thus, we hypothesized that CBA administration to SIV‐infected macaques would result in greater excitotoxic signaling and impaired BDNF‐mediated cell signaling in the PFC, suggesting a potential mechanism underlying exacerbation of HAND. Adult rhesus macaques were fitted with intragastric catheters for the infusion of alcohol (n=3, CBA/SIV, 13–14 g/kg/week) or sucrose (n=3, SUC/SIV), beginning three months prior to inoculation with SIV, and continued for the duration of the study. Animals were euthanized upon reaching end‐stage disease (13 – 23 months post SIV) and PFC tissue was isolated for mRNA and protein analysis by qPCR and Western blot, respectively. PFC tissue from non SIV‐infected macaques (SIV‐, n=3–5) was used to normalize mRNA and protein expression. PFC of CBA/SIV macaques showed increased NMDA receptor subunits NR1 and NR2B relative to dendritic marker MAP2 (1.5‐fold increase), and increased Bax:Bcl‐2 ratio (10‐fold increase), suggesting enhanced excitotoxic apoptotic signaling. Additionally, we found decreased expression of BDNF mRNA (2‐fold reduction); activated BDNF receptor (pTrkB) (15‐fold lower); and pAKT levels (3‐fold reduction) in CBA/SIV animals compared to that of SUC/SIV animals. These findings indicated that CBA exacerbates apoptotic signaling in the PFC of SIV‐infected macaques. We predict these findings implicate increased PFC neuronal apoptosis as a likely mechanism underlying accentuated neurocognitive impairment in CBA/SIV macaques.Support or Funding InformationP60AA09803, T32AA07577, F31AA024365