bluuebunny/crossref_metadata_2025_split huggingface dataset:
Repeated hypoglycemia blunts the responsiveness of glucose-inhibited GHRH neurons by remodeling neural inputs and disrupting mitochondrial structure and function
Authors: Bayne M , Alvarsson A , Devarakonda K , Li R , Jimenez-Gonzalez M , Conner K. , Varghese M , Serasinghe M N , Chipuk J E , Hof P R , Stanley S A 
Published: October 2019

AbstractHypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counter-regulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia unawareness. The mechanisms leading to these blunted effects remain incompletely understood. Here, we identify, with in situ hybridization, immunohistochemistry and the tissue clearing capability of iDisco, that GHRH neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivationin vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule, mdivi-1, we preserved hypoglycemia sensitivityin vitroandin vivo. Our findings present possible mechanisms for the blunting of the CRR, broaden significantly our understanding of the structure of GHRH neurons and for the fist time, propose that mitochondrial dynamics play an important role in hypoglycemia unawareness. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent hypoglycemia unawareness in diabetic patients.
Functional and structural properties of highly responsive somatosensory neurons in mouse barrel cortex
Authors: Barz C , Garderes PM , Ganea D , Reischauer S , Feldmeyer D , Haiss F 
Published: October 2019

SummarySparse population activity is a hallmark of supra-granular sensory neurons in neocortex. The mechanisms underlying sparseness are not well understood because a direct link between the neurons activated in vivo and their cellular properties investigated in vitro has been missing. We used two-photon calcium imaging to identify a subset of neurons in layer L2/3 (L2/3) of mouse primary somatosensory cortex that are highly active following principal whisker vibrotactile stimulation. These high responders were then tagged using photoconvertible green fluorescent protein for subsequent targeting in the brain slice using intracellular patch-clamp recordings and biocytin staining. This approach allowed us to investigate the structural and functional properties of high responders that distinguish them from less active control cells. Compared to less responsive L2/3 neurons, high responders displayed increased levels of stimulus-evoked and spontaneous activity, elevated noise and spontaneous pair-wise correlations, and stronger coupling to the population response. Intrinsic excitability was reduced in high responders, while other electrophysiological and morphological parameters were unchanged. Thus, the choice of which neurons participate in stimulus encoding may largely be determined by network connectivity rather than by cellular structure and function.
Encapsulation boosts islet-cell signature in differentiating human induced pluripotent stem cells via integrin signalling
Authors: Vethe Heidrun , Legøy Thomas Aga , Abadpour Shadab , Strand Berit L. , Scholz Hanne , Paulo Joao A. , Ræder Helge , Ghila Luiza , Chera Simona 
Published: October 2019

AbstractCell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalling cascades promoting differentiation. These analyses provide a molecular framework for understanding the confinement effects on hiPSCs differentiation while confirming its importance for this process.
Body weight at young adulthood and association with epigenetic aging and lifespan in the BXD murine family
Authors: Sandoval-Sierra Jose Vladimir , Helbing Alexandra H. B. , Williams Evan G. , Ashbrook David G. , Roy Suheeta , Williams Robert W. , Mozhui Khyobeni 
Published: October 2019

SummaryDNA methylation (DNAm) is shaped by genetic and environmental factors and modulated by aging. Here, we examine interrelations between epigenetic aging, body weight (BW), and lifespan in 12 inbred mouse strains from the BXD panel that exhibit over two-fold variation in longevity. Genome-wide DNAm was assayed in 70 liver specimens from mice ranging in age from 6 to 25 months that were maintained on normal chow or high fat diet (HFD). We defined subsets of CpG regions associated with age, BW at young adulthood, and strain-by-diet dependent life expectancy. The age associated differentially methylated CpG regions (age-DMRs) featured distinct genomic characteristics, with DNAm gains over time occurring in sites with high CpG density and low average methylation. CpG regions associated with BW were enriched in introns and generally showed lower methylation in mice with higher BW, and inversely correlated with gene expression such that mRNA was higher in mice with higher BW. Lifespan-associated regions featured no distinct genomic characteristics but were linked to genes involved in lifespan regulation, including the telomerase reverse transcriptase gene,Tert, which showed lower methylation and higher gene expression in long-lived strains. An epigenetic clock defined from the age-DMRs conveyed accelerated aging in mice belonging to strains with shorter lifespans. Both higher BW at young adulthood and HFD were associated with accelerated epigenetic aging. Our results highlight the age-accelerating effect of heavier body weight. Furthermore, the study demonstrates that the measure of epigenetic aging derived from age-DMRs can predict strain and diet-induced differences in lifespan.
circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation
Authors: Hollensen Anne Kruse , Thomsen Henriette Sylvain , Lloret-Llinares Marta , Kamstrup Andreas Bjerregaard , Jensen Jacob Malthe , Luckmann Majbritt , Birkmose Nanna , Palmfeldt Johan , Jensen Torben Heick , Hansen Thomas Birkballe , Damgaard Christian Kroun 
Published: October 2019

AbstractCircular RNAs are important for many cellular processes but their mechanisms of action remain poorly understood. Here, we map circRNA inventories of mouse embryonic stem cells, neuronal progenitor cells and differentiated neurons and identify hundreds of highly expressed circRNAs. By screening several candidate circRNAs for a potential function in neuronal differentiation, we find that circZNF827 represses expression of key neuronal markers, suggesting that this molecule negatively regulates neuronal differentiation. Among 760 tested genes linked to known neuronal pathways, knockdown of circZNF827 deregulates expression of numerous genes including nerve growth factor receptor (NGFR), which becomes transcriptionally upregulated to enhance NGF signalling. We identify a circZNF827-nucleated transcription-repressive complex containing hnRNP-K/L proteins and show that knockdown of these factors strongly augments NGFR regulation. Finally, we show that ZNF827 protein is part of the mRNP complex, suggesting a functional co-evolution of a circRNA and the protein encoded by its linear pre-mRNA host.
Alcohol induces mitochondrial fragmentation and stress responses to maintain normal muscle function in Caenorhabditis elegans
Authors: Oh Kelly H. , Kim Hongkyun 
Published: October 2019

AbstractsChronic excessive ethanol consumption produces distinct toxic and adverse effects on different tissues. In skeletal muscle ethanol causes alcoholic myopathy characterized by myofiber atrophy and loss of muscle strength. Alcoholic myopathy is more prevalent than all inherited muscle diseases combined. Current evidence indicates that ethanol directly impairs muscle organization and function. However, the underlying mechanism by which ethanol causes its toxicity to muscle is poorly understood. Here, we show that the nematode C. elegans recapitulates key aspects of alcoholic myopathy when exposed to ethanol. As in mammals, ethanol exposure impairs muscle strength and organization and induces the expression of protective genes, including oxidative stress response. In addition, ethanol exposure causes a fragmentation of mitochondrial networks aligned with myofibril lattices. This ethanol-induced mitochondrial fragmentation is dependent on mitochondrial fission factor DRP-1 (dynamin-like protein 1), and its receptor proteins on the mitochondrial outer membrane. Our data indicate that this fragmentation contributes to activation of mitochondrial unfolded protein response (UPR). We also found that robust perpetual mitochondrial UPR activation effectively counters muscle weakness caused by ethanol exposure. Our results strongly suggest that modulation of mitochondrial stress responses provides a mechanism to ameliorate alcohol toxicity and damage to muscle.SignificanceChronic alcohol abuse causes the damage and toxicity to peripheral tissues, including muscle. Alcohol perturbs the structure and function of striated skeletal and cardiac muscles. These toxic effects of alcohol on striated muscles negatively impact morbidity and mortality to alcohol misusers. Here, we demonstrate that the nematode C. elegans also exhibits key features of alcoholic myopathy when exposed to ethanol. Ethanol exposure impairs muscle organization and strength, and induces the expression of genes that cope with alcohol toxicity. Particularly, we find that ethanol toxicity is centered on mitochondria, the power plants of the cell. As an adaptive protective response to mitochondrial dysfunction, ethanol-exposed cells induce global transcriptional reprogramming to restore normal mitochondrial function. Upregulation of this transcriptional reprogramming in C. elegans effectively blocks ethanol-induced muscle weakness, a key feature of alcoholic myopathy. Thus, the modulation of mitochondrial stress responses is a potentially promising therapeutic strategy to ameliorate alcohol toxicity to muscle.
Microglial depletion with CSF1R inhibitor during chronic phase of experimental traumatic brain injury reduces neurodegeneration and neurological deficits
Authors: Henry Rebecca J. , Ritzel Rodney M. , Barrett James P. , Doran Sarah J. , Jiao Yun , Leach Jennie B. , Szeto Gregory L. , Stoica Bogdan A. , Faden Alan I. , Loane David J. 
Published: October 2019

AbstractChronic neuroinflammation with sustained microglial activation occurs following moderate-to-severe traumatic brain injury (TBI) and is believed to contribute to subsequent neurodegeneration and neurological deficits. Microglia, the primary innate immune cells in brain, are dependent on colony stimulating factor 1 receptor (CSF1R) signaling for their survival. In this translational study, we examined the effects of delayed depletion and subsequent repopulation of microglia on chronic neurodegeneration and functional recovery up to three months posttrauma. A CSF1R inhibitor, PLX5622, was administered to injured adult male C57Bl/6 mice at one month after controlled cortical impact to remove chronically activated microglia, and the inihibitor was withdrawn 1 week later to allow microglial repopulation. Following TBI, the repopulated microglia displayed a ramified morphology, similar to that of sham control uninjured animals, whereas microglia in untreated injured animals showed the typical chronic posttraumatic hypertrophic morphology. PLX5622 treatment limited TBI-associated neuropathological changes at 3 months posttrauma; these included a smaller cortical lesion, reduced neuronal cell death in the injured cortex and ipsilateral hippocampus, and decreased NOX2-dependent reactive microgliosis. Furthermore, delayed depletion of microglia led to widespread changes in the cortical transcriptome, including alterations in gene pathways involved in neuroinflammation, oxidative stress, and neuroplasticity. PLX5622 treated animals showed significantly improved neurological recovery using a variety of complementary neurobehavioral evaluations. These included beam walk and rotorod tests for sensori-motor function, as well as Y-maze, novel object recognition, and Morris water maze tests to evaluate cognitive function. Together, our findings show that chronic phase removal of neurotoxic microglia using CSF1R inhibitors after experimental TBI can markedly reduce chronic neuroinflammation and neurodegeneration, as well as related long-term motor and cognitive deficits. Thus, CSF1R inhibition may provide a clinically feasible approach to limit posttraumatic neurodegeneration and neurological dysfunction following head injury.
A Petascale Automated Imaging Pipeline for Mapping Neuronal Circuits with High-throughput Transmission Electron Microscopy
Authors: Yin Wenjing , Brittain Derrick , Borseth Jay , Scott Marie E. , Williams Derric , Perkins Jed , Own Chris , Murfitt Matt , Torres Russel M. , Kapner Daniel , Bleckert Adam , Castelli Daniel , Reid David , Lee Wei-Chung Allen , Graham Brett J. , Takeno Marc , Bumbarger Dan J. , Farrell Colin , Reid R. Clay , da Costa Nuno Macarico 
Published: October 2019

ABSTRACTSerial-section electron microscopy is the method of choice for studying cellular structure and network connectivity in the brain. We have built a pipeline of parallel imaging using transmission electron automated microscopes (piTEAM) that scales this technology and enables the acquisition of petascale datasets containing local cortical microcircuits. The distributed platform is composed of multiple transmission electron microscopes that image, in parallel, different sections from the same block of tissue, all under control of a custom acquisition software (pyTEM) that implements 24/7 continuous autonomous imaging. The suitability of this architecture for large scale electron microscopy imaging was demonstrated by acquiring a volume of more than 1 mm3 of mouse neocortex spanning four different visual areas. Over 26,500 ultrathin tissue sections were imaged, yielding a dataset of more than 2 petabytes. Our current burst imaging rate is 500 Mpixel/s (image capture only) per microscope and net imaging rate is 100 Mpixel/s (including stage movement, image capture, quality control, and post processing). This brings the combined burst acquisition rate of the pipeline to 3 Gpixel/s and the net rate to 600 Mpixel/s with six microscopes running acquisition in parallel, which allowed imaging a cubic millimeter of mouse visual cortex at synaptic resolution in less than 6 months.
Sensitive detection of protein ubiquitylation using a protein-fragment complementation assay
Authors: Boulch Marie Le , Brossard Audrey , Dez Gaëlle Le , Rabut Gwenaël 
Published: October 2019

ABSTRACTUbiquitylation is a reversible post-translational protein modification that regulates a multitude of cellular processes. Detection of ubiquitylated proteins is often challenging, because of their low abundance. Here, we present NUbiCA, a sensitive protein-fragment complementation assay to facilitate the monitoring of ubiquitylation events in cultured cells and model organisms. Using yeast as a model system, we demonstrate that NUbiCA enables to accurately monitor mono- and poly-ubiquitylation of proteins expressed at endogenous levels. We also show that it can be applied to decipher ubiquitin chain linkages. We used NUbiCA to investigate the ubiquitylation of the low abundance centromeric histone Cse4, and found that it is ubiquitylated during S-phase. Finally, we assembled a genome wide collection of yeast strains ready to investigate the ubiquitylation of proteins with this new assay. This resource will facilitate the analysis of local or transient ubiquitylation events that are difficult to detect with current methods.Summary statementWe describe a sensitive protein-fragment complementation assay to facilitate the monitoring of ubiquitylation events that take place in cultured cells or model organisms.
Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination
Authors: Hirokawa Soichi , Chure Griffin , Belliveau Nathan M. , Lovely Geoffrey A. , Anaya Michael , Schatz David G. , Baltimore David , Phillips Rob 
Published: October 2019

Developing lymphocytes in the immune system of jawed vertebrates assemble antigen-receptor genes by undergoing large-scale reorganization of spatially separated V, D, and J gene segments through a process known as V(D)J recombination. The RAG protein initiates this process by binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to quantify the formation, stability, and cleavage of the RAG-12RSS-23RSS paired complex (PC) for numerous synthetic and endogenous 12RSSs. We thoroughly investigate the sequence space around a RSS by making 40 different single-bp changes and characterizing the reaction dynamics. We reveal that single-bp changes affect RAG function based on their position: loss of cleavage function (first three positions of the heptamer); reduced propensity for forming the PC (the nonamer and last four bp of the heptamer); or variable effects on PC formation (spacer). We find that the rare usage of some endogenous gene segments can be mapped directly to their adjacent 12RSSs to which RAG binds weakly. The 12RSS, however, cannot explain the high-frequency usage of other gene segments. Finally, we find that RSS nicking, while not required for PC formation, substantially stabilizes the PC. Our findings provide detailed insights into the contribution of individual RSS positions to steps of the RAG-RSS re-action that previously have been difficult to assess quantitatively.SummaryV(D)J recombination is a genomic cut-and-paste process for generating diverse antigen-receptor repertoires. The RAG enzyme brings separate gene segments together by binding the neighboring sequences called RSSs, forming a paired complex (PC) before cutting the DNA. There are limited quantitative studies of the sequence-dependent dynamics of the crucial inter-mediate steps of PC formation and cleavage. Here, we quantify individual RAG-DNA dynamics for various RSSs. While RSSs of frequently-used segments do not comparatively enhance PC formation or cleavage, the rare use of some segments can be explained by their neighboring RSSs crippling PC formation and/or cleavage. Furthermore, PC lifetimes reveal DNA-nicking is not required for forming the PC, but PCs with nicks are more stable.
Transmembrane helix 6b links proton- and metal-release pathways to drive conformational change in an Nramp transition metal transporter
Authors: Bozzi Aaron T. , McCabe Anne L. , Barnett Benjamin C. , Gaudet Rachelle 
Published: October 2019

ABSTRACTThe natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton co-transporters found in organisms from bacteria to humans. Recent structures ofDeinococcus radiodurans(Dra)Nramp in multiple conformations revealed the intramolecular rearrangements required for alternating access. Here we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp—one lining the wide intracellular vestibule for metal release, the other forming a narrow proton-transport pathway—are essential for metal transport. We further show that mutagenic or post-translational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impedes normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, H232 and H237. Different mutagenic perturbations for H232, just below the metal-binding site along the proton-exit route, differentially affect DraNramp’s conformational state, suggesting H232 serves as a pivot point for conformational change. In contrast, any tested replacement for H237, lining the metal-exit route, locks the transporter in a transport-inactive outward-closed state. We conclude that these two histidines, and TM6b more broadly, help trigger the bulk rearrangement to the inward-open state upon metal binding and facilitate the return of the empty transporter to an outward-open state upon metal release.
Field decomposition of Bt-506 corn leaf and its effect on Collembola in the black soil region of Northeast China
Authors: Wang Baifeng , Wu Fengci , Yin Junqi , Zhang Ling , Song Xinyuan 
Published: October 2019

AbstractThe litters of Bt corn would go into the soil through straw returning and field ploughing after cultivation. To clarify whether the leaf litter decomposition rate and the non-target soil Collembola were influenced by the Bt protein or other litter properties in leaf litters of Bt corn in Northeast China, leaf litterbags of Bt-506, its near isoline Zheng 58 and a local type Zhengdan 958 were used in the field in Northeast China. The leaf decomposition rate, the leaf properties and the collembolan community in litterbags were investigated later. After seven months, only 43.5 ng/g Bt protein in Bt-506 leaf litter was left. All the investigated indices were not significantly different between Bt-506 and its near isoline Zheng 58. But when compared with local type Zhengdan 958, Bt-506 and its near isoline Zheng 58 contained lower non-structural carbohydrate content but higher total nitrogen content, and had lower litter decomposition rate and less abundance of Collembola. Collembolan abundance and litter decomposition rate were both significantly correlated with the non-structural carbohydrate and total nitrogen contents of the leaf litters. Field study results revealed Bt protein did not affect the leaf litter decomposition rate and the collembolan community in leaf litterbags in short term. The significant differences of these investigated indices among corn types were caused by the different non-structural carbohydrate and total nitrogen contents in leaf litter.