bluuebunny/crossref_metadata_2025_split huggingface dataset:
ROCK1 regulates insulin secretion from β-cells
Authors: Sung Byung-Jun , Lim Sung-Bin , Kim Jae Hyeon , Yang Won Mo , Kulkarni Rohit N. , Kim Young-Bum , Lee Moon-Kyu 
Published: June 2021

ABSTRACTObjectiveThe endocrine pancreatic β-cells play a pivotal role in the maintenance of whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate β-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homoeostasis.MethodsMice lacking ROCK1 in pancreatic β-cells (RIP-Cre; ROCK1loxP/loxP, β-ROCK1-/-) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. Insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from β-ROCK1-/-mice or β-cell lines with knockdown of ROCK1 were also evaluated. Proximity ligation assay was performed to determine the physical interactions between PK and ROCK1.ResultsMice with a deficiency of ROCK1 in pancreatic β-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, β-ROCK1-/-mice displayed progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS was markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium levels, ATP levels, and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown β-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that ROCK1 binding to PK is greatly enhanced by glucose stimulation in β-cells.ConclusionsOur findings demonstrate that β-cell ROCK1 is essential for glucose-stimulated insulin secretion and maintenance of glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.
CaProDH2-mediated modulation of proline metabolism confers tolerance to Ascochyta in chickpea under drought
Authors: Patil Mahesh , Pandey Prachi , Irrulappan Vadivelmurugan , Singh Anuradha , Verma Praveen , Ranjan Ashish , Senthil-Kumar Muthappa 
Published: June 2021

AbstractDrought and leaf blight caused by the fungus Ascochyta rabiei often co-occur in chickpea (Cicer arietinum)-producing areas. While the responses of chickpea to either drought or A. rabiei infection have been extensively studied, their combined effect on plant defense mechanisms is unknown. Fine modulation of stress-induced signaling pathways under combined stress is an important stress adaptation mechanism that warrants a better understanding. Here we show that drought facilitates resistance against A. rabiei infection in chickpea. The analysis of proline levels and gene expression profiling of its biosynthetic pathway under combined drought and A. rabiei infection revealed the gene encoding proline dehydrogenase (CaProDH2) as a strong candidate conferring resistance to A. rabiei infection. Transcript levels of CaProDH2, pyrroline-5-carboxylate (P5C) quantification, and measurement of mitochondrial reactive oxygen species (ROS) production showed that fine modulation of the proline–P5C cycle determines the observed resistance. In addition, CaProDH2-silenced plants lost basal resistance to A. rabiei infection induced by drought, while overexpression of the gene conferred higher resistance to the fungus. We suggest that the drought-induced accumulation of proline in the cytosol helps maintain cell turgor and raises mitochondrial P5C contents by a CaProDH2-mediated step, which results in ROS production that boosts plant defense responses and confers resistance to A. rabiei infection. Our findings indicate that manipulating the proline–P5C pathway may be a possible strategy for improving stress tolerance in plants suffering from combined drought and A. rabiei infection.
Multiplexed single-molecule analysis of human telomerase synthesizing DNA
Authors: Hentschel Jendrik , Choi Junhong , Bagshaw Clive R. , Lapointe Christopher P. , Wang Jinfan , Jansson Linnea I. , Badstuebner Mareike , Puglisi Joseph D. , Stone Michael D. 
Published: June 2021

AbstractGenomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we developed a real-time single-molecule RNA sequencing approach that visualizes telomerase catalysis and structural dynamics at single-nucleotide resolution using FRET and zero-mode waveguides. The method permits direct detection of dynamic steps and structural states throughout the telomerase catalytic cycle and can be generalized to other nucleic acid polymerase systems.
Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing
Authors: Brown David , Altermatt Michael , Dobreva Tatyana , Chen Sisi , Wang Alexander , Thomson Matt , Gradinaru Viviana 
Published: June 2021

AbstractEngineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at unprecedented resolution. Using our platform, we have corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Our transcriptomic analysis revealed that this neuronal bias is mainly due to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. Such ability to parallelize the characterization of AAV tropism and simultaneously measure the transcriptional response of transduction will facilitate the advancement of safe and precise gene delivery vehicles.
AP-4 regulates neuronal lysosome composition, function, and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network
Authors: Majumder Piyali , Edmison Daisy , Rodger Catherine , Patel Sruchi , Reid Evan , Gowrishankar Swetha 
Published: June 2021

AbstractThe adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimer’s disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP.
Cytoplasmic Colocalization of Granulins and TDP-43 Prion-like Domain Involves Electrostatically Driven Coacervation Tuned by the Redox State of Cysteines
Authors: Bhopatkar Anukool A. , Dhakal Shailendra , Rangachari Vijayaraghavan 
Published: June 2021

AbstractCytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. We were interested in understanding if and how cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of a genetic risk factor in FTLD called progranulin, interact with TDP-43. We show that extracellular GRNs internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show no presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Identification of the sequence determinants within GRNs for the observed phase transitions reveal the negative charges to be the drivers of LLPS modulated by the positive charges and the redox state of cysteines. Furthermore, RNA and GRNs compete and expunge one another from PrLD condensates, providing a basis for GRN’s absence in SGs. Together, the results illustrate the potential mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize, and modulate cytoplasmic TDP-43 inclusions in proteinopathies.
Mitogen Kinase Kinase (MKK7) controls cytokine production in vitro and in vivo in mice
Authors: Caliz Amada D. , Yoo Hyung-Jin , Vertii Anastassiia , Tournier Cathy , Davis Roger J. , Keaney John F. , Kant Shashi 
Published: June 2021

AbstractMitogen kinase kinase 4 (MKK4) and Mitogen kinase kinase 7 (MKK7) are members of the MAP2K family which can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both, c-Jun N-terminal Kinase (JNK) and p38 MAPK, whereas MKK7 only activates JNK in response to different stimuli. The stimuli as well as cell type determine the choice of MAP2K member that mediates the response. In a variety of cell types, the MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK7 and MKK4 contributes to innate immune response in macrophages as well as during inflammation in vivo. To address this question and elucidate the role of MKK7 and MKK4 in macrophage and in vivo, we developed MKK7- and MKK4-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for LPS induced cytokine production and migration which appears to be a major contributor to the inflammatory response in vivo. Whereas MKK4 plays a significant but minor role in cytokine production in vivo.
MassGenie: a transformer-based deep learning method for identifying small molecules from their mass spectra
Authors: Shrivastava Aditya Divyakant , Swainston Neil , Samanta Soumitra , Roberts Ivayla , Muelas Marina Wright , Kell Douglas B. 
Published: June 2021

AbstractThe ‘inverse problem’ of mass spectrometric molecular identification (‘given a mass spectrum, calculate the molecule whence it came’) is largely unsolved, and is especially acute in metabolomics where many small molecules remain unidentified. This is largely because the number of experimentally available electrospray mass spectra of small molecules is quite limited. However, the forward problem (‘calculate a small molecule’s likely fragmentation and hence at least some of its mass spectrum from its structure alone’) is much more tractable, because the strengths of different chemical bonds are roughly known. This kind of molecular identification problem may be cast as a language translation problem in which the source language is a list of high-resolution mass spectral peaks and the ‘translation’ a representation (for instance in SMILES) of the molecule. It is thus suitable for attack using the deep neural networks known as transformers. We here present MassGenie, a method that uses a transformer-based deep neural network, trained on ~6 million chemical structures with augmented SMILES encoding and their paired molecular fragments as generatedin silico, explicitly including the protonated molecular ion. This architecture (containing some 400 million elements) is used to predict the structure of a molecule from the various fragments that may be expected to be observed when some of its bonds are broken. Despite being given essentially no detailed nor explicit rules about molecular fragmentation methods, isotope patterns, rearrangements, neutral losses, and the like, MassGenie learns the effective properties of the mass spectral fragment and valency space, and can generate candidate molecular structures that are very close or identical to those of the ‘true’ molecules. We also use VAE-Sim, a previously published variational autoencoder, to generate candidate molecules that are ‘similar’ to the top hit. In addition to using the ‘top hits’ directly, we can produce a rank order of these by ‘round-tripping’ candidate molecules and comparing them with the true molecules, where known. As a proof of principle, we confine ourselves to positive electrospray mass spectra from molecules with a molecular mass of 500Da or lower. The transformer method, applied here for the first time to mass spectral interpretation, works extremely effectively both for mass spectra generatedin silicoand on experimentally obtained mass spectra from pure compounds. The ability to create and to ‘learn’ millions of fragmentation patternsin silico, and therefrom generate candidate structures (that do not have to be in existing libraries)directly, thus opens up entirely the field ofde novosmall molecule structure prediction from experimental mass spectra.
Xrn1 is a deNADding Enzyme Modulating Mitochondrial NAD Levels
Authors: Sharma Sunny , Yang Jun , Grudzien-Nogalska Ewa , Kiledjian Megerditch 
Published: June 2021

ABSTRACTThe existence of non-canonical nicotinamide adenine diphosphate (NAD) 5’-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5’-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that still retains its 5’-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and in turn intramitochondrial NAD levels. Our findings uncover a functional role for mitochondrial NAD-capped RNAs as a reservoir to maintain overall NAD homeostasis. We propose NAD-capped RNAs function as a cistern for mitochondrial NAD with Xrn1 serving as a rheostat for NAD-capped RNAs.
Inositolphosphate glycans and a fucosylated xyloglucan oligosaccharide are accumulated upon Arabidopsis thaliana/ Botrytis cinerea infection
Authors: Voxeur Aline , Sechet Julien , Vernhettes Samantha 
Published: June 2021

AbstractIn mammals, insulin is involved in controlling blood glucose levels and its role in modulating immunity is being more and more documented. This hormone promotes the release of inositolphosphate glycans (IPG) which act as mediators. In plants, one IG has already been identified in plant culture cells (Smith and Fry, 1999; Smith et al., 1999) but, to our knowledge, no IPG have been yet identified. Here, we discovered 7 IPG that are accumulated upon Arabidopsis thaliana-Botrytis cinerea interaction, concomitantly with oligogalacturonides and a fucosylated xyloglucan oligosaccharide. Further structural characterization showed that they come from the hydrolysis of polar heads of Serie A to H glycosylinositol phosphorylceramides presumably via a phospholipase C activity. Taken together with the emerging role of insulin as immune regulator, these results question the role of IPG as damage associated molecular pattern both in animal and plant kingdoms.
A metabolically stable PET tracer for imaging synaptic vesicle protein 2A: Synthesis and preclinical characterization of [18F]SDM-16
Authors: Zheng Chao , Holden Daniel , Zheng Ming-Qiang , Pracitto Richard , Wilcox Kyle C. , Lindemann Marcel , Felchner Zachary , Zhang Li , Tong Jie , Fowles Krista , Finnema Sjoerd J. , Nabulsi Nabeel , Carson Richard E. , Huang Yiyun , Cai Zhengxin 
Published: June 2021

ABSTRACTPurposeTo investigate the synaptic vesicle glycoprotein 2A (SV2A) expression in the whole central nervous system and peripheral tissues, a metabolically stable SV2A radiotracer is desirable to minimize a potential confounding effect of radiometabolites. The aim of this study was to develop and evaluate a metabolically stable SV2A radiotracer, [18F]SDM-16, in nonhuman primate brains.MethodsThe racemic SDM-16 (4-(3,5-difluorophenyl)-1-((2-methyl-1H-imidazol-1-yl)methyl)pyrrolidin-2-one) was synthesized and assayed for in vitro SV2A binding affinity. We synthesized the enantiopure [18F]SDM-16 using the corresponding arylstannane precursor. Nonhuman primate brain PET was performed on a FOCUS 220 system. Arterial blood was drawn for metabolite analysis and construction of plasma input function. Regional time-activity curves (TACs) were evaluated with the one-tissue compartment (1TC) model to obtain the volume of distribution (VT). Binding potential (BPND) was calculated using either the nondisplaceable volume of distribution (VND) or the centrum semiovale (CS) as the reference region.ResultsRacemic SDM-16 was synthesized in 3 steps with 44% overall yield and has high affinity (Ki = 3.7 nM) to human SV2A. [18F]SDM-16 was prepared in greater than 99% radiochemical and enantiomeric purity. This radiotracer displayed high specific binding in brain and was metabolically more stable than other SV2A PET tracers. The plasma free fraction (fP) of [18F]SDM-16 was 69%, which was higher than those of [11C]UCB-J (46%), [18F]SynVesT-1 (43%), [18F]SynVesT-2 (41%), and [18F]UCB-H (43%). The TACs were well described with the 1TC. The averaged test-retest variability (TRV) was −9±8%, and averaged absolute TRV (aTRV) was 10±7% for all analyzed brain regions.ConclusionWe have successfully synthesized a metabolically stable and high affinity SV2A PET tracer, [18F]SDM-16, which showed high specific and reversible binding in the NHP brain. [18F]SDM-16 may have potential application in the visualization and quantification of SV2A beyond the brain.
Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM
Authors: Zhao Jianhua , Makhija Suraj , Huang Bo , Cheng Yifan 
Published: June 2021

ABSTRACTA major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.
FRET and LRET Biosensors for Cell-based Imaging and Screening of Rac1 Activation
Authors: Pham Ha , Soflaee Mona Hoseini , Karginov Andrei V , Miller Lawrence W. 
Published: June 2021

ABSTRACTRac1 is a key regulator of several cell signaling pathways and dysregulated Rac1 activation has been implicated in cancer. Genetically encoded Förster resonance energy transfer (FRET) biosensors with enhanced dynamic range enabled live cell fluorescence imaging of Rac1 activity and a cell lysate-based assay of Rac1 inhibition in 96-well plates. We prepared HEK293 cell lines that stably expressed polypeptides with a general domain sequence (N- to C-terminus) of 1) FRET acceptor; 2) Rac/Cdc42 binding domain of human p21 protein kinase A (residues 68-150); 3) a linker domain; 4) FRET donor; and 5) full-length Rac1. Activated Rac1 binds to the protein kinase A domain, bringing donors and acceptors close together to increase FRET. We evaluated the effects on FRET signal dynamic range of alpha helical linkers comprised of alternating repeats of roughly four glutamate and four arginine or lysine residues. So-called ER/K linkers had limited effects on conventional FRET biosensors that incorporated the fluorescent protein (FP) pairs mCerulean/YPet, or mTFP1(cp227)/mVenus(cp229). Fluorometric measurements of cells that co-expresssed biosensors with positive (TIAM1) or negative (RhoGDI) Rac1 regulators revealed significant dynamic range enhancement in only one FP construct (mCerulean/YPet with 20 nm ER/K linker) relative to an analogous structure that incorporated an unstructured linker. We transfected this construct into a cell line that stably expressed a rapamycin-inducible c-Src analog (RapR-Src) and observed activation of Rac1 at protruding edges following rapamycin stimulation. In cells that expressed lanthanide-based FRET (LRET biosensors) that incorporated a luminescent terbium complex donor and GFP fluorescent acceptor, time-gated luminescence (TGL) measurements showed substantial gains in dynamic range that increased with linker length (up to 1200%). We robustly detected small molecule Rac1 inhibition following lysis of LRET biosensor-expressing cells grown directly in 96-well plates. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.