bluuebunny/crossref_metadata_2025_split huggingface dataset:
Self-assembled nanoparticles enable delivery of VCP inhibitor, resulting in enhanced photoreceptor protection in retinal explants
Authors: Sen Merve , Al-Amin MD , Kicková Eva , Sadeghi Amir , Puranen Jooseppi , Urtti Arto , Caliceti Paolo , Salmaso Stefano , Arango-Gonzalez Blanca , Ueffing Marius 
Published: June 2021

1.AbstractMutations in rhodopsin lead to its misfolding resulting in autosomal dominant retinitis pigmentosa (adRP). Pharmacological inhibition of the ATP-driven chaperone valosin- containing protein (VCP), a molecular checkpoint for protein quality control, slows down retinal degeneration in animal models. However, poor water-solubility of VCP inhibitors poses a challenge to their clinical translation as intravitreal injections for retinal treatment. In order to enable the delivery of VCP inhibitors, we have developed and investigated two formulations for the VCP inhibitor ML240. Nanoformulations of ML240 were obtained by using amphiphilic polymers methoxy-poly (ethylene glycol)5kDa-cholane (mPEG5kDa-cholane) and methoxy-poly (ethylene glycol)5kDa- cholesterol (mPEG5kDa-cholesterol). Both formulations increased the water-solubility of ML240 by two orders of magnitude and prolonged the drug released over ten days. Encapsulation of ML240 in mPEG5kDa-cholane showed superior photoreceptor protection at lower drug concentrations, normalized rhodopsin localization, and alleviated inflammatory microglial responses in an ex vivo rat model of retinal degeneration. The study demonstrates the potential of VCP inhibitor nanoformulations to treat adRP, a pharmacologically orphan disease.
Cerebral hemodynamics: a mathematical model including autoregulation, baroreflex and extracranial peripheral circulation
Authors: Garcia Francisco A. , Spavieri Deusdedit L. , Linninger Andreas 
Published: June 2021

ABSTRACTCerebral autoregulation, the physiological capability to regulate cerebral blood flow, may be assisted by short-term mean arterial pressure control via baroreflex, which, among several effects, modulates total peripheral resistance. It is unclear, however, whether the resistance of the head and neck vasculatures is also affected by baroreflex and whether these extracranial vessels assist autoregulation. Since sensing technologies such as functional Near-Infrared Spectroscopy and noninvasive intracranial pressure monitoring by strain gauge may be influenced by superficial tissue, it is clinically relevant to understand the relations between intracranial and extracranial hemodynamics. Therefore, we created an autoregulation model consisting of arteries and arterioles regulated by the intralumial pressure and microcirculation regulated by local blood flow. As the first critical step to quantify the signal deterioration introduced by the extracranial circulation on superficial sensors, the extracranial peripheral circulation of the head and neck and baroreflex regulation of the peripheral vasculature and of heart rate were also included. During simulations of a bout of acute hypotension, the model predicts a rapid return of cerebral blood flow to baseline levels and a prolonged suppression of the blood flow to the external carotid vasculature, in accordance with experimental evidence. The inclusion of peripheral control via baroreflex at the external carotid vasculature did not assist cerebral autoregulation, thus we raise the hypothesis that baroreflex may act on the head and neck vasculatures but this action has negligible effects on regulation of cerebral blood flow. When autoregulation is impaired, results suggest that the blood flow of the brain and of the head and neck present similar dynamics, while they are weakly coupled when autoregulation is intact. The model also provides a mechanistic explanation of the protection brought by cerebral autoregulation to the microvasculature and to the brain parenchyma. Our model forms the foundation for predicting the interference introduced by the superficial tissue to nonivasive sensors.
The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei
Authors: Bakari-Soale Majeed , Ikenga Nonso Josephat , Scheibe Marion , Butter Falk , Jones Nicola G. , Kramer Susanne , Engstler Markus 
Published: June 2021

ABSTRACTThe biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.
Recirculation of H2, CO2, and ethylene improves carbon fixation and carboxylate yields in anaerobic fermentation
Authors: Baleeiro Flávio C. F. , Kleinsteuber Sabine , Sträuber Heike 
Published: June 2021

ABSTRACTAnaerobic fermentation with mixed cultures has gained momentum as a bioprocess for its promise to produce platform carboxylates from low-value biomass feedstocks. Anaerobic fermenters are net carbon emitters and their carboxylate yields are limited by electron donor availability. In a new approach to tackle these two disadvantages, we operated two bioreactors fed with acetate and lactate as a model feedstock while recirculating H2/CO2 to stimulate concomitant autotrophic activity. After 42 days of operation, hydrogenotrophic methanogenesis was predominant and ethylene (≥1.3 kPa) was added to one of the reactors, inhibiting methanogenesis completely and recovering net carbon fixation (0.20 g CO2 L−1 d−1). When methanogenesis was inhibited, exogenous H2 accounted for 17% of the consumed electron donors. Lactate-to-butyrate selectivity was 101% (88% in the control without ethylene) and lactate-to-caproate selectivity was 17% (2.3% in the control). Community analysis revealed that ethylene caused Methanobacterium to be washed out, giving room to acetogenic bacteria. In contrast to 2-bromoethanosulfonate, ethylene is a scalable methanogenesis inhibition strategy that did not collaterally block i-butyrate formation. By favoring the bacterial share of the community to become mixotrophic, the concept offers a way to simultaneously increase selectivity to medium-chain carboxylates and to develop a carbon-fixing chain elongation process.
Reduced Hippocampal Inhibition and Enhanced Autism-Epilepsy Comorbidity in Mice Lacking Neuropilin 2
Authors: Eisenberg Carol , Subramanian Deepak , Afrasiabi Milad , Ziobro Patryk , DeLucia Jack , Hirschberg Pamela R. , Shiflett Michael W. , Santhakumar Vijayalakshmi , Tran Tracy S. 
Published: June 2021

AbstractThe neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2 deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2-/- mice have reduced number of parvalbumin, somatostatin and Neuropeptide Y cells, mainly in CA1. Whole cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/- and Nrp2-/- mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2 deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration and higher severity in Nrp2-/- compared to Nrp2+/+ animals. Finally, Nrp2+/- and Nrp2-/-, but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2-/- mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.
Holosteans contextualize the role of the teleost genome duplication in promoting the rise of evolutionary novelties in the ray-finned fish innate immune system
Authors: Dornburg Alex , Wcisel Dustin J. , Zapfe Katerina , Ferraro Emma , Roupe-Abrams Lindsay , Thompson Andrew W. , Braasch Ingo , Ota Tatsuya , Yoder Jeffrey A. 
Published: June 2021

AbstractOver 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrates that have diversified across virtually all fresh and saltwater ecosystems. This ecological diversity raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the genomic substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.
Structure of ABCB1/P-glycoprotein bound to the CFTR potentiator ivacaftor
Authors: Barbieri Alessandro , Thonghin Nopnithi , Shafi Talha , Prince Stephen M. , Collins Richard F. , Ford Robert C. 
Published: June 2021

AbstractABCB1 (P-glycoprotein) is an ATP binding cassette transporter that is involved in the clearance of xenobiotics and it affects the disposition of many drugs in the body. Here we have studied ABCB1 in the drug-bound and drug-free states, simultaneously, using high contrast cryo-electron microscopy imaging and a Volta phase plate. The binding of the potent CFTR potentiator, ivacaftor, at a site in the central aqueous cavity is mediated by transmembrane α-helices 3,6,10,11 & 12. Binding is associated with a wider separation of the two halves of the transporter in the inward-facing state. Induced-fit changes the nucleotide binding domains in a way that may explain their increased affinity for ATP when drug is bound. Comparison of ivacaftor-bound structures of CFTR and ABCB1 suggests common features in the binding modes.
Analysis of the Mechanosensor Channel Functionality of TACAN
Authors: Niu Yiming , Tao Xiao , Vaisey George , Olinares Paul Dominic B. , Alwaseem Hanan , Chait Brian T. , MacKinnon Roderick 
Published: June 2021

AbstractMechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods we found that TACAN, at high protein concentrations, produces non-selective, heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single particle cryo-EM and observe that it forms a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme-A co-factor, confirmed by mass spectrometry. The TACAN protomers are related in 3-dimensional structure to a fatty acid elongase, ELOVL. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.
Proximity-based labeling reveals DNA damage-induced N-terminal phosphorylation of fused in sarcoma (FUS) leads to distinct changes in the FUS protein interactome
Authors: Johnson Michelle A. , Nuckols Thomas A. , Merino Paola , Bagchi Pritha , Nandy Srijita , Root Jessica , Taylor Georgia , Seyfried Nicholas T. , Kukar Thomas 
Published: June 2021

AbstractCytoplasmic accumulation of the RNA/DNA binding protein, fused in sarcoma (FUS), into inclusions is a common hallmark of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) pathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of an N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 (APEX2) paired with mass-spectrometry (MS) to investigate whether N-terminal phosphorylation shifts the FUS protein-protein interaction network (interactome), and subsequently, its function. We report the first comparative analysis of the interactomes for three FUS variants: homeostatic wild-type FUS (FUS WT), a phosphomimetic variant of FUS (a proxy for N-terminally phosphorylated FUS, FUS PM), and a toxic FUS P525L mutant (a mutation that causes juvenile ALS, FUS P525L). Data are available via ProteomeXchange with identifier PXD026578. We demonstrate that compared to FUS WT and FUS P525L, the FUS PM interactome uniquely enriches for a set of cytoplasmic proteins that mediate mRNA metabolism and translation and nuclear proteins involved in spliceosome and DNA repair functions, respectively. We further identify and validate three proteins, VPS35, MOV10, and CLTA, as novel interacting partners of all three FUS variants. Lastly, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the FUS interactome and function.
An interactive deep learning-based approach reveals mitochondrial cristae topologies
Authors: Suga Shogo , Nakamura Koki , Humbel Bruno M. , Kawai Hiroki , Hirabayashi Yusuke 
Published: June 2021

SummaryOuter and inner mitochondrial membranes are highly specialized structures with distinct functional properties. Reconstructing complex 3D ultrastructural features of mitochondrial membranes at the nanoscale requires analysis of large volumes of serial scanning electron tomography data. While deep-learning-based methods improved in sophistication recently, time-consuming human intervention processes remain major roadblocks for efficient and accurate analysis of organelle ultrastructure. In order to overcome this limitation, we developed a deep-learning image analysis platform called Python-based Human-In-the-LOop Workflows (PHILOW). Our implementation of an iterative segmentation algorithm and Three-Axis-Prediction method not only improved segmentation speed, but also provided unprecedented ultrastructural detail of whole mitochondria and cristae. Using PHILOW, we found that 42% of cristae surface exhibits tubular structures that are not recognizable in light microscopy and 2D electron microscopy. Furthermore, we unraveled a fundamental new regulatory function for the dynamin-related GTPase Optic Atrophy 1 (OPA1) in controlling the balance between lamellar versus tubular cristae subdomains.
TopFlash transgenic quail reveals dynamic TCF/β-catenin signaling during avian embryonic development
Authors: Barzilai-Tutsch Hila , Morin Valerie , Toulouse Gauthier , Firth Stephen , Marcelle Christophe , Serralbo Olivier 
Published: June 2021

ABSTRACTThe Wnt/β-catenin signaling pathway is highly conserved throughout evolution and it plays crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway’s activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12x or 16x) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterisation of TCF/β-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.
Aminoglycoside-induced premature termination codon readthrough of COL4A5 nonsense mutations that cause Alport syndrome
Authors: Omachi Kohei , Kai Hirofumi , Roberge Michel , Miner Jeffrey H. 
Published: June 2021

ABSTRACTAlport syndrome (AS) is characterized by glomerular basement membrane (GBM) abnormalities leading to progressive glomerulosclerosis. Mutations in the COL4A3, COL4A4 or COL4A5 genes encoding type IV collagen α3α4α5 cause AS. Truncated α3, α4, and α5 chains lacking an intact COOH-terminal noncollagenous domain due to a premature termination codon (PTC) cannot assemble into heterotrimers or incorporate into the GBM. Therefore, achieving full-length protein expression is a potential therapy for AS caused by truncating nonsense mutations. Small molecule-based PTC readthrough (PTC-RT) therapy has been well studied in other genetic diseases, but whether PTC-RT is applicable to AS is unexplored. To investigate the feasibility of PTC-RT therapy in AS, we made a cDNA to express COL4A5 fused to a C-terminal NanoLuc luciferase (NLuc) to monitor full-length translation. Full-length COL4A5-NLuc produces luminescence, but mutants truncated due to a PTC do not. To screen for COL4A5 nonsense mutants susceptible to PTC-RT, we introduced 49 individual nonsense mutations found in AS patients into the COL4A5-NLuc cDNA. Luciferase assays revealed that 11 mutations (C29X, S36X, E130X, C1521X, R1563X, C1567X, W1594X, S1632X, R1683X, C1684X and K1689X) were susceptible to PTC-RT induced by G418, which is known to have high readthrough activity. Moreover, we found that some next-generation “designer” PTC-RT drugs induced RT, and RT enhancer compounds increased the efficacy of PTC-RT in a G418-susceptible PTC mutant. These results suggest that PTC-RT therapy is a feasible approach for some patients with AS. Our luciferase-based COL4A5 translation reporter system will contribute to further development of PTC-RT therapies in a personalized medicine approach to treating AS.
In vitro inhibition of H. pylori in a preferential manner using bioengineered L. lactis releasing guided Antimicrobial peptides
Authors: Choudhury Ankan , Ortiz Patrick , Kearney Christopher M. 
Published: June 2021

AbstractObjectivesTargeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. This is even more important when a pathogen like H. pylori resides in stomach, a sensitive microbial ecosystem. Using a probiotic like Lactococcus lactis and bioengineering it to release a guided Antimicrobial Peptide (AMP) targeted towards the pathogen offers a pathway to specifically knock-out the deleterious species and not disturbing the stomach microbiome.ResultsThree AMPs, Alyteserin, CRAMP and Laterosporulin, were genetically fused to a guiding peptide MM1, which selectively binds to Vacuolating Toxin A (VacA) of H. pylori and cloned into an excretory vector pTKR inside L. lactis. When cultured together in vitro, the L. lactis bioengineered with guided AMPs selectively killed H. pylori when compared to E. coli or Lactobacillus plantarum, as determined by qPCR. Chemically synthesized Alyteserin and MM1-Alyteserin showed similar preferential inhibition of H. pylori when compared against E. coli, with the MIC of MM1-Alyteserin becoming significantly higher for E. coli than Alytserin whereas no such effet was observed against H. pylori.ConclusionsProbiotics bioengineered to excrete guided AMPs can be a novel and useful approach for combating pathogens without endangering the natural microbial flora. Given the wealth of AMPs and guiding ligands, both natural and synthetic, this approach can be adapted to develop a diverse array of chimeric guided AMPs and can be cloned into probiotics to create a safe and effective alternative to conventional chemical antibiotics.
Structural Basis for Cytoplasmic Dynein-1 Regulation by Lis1
Authors: Gillies John P. , Reimer Janice M. , Karasmanis Eva P. , Lahiri Indrajit , Htet Zaw Min , Leschziner Andres E. , Reck-Peterson Samara L. 
Published: June 2021

AbstractThe lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator that binds directly to dynein’s motor domain, and by doing so alters dynein’s mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein–Lis1 complex. Our 3.1Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1’s ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1’s ability to form fully active human dynein complexes and to regulate yeast dynein’s mechanochemistry and in vivo function. We present a model for the conserved role of Lis1 in regulating dynein from yeast to humans.