bluuebunny/crossref_metadata_2025_split huggingface dataset:
PTEN deletion in spinal pathways via retrograde transduction with AAV-rg enhances forelimb motor recovery after cervical spinal cord injury; sex differences and late-onset pathophysiologies
Authors: Metcalfe Mariajose , Steward Oswald 
Published: March 2023

ABSTRACTSpinal cord injuries (SCI) cause permanent functional impairments due to interruption of motor and sensory pathways. Regeneration of axons does not occur due to lack of intrinsic growth capacity of adult neurons and extrinsic inhibitory factors, especially at the injury site. However, some regeneration can be achieved via deletion of the phosphatase and tensin homolog (PTEN) in cells of origin of spinal pathways. Here, we deployed an AAV variant that is retrogradely transported (AAV-rg) to deliver gene modifying cargos to the cells of origin of multiple pathways interrupted by SCI, testing whether this promoted recovery of motor function. PTENf/f;RosatdTomatomice and control RosatdTomatomice received injections of different doses (number of genome copies, GCs) of AAV-rg/Cre into the cervical spinal cord at the time of a C5 dorsal hemisection injury. Forelimb grip strength was tested over time using a grip strength meter. PTENf/f;RosatdTomatomice with AAV-rg/Cre (PTEN-deleted) exhibited substantial improvements in forelimb gripping ability in comparison to controls. Of note, there were major sex differences in the extent of recovery, with male mice exhibiting greater recovery than females. However, at around 5-7 weeks post-injury/injection, many mice with SCI and AAV-rg-mediated PTEN deletion began to exhibit pathophysiologies involving excessive scratching of the ears and back of the neck and rigid forward extension of the hindlimbs. These pathophysiologies increased in incidence and severity over time. Our results reveal that although intra-spinal injections of AAV-rg/Cre in PTENf/f;RosatdTomatomice can enhance forelimb motor recovery after SCI, late-developing functional abnormalities occur with the experimental conditions used here. Mechanisms underlying late-developing pathophysiologies remain to be defined.
Depletion of bleomycin hydrolase (Blmh) downregulates histone demethylase Phf8, impairs mTOR signaling/autophagy, accelerates amyloid beta accumulation, and induces neurological deficits in mice
Authors: Witucki Łukasz , Borowczyk Kamila , Suszyńska-Zajczyk Joanna , Warzych Ewelina , Pawlak Piotr , Jakubowski Hieronim 
Published: March 2023

ABSTRACTBleomycin hydrolase (BLMH), a homocysteine (Hcy)-thiolactone detoxifying enzyme, is attenuated in brains of Alzheimer’s disease patients. In mice, Blmh depletion causes astrogliosis and behavioral changes. Depletion of histone demethylase PHF8, which controls mTOR signaling by demethylating H4K20me1, causes neuropathy in humans and mice. Here we examined how Blmh depletion affects the Phf8/H4K20me1/mTOR signaling/autophagy pathway and amyloid beta (Aβ) accumulation and cognitive/neuromotor performance in mice. We found that Phf8 was significantly downregulated in brains ofBlmh-/-micevs.Blmh+/+sibling controls. H4K20me1, mTOR, phospho-mTOR, and App were upregulated while autophagy markers Bcln1, Atg5, and Atg7 were downregulated inBlmh-/-brains. Blmh depletion caused similar biochemical changes and significantly elevated Aβ inBlmh-/-5xFADvs.Blmh+/+5xFAD brains. Behavioral testing identified cognitive/neuromotor deficits inBlmh-/-andBlmh-/-5xFAD mice. In Blmh-depleted N2a-APPswecells, Phf8 was downregulated, while APP, total H4K20me1, and H4K20me1-mTORpromoter binding were elevated. This led to mTOR upregulation, autophagy downregulation, and significantly increased APP and Aβ levels. Phf8 depletion or treatments with Hcy-thiolactone orN-Hcy-protein, metabolites that are elevated in Blmh-depleted mice, induced similar biochemical changes in N2a-APPswecells, akin to those in induced by Blmh depletion. Taken together, our findings indicate that Blmh interacts with APP and the Phf8/H4K20me1/mTOR/autophagy pathway and show that disruption of these interactions lead to Aβ accumulation and cognitive and neuromotor deficits.
Nanograms of SARS-CoV-2 Spike Protein Delivered by Exosomes Induce Potent Neutralization of Both Delta and Omicron Variants
Authors: Cacciottolo Mafalda , Li Yujia , Nice Justin B , LeClaire Michael J. , Twaddle Ryan , Mora Ciana L. , Adachi Stephanie Y. , Young Meredith , Angeles Jenna , Elliott Kristi , Sun Minghao 
Published: March 2023

ABSTRACTExosomes are emerging as potent and safe delivery carriers for use in vaccinology and therapeutics. A better vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to provide improved, broader, longer lasting neutralization of SARS-CoV-2, a more robust T cell response, enable widespread global usage, and further enhance the safety profile of vaccines given the likelihood of repeated booster vaccinations. Here, we use Capricor’s StealthX™ platform to engineer exosomes to express native SARS-CoV-2 spike Delta variant (STX-S) protein on the surface for the delivery of a protein-based vaccine for immunization against SARS-CoV-2 infection. The STX-S vaccine induced a strong immunization with the production of a potent humoral immune response as demonstrated by high levels of neutralizing antibody not only against the delta SARS-CoV-2 virus but also two Omicron variants (BA.1 and BA.5), providing broader protection than current mRNA vaccines. Additionally, both CD4+and CD8+T cell responses were increased significantly after treatment. Quantification of spike protein by ELISA showed that only nanograms of protein were needed to induce a potent immune response. This is a significantly lower dose than traditional recombinant protein vaccines with no adjuvant required, which makes the StealthX™ exosome platform ideal for the development of multivalent vaccines with a better safety profile. Importantly, our exosome platform allows novel proteins, or variants in the case of SARS-CoV-2, to be engineered onto the surface of exosomes in a matter of weeks, comparable with mRNA vaccine technology, but without the cold storage requirements. The ability to utilize exosomes for cellular delivery of proteins, as demonstrated by STX-S, has enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation at an extremely low dose resulting in a potent, broad antibody response.
Nucleolar Essential Protein 1 (Nep1): Elucidation of Enzymatic Catalysis Mechanism by Combined Molecular Dynamics Simulation and Quantum Chemical Calculations
Authors: Jedrzejewski Mateusz , Bełza Barbara , Lewandowska Iwona , Sadlej Marta , Perlinska Agata P. , Augustyniak Rafal , Christian Thomas , Hou Ya–Ming , Kalek Marcin , Sulkowska Joanna I. 
Published: March 2023

AbstractNep1 is a protein essential for the formation of the eukaryotic and archaeal small ribosomal subunit. It is an enzyme responsible for the site–specific SAM–dependent methylation of pseudouridine (Ψ) during the pre–rRNA processing. It possesses a non–trivial topology, namely, a 31knot in the active site. Herein, we investigate the structure and mechanism of catalysis of Nep1 using a combination of bioinformatics, computational, and experimental methods. In particular, we address the issue of seemingly unfeasible deprotonation of Ψ nucleobase in the active site of Nep1 by a distant aspartate residue (e.g., D101 in Nep1 ofS. cerevisiae). Sequence alignment analysis across different organisms identifies a conserved serine/threonine residue that may play a role of a proton–transfer mediator (e.g., S233 in Nep1 fromS. cerevisiae), facilitating the reaction. Two enzyme–substrate complexes, one based on an available crystal structure and the other generated by molecular docking, of representative eukaryotic (fromS. cerevisiae) and archaeal (fromA. fulgidus) Nep1 homologs are subjected to molecular dynamics (MD) simulations. The resulting trajectories confirm that the hydroxyl–containing amino acid can indeed adopt a position suitable for proton–shuttling, with the OH group located in between the proton donor and acceptor. However, during the MD simulations, a water molecule emerges from arrangements of the active site, which can assume the role of the proton–transfer mediator instead. To discern between these two alternative pathways, we evaluate the possible methylation mechanisms by quantum–chemical calculations based on density functional theory, using the cluster approach. The obtained energy profiles indicate that the most facile course of the reaction for both the yeast and archaeal enzymes is to engage the water molecule. These results are corroborated by agreement of the computed energy barriers with experimentally measured enzyme kinetics. Moreover, mutational studies show that, while aspartate D101 is crucial for the catalytic activity, serine S233 is irrelevant in this context, indirectly supporting the water–mediated proton transfer. Our findings comprehensively elucidate the mode of action of Nep1 and provide implication for understanding the catalytic mechanisms of other enzymes that involve a proton transfer in the active site over extended distances.
HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages
Authors: Virgilio Maria C. , Ramnani Barka , Chen Thomas , Disbennett W. Miguel , Lubow Jay , Welch Joshua D. , Collins Kathleen L. 
Published: March 2023

ABSTRACTHIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.
Dynamic lid domain ofChloroflexus aurantiacusMalonyl-CoA Reductase controls the reaction
Authors: Kabasakal Burak V. , Cotton Charles A. R. , Murray James W. 
Published: March 2023

AbstractMalonyl-Coenzyme A Reductase (MCR) inChloroflexus aurantiacus, a characteristic enzyme of the 3-hydroxypropionate (3-HP) cycle, catalyses the reduction of malonyl-CoA to 3-HP. MCR is a bi-functional enzyme; in the first step, malonyl-CoA is reduced to the free intermediate malonate semialdehyde by the C-terminal region of MCR, and further reduced to 3-HP by the N-terminal region of MCR. Here we present the crystal structures of both N-terminal and C-terminal regions of the split MCR fromC. aurantiacus. A catalytic mechanism is suggested by ligand and substrate bound structures, and structural and kinetic studies of MCR variants. Both MCR structures reveal one catalytic, and one non-catalytic SDR (short chain dehydrogenase/reductase) domain. C-terminal MCR has a lid domain which undergoes a conformational change and controls the reaction. In the proposed mechanism of the C-terminal MCR, the conversion of malonyl-CoA to malonate semialdehyde is based on the reduction of malonyl-CoA by NADPH, followed by the decomposition of the hemithioacetal to produce malonate semialdehyde and coenzyme A. Conserved arginines, Arg734 and Arg773 are proposed to play key roles in the mechanism and conserved Ser719, and Tyr737 are other essential residues forming an oxyanion hole for the substrate intermediates.
Induction of osteogenic differentiation of MSCs by GSK3β knockdown through GSK3β siRNAs transfection
Authors: Galitsyna Elena V. , Buianova Anastasiia A. , Bukharova Tatiana B. , Krivosheeva Irina A. , Skoblov Mikhail Yu. , Goldshtein Dmitriy V. 
Published: March 2023

AbstractThe development of effective strategies for the treatment of bone defects is based on gene therapy methods aimed at regulating the differentiation of osteoprogenitor cells. One approach is the development of knockdown systems of inhibitory genes of osteogenic cell differentiation using siRNA molecules. In this work, we developed approaches to induce osteogenic differentiation of mesenchymal stem cells (MSCs) by knockdown ofGSK3βusing siRNAs in cultures of MSCs derived from human adipose tissue (AD-MSCs). For this purpose, we performed a comparative evaluation of the efficacy of lipoplexes and polyplexes formed with one of the 4 siRNA molecules and 5 commercial transfection agents most commonly used in laboratory practice. The most effective transfection agent appeared to be PEI, which demonstrated high cytocompatibility in free form and as part of polyplexes even when maximum concentrations were used. Using the polyplexes formed by siRNA molecule designed for the first time and PEI, we developed a highly efficientGSK3βgene knockdown system, which showed its effectiveness in cultures of AD-MSCs. As a result, we demonstrated the osteoinductive properties of GSK3β siRNA molecules in these cultures. The results obtained can be applied in the development of gene therapy strategies based on siRNA molecules in human bone tissue diseases.
The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication
Authors: Dupont Maud , Krischuns Tim , Giai-Gianetto Quentin , Paisant Sylvain , Bonazza Stefano , Brault Jean-Baptiste , Douché Thibaut , Perez-Perri Joel I , Hentze Matthias W , Cusack Stephen , Matondo Mariette , Isel Catherine , Courtney David G , Naffakh Nadia 
Published: March 2023

ABSTRACTRecent technical advances have significantly improved our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells, with a particular focus on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, notably SARS-CoV-2, revealing both similarities and differences between the RBP profiles of viral and cellular mRNAs. Herein, we comprehensively identified the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, which associates with the viral mRNAs, results in lower levels of viral mRNAs within infected cells, and a decreased yield of infectious viral particles. Overall, our results uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs, and point to a role of the viral polymerase in orchestrating the assembly of viral mRNPs.
Multimodal acoustic-electric trigeminal nerve stimulation modulates conscious perception
Authors: Wu Min , Auksztulewicz Ryszard , Riecke Lars 
Published: March 2023

AbstractMultimodal stimulation has the potential to reverse pathological neural activity and alleviate symptoms in neuropsychiatric diseases. However, the reliability of this approach and the mechanisms through which it improves consciousness remain largely unknown. We investigated the effects of multimodal stimulation combining music stimulation with electrical trigeminal nerve stimulation in healthy human participants. We assessed conscious perception before and after acoustic-electric stimulation and investigated the mechanisms underlying the putative stimulation effects. Our results show that (1) acoustic-electric stimulation improves conscious tactile perception in healthy human participants without a concomitant change in auditory perception, (2) this improvement is caused by the interplay of the acoustic and electric stimulation rather than any of the unimodal stimulation alone, and (3) the effect of acoustic-electric stimulation on conscious perception correlates with inter-regional connection changes in a recurrent neural processing model. These findings provide evidence that multimodal acoustic-electric stimulation can promote conscious perception and offer insights into its underlying mechanisms.
Structure of Dimeric Lipoprotein Lipase Reveals a Pore for Hydrolysis of Acyl Chains
Authors: Gunn Kathryn H. , Neher Saskia B. 
Published: March 2023

AbstractLipoprotein lipase (LPL) hydrolyzes triglycerides from circulating lipoproteins, releasing free fatty acids. Active LPL is needed to prevent hypertriglyceridemia, which is a risk factor for cardiovascular disease (CVD). Using cryogenic electron microscopy (cryoEM), we determined the structure of an active LPL dimer at 3.9 Å resolution. This is the first structure of a mammalian lipase with an open, hydrophobic pore adjacent to the active site. We demonstrate that the pore can accommodate an acyl chain from a triglyceride. Previously, it was thought that an open lipase conformation was defined by a displaced lid peptide, exposing the hydrophobic pocket surrounding the active site. With these previous models after the lid opened, the substrate would enter the active site, be hydrolyzed and then released in a bidirectional manner. It was assumed that the hydrophobic pocket provided the only ligand selectivity. Based on our structure, we propose a new model for lipid hydrolysis, in which the free fatty acid product travels unidirectionally through the active site pore, entering and exiting opposite sides of the protein. By this new model, the hydrophobic pore provides additional substrate specificity and provides insight into how LPL mutations in the active site pore may negatively impact LPL activity, leading to chylomicronemia. Structural similarity of LPL to other human lipases suggests that this unidirectional mechanism could be conserved but has not been observed due to the difficulty of studying lipase structure in the presence of an activating substrate. We hypothesize that the air/water interface formed during creation of samples for cryoEM triggered interfacial activation, allowing us to capture, for the first time, a fully open state of a mammalian lipase. Our new structure also revises previous models on how LPL dimerizes, revealing an unexpected C-terminal to C-terminal interface. The elucidation of a dimeric LPL structure highlights the oligomeric diversity of LPL, as now LPL homodimer, heterodimer, and helical filament structures have been elucidated. This diversity of oligomerization may provide a form of regulation as LPL travels from secretory vesicles in the cell, to the capillary, and eventually to the liver for lipoprotein remnant uptake. We hypothesize that LPL dimerizes in this active C-terminal to C-terminal conformation when associated with mobile lipoproteins in the capillary.
RNase R Controls Membrane Fatty Acid Composition inStreptococcus pneumoniae
Authors: Filipe Alípio André , Bárria Cátia , Pobre Vânia , Matos Rita , Amblar Mónica , Maria Arraiano Cecília , Domingues Susana 
Published: March 2023

AbstractPrevious studies on RNase R have highlighted significant effects of this ribonuclease in several processes ofStreptococcus pneumoniaebiology. In this work we have studied the global impact of RNase R by comparing the transcriptional landscape of a deleted RNase R mutant to that of the wild-type strain, and this led us investigate specific targets affected by RNase R. RNA-Seq showed that RNase R deletion affects transcripts from several different biological processes. Of particular interest, elimination of RNase R results in overexpression of most of the genes encoding the components of type II fatty acid biosynthesis (FAS-II) cluster. We demonstrate that RNase R governs the turnover of most of genes from this pathway, affecting the outcome of the whole FAS-II cluster, and leading to an unbalanced membrane fatty acid composition. Our results show that the membrane of the deleted strain contains a higher proportion of unsaturated and long-chained fatty acids than the wild type strain. This leads to a higher fluidity of the Δrnrmutant membrane, which is probably related with the increased sensitivity to detergent observed in this strain. We demonstrate that RNase R expression is induced in cells challenged with H2O2, which is suggestive of a role for this ribonuclease on the regulation of membrane homeostasis under oxidative stress. Reprogramming of membrane fluidity is an adaptative cell response crucial for bacterial survival in constantly changing environmental conditions. The fact that RNase R controls the expression of several essential genes to the fatty acid synthesis unveils a new important function of this enzyme.
BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine
Authors: Perera Dilhan J. , Domenech Pilar , Babuadze George Giorgi , Naghibosadat Maedeh , Alvarez Fernando , Koger-Pease Cal , Labrie Lydia , Stuible Matthew , Durocher Yves , Piccirillo Ciriaco A. , Lametti André , Fiset Pierre Olivier , Elahi Seyyed Mehdy , Kobinger Gary P. , Gilbert Rénald , Olivier Martin , Kozak Robert , Reed Michael B. , Ndao Momar 
Published: March 2023

AbstractDespite medical interventions and several approved vaccines, the COVID-19 pandemic is continuing into its third year. Recent publications have explored single-dose intranasal (i.n.) adenovirus-based vaccines as an effective strategy for curbing SARS-CoV-2 in naïve animal models. However, the effects of prior immunizations and infections have yet to be considered within these models. Here, we investigate the immunomodulatory effects ofMycobacterium bovisBCG pre-immunization on a subsequent S-protein expressing i.n. Ad vaccination, termed Ad(Spike). We found that Ad(Spike) alone conferred long-term protection from severe SARS-CoV-2 pathology within a mouse model, yet it was unable to limit initial infection 6 months post-vaccination. While i.n. Ad(Spike) retains some protective effect after 6 months, a single administration of BCG-Danish prior to Ad(Spike) vaccination potentiates its ability to control viral replication of the B.1.351 SARS-CoV-2 variant within the respiratory tract. Though BCG-Danish had no effect on the ability of Ad(Spike) to generate and maintain humoral immunity, it promoted the generation of cytotoxic and Th1 responses over suppressive FoxP3+TREGcells in the lungs of infected mice. These data demonstrate a novel vaccination strategy that may prove useful in limiting future viral pandemics by potentiating the long-term efficacy of next generation mucosal vaccines within the context of the safe and widely distributed BCG vaccine.One sentence summaryBCG enhances anti-SARS-CoV-2 immunity and protection afforded by a novel adenovirus-vectored vaccine.