bluuebunny/crossref_metadata_2025_split huggingface dataset:
A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2
Authors: Yao Hebang , Cai Hongmin , Li Tingting , Zhou Bingjie , Qin Wenming , Lavillette Dimitri , Li Dianfan 
Published: September 2020

ABSTRACTA key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.
Disinhibitory circuitry gates associative synaptic plasticity in olfactory cortex
Authors: Canto-Bustos Martha , Friason F. Kathryn , Bassi Constanza , Oswald Anne-Marie M. 
Published: September 2020

AbstractInhibitory microcircuits play an essential role in regulating cortical responses to sensory stimuli. Interneurons that inhibit dendritic or somatic integration in pyramidal neurons act as gatekeepers for neural activity, synaptic plasticity and the formation of sensory representations. Conversely, interneurons that specifically inhibit other interneurons can open gates through disinhibition. In the rodent piriform cortex, relief of dendritic inhibition permits long-term potentiation (LTP) of the recurrent synapses between pyramidal neurons (PNs) thought to underlie ensemble odor representations. We used an optogenetic approach to identify the inhibitory interneurons and disinhibitory circuits that regulate LTP. We focused on three prominent inhibitory neuron classes-somatostatin (SST), parvalbumin (PV), and vasoactive intestinal polypeptide (VIP) interneurons. We find that LTP is gated by the inactivation SST or PV interneurons or by activation of VIP interneurons. Further, activation of VIP interneurons strongly inhibits putative SST-cells during LTP induction, but only weakly inhibit PV-interneurons. Taken together, these findings suggest that VIP-interneurons mediate a disinhibitory circuit that can regulate synaptic plasticity during olfactory processing.
Increased oxidative stress tolerance of a spontaneously-occurringperRgene mutation inStreptococcus mutansUA159
Authors: Kajfasz Jessica K. , Zuber Peter , Ganguly Tridib , Abranches Jacqueline , Lemos José A. 
Published: September 2020

AbstractThe ability of bacteria such as the dental pathogenStreptococcus mutansto coordinate a response against damage-inducing oxidants is a critical aspect of their pathogenicity. The oxidative stress regulator SpxA1 has been proven to be a major player in the ability ofS. mutansto withstand both disulfide and peroxide stresses. While studying spontaneously-occurring variants of anS. mutansΔspxA1strain, we serendipitously discovered that ourS. mutansUA159 host strain bore a single nucleotide deletion within the coding region ofperR, resulting in a premature truncation of the encoded protein. PerR is a metal-dependent transcriptional repressor that senses and responds to peroxide stress such that loss of PerR activity results in activation of oxidative stress responses. To determine the impact of loss of PerR regulation, we obtained a UA159 isolated bearing an intactperRcopy and created a cleanperRdeletion mutant. Our findings indicate that loss of PerR activity results in a strain that is primed to tolerate oxidative stresses in the laboratory setting. Interestingly, RNA-Seq and targeted transcriptional expression analyses reveal that PerR has a minor contribution to the ability ofS. mutansto orchestrate a transcriptional response to peroxide stress. Furthermore, we detected loss-of-functionperRmutations in two other commonly used laboratory strains ofS. mutanssuggesting that this may be not be an uncommon occurrence. This report serves as a cautionary warning regarding the so-called domestication of laboratory strains and advocates for the implementation of more stringent strain authentication practices.ImportanceA resident of the human oral biofilm,Streptococcus mutansis one of the major bacterial pathogens associated with dental caries. This report highlights a spontaneously-occurring mutation within the laboratory strainS. mutansUA159, found in the coding region ofperR, a gene encoding a transcriptional repressor associated with peroxide tolerance. ThoughperRmutant strains ofS. mutansshowed a distinct growth advantage and enhanced tolerance toward H2O2, a ΔperRdeletion strain showed a small number of differentially expressed genes as compared to the parent strain, suggesting few direct regulatory targets. In addition to characterizing the role of PerR inS. mutans, our findings serve as a warning to laboratory researchers regarding bacterial adaptation toin vitrogrowth conditions.
TgIF2K-B is an eIF2α kinase inToxoplasma gondiithat responds to oxidative stress and optimizes pathogenicity
Authors: Augusto Leonardo , Martynowicz Jennifer , Amin Parth H. , Carlson Kenneth R. , Wek Ronald C. , Sullivan William J. 
Published: September 2020

AbstractToxoplasma gondiiis an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered byToxoplasmais suggested to impair parasite replication and dissemination. However, the strategies by whichToxoplasmamitigates oxidative stress are not completely clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eIF2. Here, we show that theToxoplasmaeIF2 kinase, TgIF2K-B, is activated in response to oxidative stress and affords protection. Knockout of TgIF2K-B, Δtgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in anin vivomodel of infection. Our results establish that TgIF2K-B is essential forToxoplasmaresponses to oxidative stress, which are important for the parasite’s ability to establish persistent infection in its host.IMPORTANCEToxoplasma gondiiis a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor, TgIF2α. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasite’s oxidative stress response and its ability to persist in the host as a latent infection.
Temporal stability and genetic diversity of 48-year old T-series phages
Authors: Subedi Dinesh , Barr Jeremy J. 
Published: September 2020

AbstractT-series phages have been model organisms for molecular biology since the 1940s. Given that these phages have been stocked, distributed, and propagated for decades across the globe, there exists the potential for genetic drift to accumulate between stocks over time. Here we compared the temporal stability and genetic relatedness of laboratory-maintained phage stocks with a T-series collection from 1972. Only the T-even phages produced viable virions. We obtained complete genomes of these T-even phages, along with two contemporary T4 stocks. Performing comparative genomics, we found 12 and 16 nucleotide variations, respectively, in the genomes of T2 and T6; whereas there were ~172 nucleotide variations between T4-sublines when compared with NCBI RefSeq genome. To account for the possibility of artefacts in the NCBI RefSeq, we used the 1972 T4 stock as a reference and compared genetic and phenotypic variations between T4-sublines. Genomic analysis predicted nucleotide variations in genes associated with DNA metabolism and structural proteins. We did not however, observe any differences in growth characteristics or host range between the T4-sublines. Our study highlights the potential for genetic drift between individually maintained T-series phage stocks, yet after 48-years, this has not resulted in phenotypic alternations in these important model organisms.ImportanceT-series bacteriophages have been used throughout the world for various molecular biology researches, which were critical for establishing the fundamentals of molecular biology – from the structure of DNA to advanced gene-editing tools. These model bacteriophages help keep research data consistent and comparable between laboratories. However, we observed genetic variability when compared contemporary sublines of T4-phages to a 48-year old stock of T4. This may have effects in the comparability of results obtained using T4 phage. Here, we highlighted the genomic differences between T4 sublines and examined phenotypic differences in phage replication parameters. We observed limited genomic changes but no phenotypic variations between T4 sublines. Our research highlights the possibility of genetic drift in model bacteriophages.
The level of oncogenic Ras controls the malignant transformation of Lkb1 mutant tissue in vivo
Authors: Rackley Briana , Seong Chang-Soo , Kiely Evan , Parker Rebecca E. , Rupji Manali , Dwivedi Bhakti , Heddleston John M. , Giang William , Anthony Neil , Chew Teng-Leong , Gilbert-Ross Melissa 
Published: September 2020

AbstractThe genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate aDrosophila melanogastermodel of Ras/Lkb1mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLo) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHi) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes ofRas/Lkb1tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignantRas/Lkb1tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncover a novel vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.One Sentence SummaryA multivariable Ras-drivenDrosophilamodel reveals a novel LKB1 mutant lung adenocarcinoma patient subpopulation and targetable effector pathway.
Structure and substrate specificity determinants of the taurine biosynthetic enzyme cysteine sulphinic acid decarboxylase
Authors: Mahootchi Elaheh , Raasakka Arne , Luan Weisha , Muruganandam Gopinath , Loris Remy , Haavik Jan , Kursula Petri 
Published: September 2020

AbstractPyridoxal 5′-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine,γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by non-enzymatic oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that similarly to its closest homologue, GADL1, CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites.
Role of the SUT1 and SUT2 sugar transporters during stolbur phytoplasma infection in tomato
Authors: Marco Federica De , Batailler Brigitte , Thorpe Michael R. , Razan Frédérique , Hir Rozenn Le , Vilaine Françoise , Bouchereau Alain , Martin-Magniette Marie-Laure , Eveillard Sandrine , Dinant Sylvie 
Published: September 2020

SummaryPhytoplasmas inhabit phloem sieve elements and cause abnormal growth and altered sugar partitioning. But how they interact with phloem functions is not clearly known. The phloem responses were investigated in tomato infected by ‘CandidatusPhytoplasma solani’, at the beginning of the symptomatic stage of infection, both in symptomatic and asymptomatic leaves, the first symptoms appearing in the sink top leaf at the stem apex. Antisense lines impaired in the phloem sucrose transporters SUT1 and SUT2 were included. The infection in source leaves was not associated with symptoms. In the symptomatic, sink leaf, yellowing and leaf curling was associated with higher starch accumulation and expression of defense genes. The transcriptional analysis of symptomatic leaf midribs indicated that transcript levels for genes acting in the glycolysis and peroxisome metabolism in infected plants differed from these in non-infected plants. Phytoplasma multiplied actively in at least three additional lower leaves although they were symptomless, with no sign of activation of defense markers, although the rate of exudation of sucrose from these symptomless, source leaves was lower for infected plants. A few metabolites in phloem-enriched exudate were affected by the infection, such as glycolate and aspartate, and some of them were also affected in the controlSUT1- andSUT2- antisense lines, in which sucrose retrieval or release in the sieve elements are impaired. A metabolic switch could explain the delivery of more glycolate into the sieve elements of infected plants. The findings suggest a link between sugar transport and redox homeostasis.One sentence summaryAn impairment of sucrose retrieval and release in the sieve elements occurs during phytoplasma infection, associated with changes in sugar and peroxisome metabolism
Prophylactic intranasal administration of a TLR2 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model
Authors: Proud Pamela C. , Tsitoura Daphne , Watson Robert J. , Chua Brendon Y , Aram Marilyn J. , Bewley Kevin R. , Cavell Breeze E. , Cobb Rebecca , Dowall Stuart , Fotheringham Susan A. , Ho Catherine M. K. , Lucas Vanessa , Ngabo Didier , Rayner Emma , Ryan Kathryn A. , Slack Gillian S. , Thomas Stephen , Wand Nadina I. , Yeates Paul , Demaison Christophe , Jackson David C. , Bartlett Nathan W. , Mercuri Francesca , Carroll Miles W. 
Published: September 2020

AbstractRespiratory viruses such as coronaviruses represent major ongoing global threats, causing epidemics and pandemics with huge economic burden. Rapid spread of virus through populations poses an enormous challenge for outbreak control. Like all respiratory viruses, the most recent novel human coronavirus SARS-CoV-2, initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission.We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT to reduce SARS-CoV-2 transmission and provide protection against COVID-19.
Molecular basis of F-actin regulation and sarcomere assemblyviamyotilin
Authors: Kostan Julius , Pavšič Miha , Puž Vid , Schwarz Thomas C. , Drepper Friedel , Molt Sibylle , Graewert Melissa Ann , Schreiner Claudia , Sajko Sara , van der Ven Peter F.M. , Svergun Dmitri I. , Warscheid Bettina , Konrat Robert , Fürst Dieter O. , Lenarčič Brigita , Djinović-Carugo Kristina 
Published: September 2020

AbstractSarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain containing protein myotilin provides structural integrity to Z-discs - the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. We used a combination of small angle X-ray scattering, cross-linking mass spectrometry, biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actinviaits Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin’s structural role in Z-discs.Significance StatementSarcomeres are the primary structural and functional unit of striated muscles, conferring movement in all animals. The Z-disk is the boundary between adjacent sarcomeres, where actin filaments (F-actin) are anchored. Z-disc protein myotilin, is a scaffold protein, which provides structural integrity to the Z-disc by multiple interactions to its central components, including F-actin and α-actinin-2. Here we provide the structure of myotilin, revealing its structural plasticity in solution and the first integrative structural model of its complex with F-actin. We further show that myotilin displaces tropomyosin from F-actin, implying a novel role of myotilin in sarcomere biogenesis beyond being an interaction hub for Z-disk partners.Highlights፧Myotilin is structurally described as a dynamic ensemble፧Flanking regions enhance F-acting binding to tandem Ig domains፧Integrative structural model of myotilin bound to F-actin፧Myotilin displaces tropomyosin from F-actin, suggesting an organisational role in Z-disc
Type III-A CRISPR systems as a versatile gene knockdown technology
Authors: Woodside Walter T. , Vantsev Nikita , Terns Michael P. 
Published: September 2020

AbstractCRISPR-Cas systems are functionally diverse prokaryotic anti-viral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidisandS. thermophilus) were each expressed on a single plasmid inE. coliand the efficiency and specificity of gene knockdown was assessed by Northern blot analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and non-coding RNAsin vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.