bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of aMonascussp. red yeast rice extract on germination of bacterial spores
Authors: Marketa Husakova , Michaela Plechata , Barbora Branska , Petra Patakova 
Published: March 2021

AbstractThe pink-red colour of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant colour of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungusMonascusis known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for colouring foods, including meat products. Although, different biological activities ofMonascuspigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination ofClostridiumandBacillusspores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination ofC. beijerinckiispores but had no effect onB. subtilisspores. In contrast, total suppression ofC. beijerinckiispore germination was reached after addition of RYR extract to the medium at a concentration of 2 % v/v. ForB. subtilis, total inhibition of spore germination was observed only after addition of 4 % v/v RYR extract to the medium containing 1.3% w/w NaCl.
Comparison of SARS-CoV-2 VOC 202012/01 (UK variant) and D614G variant transmission by different routes in Syrian hamsters
Authors: Mohandas Sreelekshmy , Yadav Pragya D , Nyayanit Dimpal , Shete-Aich Anita , Sarkale Prasad , Hundekar Supriya , Kumar Sanjay , Lole Kavita 
Published: March 2021

AbstractMany SARS-CoV-2 variants of concern has been reported recently which were linked to increased transmission. In our earlier study on virus shedding using VOC 202012/01(UK variant) and D614G variant in hamster model, we observed significantly higher viral RNA shedding through nasal wash in case of UK variant. Hence, we compared the transmission of both the UK and D614G variant by various routes in Syrian hamsters to understand whether the high viral RNA shedding could enhance the transmission efficiency of the variant. The current study demonstrated comparable transmission efficiency of both UK and D614G variants of SARS-CoV-2 in Syrian hamsters.
In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M
Authors: Gao Li , Meiring Joyce C.M. , Varady Adam , Ruider Iris E. , Heise Constanze , Wranik Maximilian , Velasco Cecilia D. , Taylor Jennifer A. , Terni Beatrice , Standfuss Jörg , Cabernard Clemens C. , Llobet Artur , Steinmetz Michel O. , Bausch Andreas R. , Distel Martin , Thorn-Seshold Julia , Akhmanova Anna , Thorn-Seshold Oliver 
Published: March 2021

AbstractPhotoswitchable reagents to modulate microtubule stability and dynamics are an exciting tool approach towards micron- and millisecond-scale control over endogenous cytoskeleton-dependent processes. When these reagents are globally administered yet locally photoactivated in 2D cell culture, they can exert precise biological control that would have great potential forin vivotranslation across a variety of research fields and for all eukaryotes. However, photopharmacology’s reliance on the azobenzene photoswitch scaffold has been accompanied by a failure to translate this temporally- and cellularly-resolved control to 3D models or toin vivoapplications in multi-organ animals, which we attribute substantially to the metabolic liabilities of azobenzenes.Here, we optimised the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based instead on the styrylbenzothiazole (SBT) photoswitch scaffold, that are non-responsive to the major fluorescent protein imaging channels and so enable multiplexed imaging studies. We applied these reagents to 3D systems (organoids, tissue explants) and classic model organisms (zebrafish, clawed frog) with one- and two-protein imaging experiments. We successfully used systemic treatment plus spatiotemporally-localised illuminationsin vivoto photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in these systems with cellular precision and second-level resolution. These nanomolar,in vivo-capable photoswitchable reagents can prove a game-changer for high-precision cytoskeleton research in cargo transport, cell motility, cell division and development. More broadly, their straightforward design can also inspire the development of similarly capable optical reagents for a range of protein targets, so bringing generalin vivophotopharmacology one step closer to productive realisation.
Thriving of hyperthermophilic microbial communities from a deep-sea sulfidic hydrothermal chimney under electrolithoautotrophic conditions with nitrate as electron acceptor
Authors: Pillot G. , Davidson S. , Shintu L. , Tanet L. , Combet-Blanc Y. , Godfroy A. , Bonin P. , Liebgott P.-P. 
Published: March 2021

AbstractRecent studies have shown the presence of an abiotic electrical current across the walls of deep-sea hydrothermal chimneys, allowing the growth of electroautotrophic microbial communities. To understand the role of the different phylogenetic groups and metabolisms involved, this study focused on an electrotrophic enrichment, with nitrate as electron acceptor. The biofilm density, the community composition, the organic products released, and the electrical consumption were monitored by FISH confocal microscopy, qPCR, Metabarcoding, MNR and potentiostat measurements. A statistic analysis by PCA showed the correlation between the different parameters in 3 distinct temporal phases. TheArchaeoglobaleshave been shown to play a key role in the development of the community, as first colonizers and producing pyruvate, therefor used as organic source for heterotrophs. SomeThermococcalesshowed the ability to perform electrofermentation of this pyruvate into acetate and H2. Finally, through subcultures of the community, we showed the development of a larger biodiversity over time. This observed phenomenon could explain the biodiversity development in hydrothermal context where energy sources are transient and unstable.
Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon
Authors: Kamel Michael , Löwe Maryna , Schott-Verdugo Stephan , Gohlke Holger , Kedrov Alexej 
Published: March 2021

AbstractThe translocon SecYEG forms the primary protein-conducting channel in the cytoplasmic membrane of bacteria, and the associated ATPase SecA provides the energy for the transport of secretory and cell envelope protein precursors. The translocation requires negative charge at the lipid membrane surface, but its dependence on the properties of the membrane hydrophobic core is not known. Here, we demonstrate that SecA:SecYEG-mediated protein transport is immensely stimulated by unsaturated fatty acids (UFAs). Furthermore, UFA-rich tetraoleoyl-cardiolipin, but not bis(palmitoyloleoyl)-cardiolipin, facilitate the translocation via the monomeric translocon. Biophysical analysis and molecular dynamics simulations show that UFAs determine the loosely packed membrane interface, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced manifold at elevated ionic strength. Tight SecA:lipid interactions convert into the augmented translocation. As bacterial cells actively change their membrane composition in response to their habitat, the modulation of SecA:SecYEG activity via the fatty acids may be crucial for protein secretion over a broad range of environmental conditions.
The RNA sensor MDA5 detects SARS-CoV-2 infection
Authors: Sampaio Natalia G. , Chauveau Lise , Hertzog Jonny , Bridgeman Anne , Fowler Gerissa , Moonen Jurgen P. , Dupont Maeva , Russell Rebecca A. , Noerenberg Marko , Rehwinkel Jan 
Published: March 2021

AbstractHuman cells respond to infection by SARS-CoV-2, the virus that causes COVID-19, by producing cytokines including type I and III interferons (IFNs) and proinflammatory factors such as IL6 and TNF. IFNs can limit SARS-CoV-2 replication but cytokine imbalance contributes to severe COVID-19. We studied how cells detect SARS-CoV-2 infection. We report that the cytosolic RNA sensor MDA5 was required for type I and III IFN induction in the lung cancer cell line Calu-3 upon SARS-CoV-2 infection. Type I and III IFN induction further required MAVS and IRF3. In contrast, induction of IL6 and TNF was independent of the MDA5-MAVS-IRF3 axis in this setting. We further found that SARS-CoV-2 infection inhibited the ability of cells to respond to IFNs. In sum, we identified MDA5 as a cellular sensor for SARS-CoV-2 infection that induced type I and III IFNs.
Genetic Variant in 3’ Untranslated Region of the MousePycardGene Regulates Inflammasome Activity
Authors: Ritchey Brian , Hai Qimin , Han Juying , Barnard John , Smith Jonathan D. 
Published: March 2021

AbstractQuantitative trait locus mapping for interleukin-1β release after inflammasome priming and activation was performed on bone marrow-derived macrophages (BMDM) from an AKRxDBA/2 strain intercross. The strongest associated locus mapped very close to thePycardgene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKRPycardgenes only differ at single nucleotide polymorphism (SNP) in their 3’ untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels ofPycardmRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increasedPycardmRNA stability without an increased transcription rate. CRIPSR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change thePycard3’UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reducedPycardexpression and inflammasome activity after cells were differentiated into macrophages due to reducedPycardmRNA stability.
Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles
Authors: Sitarska Ewa , Almeida Silvia Dias , Beckwith Marianne Sandvold , Stopp Julian , Schwab Yannick , Sixt Michael , Kreshuk Anna , Erzberger Anna , Diz-Muñoz Alba 
Published: March 2021

AbstractCell migration is a hallmark out-of-equilibrium process in biology. In addition to persistent self-propelled motion, many cells display remarkable adaptive behaviors when they navigate complex environments within the body. Combining theory and experiments, we identify a curvature-sensing mechanism underlying obstacle avoidance in immune-like cells. The genetic perturbation of this machinery leads to a reduced capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. We propose that the active polymerization of the actin cytoskeleton at the advancing edge of migrating cells is locally inhibited by the curvature-sensitive BAR protein Snx33 in regions with inward plasma membrane curvature. This coupling between actin and membrane dynamics leads to a mechanochemical instability that generates complex protrusive patterns at the cellular front. Adaptive motility thus arises from two simultaneous curvature-dependent effects, i) the specific reduction of propulsion in regions where external objects deform the plasma membrane and ii) the intrinsic patterning capacity due to the membrane-actin coupling that promotes spontaneous changes in the cell’s protrusions. Our results show how cells utilize actin- and plasma membrane biophysics to sense their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment.
Structures ofBdellovibrio bacteriovorusphosphoglucose isomerase reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding
Authors: Meek R.W. , Cadby I.T. , Lovering A.L. 
Published: March 2021

AbstractGlycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI) which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate (F6P). The predatory bacteriumBdellovibrio bacteriovorusleads a complex lifecycle, switching between intraperiplasmic replicative and extracellular “hunter” attack-phase stages. Passage through this complex lifecycle involves different metabolic states. Here we present the unliganded and substrate bound structures of theBdellovibrio bacteriovorusPGI, solved to 1.74Å and 1.67 Å, respectively. These structures reveal that an induced-fit conformational change within the active site is not a pre-requisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for a glycine inBdellovibrioand other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterised to date and likely represents the minimal requirements for a functional PGI.
Development and Validation of Fluorescently Labeled, Functional Type I Collagen Molecules
Authors: Siadat Seyed Mohammad , Susilo Monica E. , Paten Jeffrey A. , Silverman Alexandra A. , DiMarzio Charles A. , Ruberti Jeffrey W. 
Published: March 2021

AbstractWhile de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are quantified for the first time. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ~1.2 labels preserves them. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labelled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development, is endocytosed rapidly by cells and is translocated into synthesized matrix networks within 24 hours.
Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs inChlamydomonas
Authors: Akella Soujanya , Ma Xinrong , Bacova Romana , Harmer Zachary P. , Kolackova Martina , Wen Xiaoxue , Wright David A. , Spalding Martin H. , Weeks Donald P. , Cerutti Heriberto 
Published: March 2021

AbstractProgrammable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas. However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. In this study, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We usedPPX1(encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precisely, scarless editedFTSYorWDTC1genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures.One sentence summaryCo-targeting two genes by co-electroporation of CRISPR/Cas9 RNPs and ssODN repair templates allows concomitant genome editing to create a selectable marker gene and to introduce precise modifications in another gene of interest.
Beyond fish eDNA metabarcoding: Field replicates disproportionately improve the detection of stream associated vertebrate species
Authors: Macher Till-Hendrik , Schütz Robin , Arle Jens , Beermann Arne J. , Koschorreck Jan , Leese Florian 
Published: March 2021

AbstractFast, reliable, and comprehensive biodiversity monitoring data are needed for environmental decision making and management. Recent work on fish environmental DNA (eDNA) metabarcoding shows that aquatic diversity can be captured fast, reliably, and non-invasively at moderate costs. Because water in a catchment flows to the lowest point in the landscape, often a stream, it can often collect traces of terrestrial species via surface or subsurface runoff along its way or when specimens come into direct contact with water (e.g., for drinking purposes). Thus, fish eDNA metabarcoding data can provide information on fish but also on other vertebrate species that live in riparian habitats. This additional data may offer a much more comprehensive approach for assessing vertebrate diversity at no additional costs. Studies on how the sampling strategy affects species detection especially of stream-associated communities, however, are scarce. We therefore performed an analysis on the effects of biological replication on both fish as well as (semi-)terrestrial species detection. Along a 2 km stretch of the river Mulde (Germany), we collected 18 1-L water samples and analyzed the relation of detected species richness and quantity of biological replicates taken. We detected 58 vertebrate species, of which 25 were fish and lamprey, 18 mammals, and 15 birds, which account for 50%, 24%, and 7% of all native species to the German federal state of Saxony-Anhalt. However, while increasing the number of biological replicates resulted in only 25% more detected fish and lamprey species, mammal, and bird species richness increased disproportionately by 69% and 84%, respectively. Contrary, PCR replicates showed little stochasticity. We thus emphasize to increase the number of biological replicates when the aim is to improve general species detections. This holds especially true, when the focus is on rare aquatic taxa or on (semi-)terrestrial species, the so-called ‘bycatch’. As a clear advantage, this information can be obtained without any additional sampling or laboratory effort when the sampling strategy is chosen carefully. With the increased use of eDNA metabarcoding as part of national fish bioassessment and monitoring programs, the complimentary information provided on bycatch can be used for biodiversity monitoring and conservation on a much broader scale.
Systematic Profiling of Temperature- and Retinal-Sensitive Rhodopsin Variants by Deep Mutational Scanning
Authors: McKee Andrew G. , Kuntz Charles P. , Ortega Joseph T. , Woods Hope , Roushar Francis J. , Meiler Jens , Jastrzebska Beata , Schlebach Jonathan P. 
Published: March 2021

ABSTRACTMembrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of “temperature-sensitive” variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and an investigational corrector (9-cis-retinal) on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures is correlated with the response to retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants within this helix appear to disrupt a network of hydrogen bonds that stabilizes a native helical kink. By comparison, mutants that alter a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest this insensitivity primarily arises from an abundance of mutations that enhance its membrane integration, stabilize its native conformation, and/ or perturb the retinal binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings elucidate various factors that mediate the sensitivity of genetic variants to temperature and to small molecule correctors.