bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of herboxidiene features that mediate conformation-dependent SF3B1 interactions to inhibit splicing
Authors: Lopez Adriana Gamboa , Allu Srinivasa Rao , Mendez Patricia , Reddy Guddeti Chandrashekar , Maul-Newby Hannah M. , Ghosh Arun K. , Jurica Melissa S.
Published: November 2020

Small molecules that target the spliceosome SF3B complex are potent inhibitors of cancer cell growth. The compounds affect an early stage of spliceosome assembly when U2 snRNP first engages the branch point sequence of an intron. Recent cryo-EM models of U2 snRNP before and after intron recognition suggest several large-scale rearrangements of RNA and protein interactions involving SF3B. Employing an inactive herboxidiene analog as a competitor with SF3B inhibitors, we present evidence for multiple conformations of SF3B in the U2 snRNP, only some of which are available for productive inhibitor interactions. We propose that both thermodynamics and an ATP-binding event promote the conformation conducive to SF3B inhibitor interactions. However, SF3B inhibitors do not impact an ATP-dependent rearrangement in U2 snRNP that exposes the branch binding sequence for base pairing. We also report extended structure activity relationship analysis of herboxidiene, which identified features of the tetrahydropyran ring that mediate its interactions with SF3B and its ability to interfere with splicing. In combination with structural models of SF3B interactions with inhibitors, our data leads us to extend the model for early spliceosome assembly and inhibitor mechanism. We postulate that interactions between a carboxylic acid substituent of herboxidiene and positively charged SF3B1 sidechains in the inhibitor binding channel are required to maintain inhibitor occupancy and counteract the SF3B transition to a closed state that is promoted by stable U2 snRNA interactions with the intron.
γ-secretase promotes postsynaptic maturation through the cleavage of a Wnt receptor
Authors: Restrepo Lucas , DePew Alison , Moese Elizabeth , Tymanskyj Stephen , Parisi Michael , Aimino Michael , Duhart Juan Carlos , Fei Hong , Mosca Timothy J.
Published: November 2020

An emerging feature of neurodegenerative disease is synaptic dysfunction and loss, leading to the suggestion that mechanisms required for synaptic maturation may be linked to disease. Synaptic maturation requires the transmission of signals between nascent synaptic sites and the nucleus, but how these signals are generated is not well understood. We posit that proteolytic cleavage of receptors, which enables their translocation to the nucleus, may be a shared molecular mechanism between the events that promote synaptic maturation and those linked to later-onset disorders of the nervous system, including neurodegenerative disease. Here we show during synaptic development, that cleavage of synaptic maturation molecules requires γ-secretase, a protein complex linked to Alzheimer’s Disease, a devastating neurodegenerative condition, is required for postsynaptic maturation. In the absence of γ-secretase,Drosophilaneuromuscular synapses fail to appropriately recruit postsynaptic scaffolding and cytoskeletal proteins, and mutant larvae display behavioral deficits. At the NMJ, γ-secretase promotes synaptic maturation through the cleavage of the Wnt receptor Fz2, and the subsequent entry of its C-terminus into the nucleus. A developmental synaptic role for γ-secretase is also conserved in both theDrosophilacentral nervous system and mammalian cortical neuron dendrites. Finally, we found that similar maturation defects are evident in fly models for ALS, Alzheimer’s, Huntington’s, and Parkinson’s Diseases. The previously unknown, but conserved, role for γ-secretase coupled with its well-known role in neurodegenerative disease suggest that neurodevelopmental defects may be common to diverse neurodegenerative disease models.
Substrate traits shape the structure of microbial community engaged in metabolic division of labor
Authors: Wang Miaoxiao , Chen Xiaoli , Tang Yue-Qin , Nie Yong , Wu Xiao-Lei
Published: November 2020

Metabolic division of labor (MDOL) is widespread in nature, whereby a complex metabolic pathway is shared between different strains within a community for mutual benefit. However, little is known about how the mutual interactions in the microbial community engaged in MDOL are regulated. We hypothesized that when degradation of an organic compound is carried out via MDOL, the substrate traits (i.e., concentration and its toxicity) modulate the benefit allocation between the two microbial populations, thus affecting the structure of this community. We tested this hypothesis by combining mathematical modelling with experiments using engineered synthetic microbial consortia. Numerous modelling analyses suggested that the proportion of the population executing the first metabolic step can be simply estimated by Monod-like formulas governed by substrate traits. The model and the proposed formula quantitatively predicted the structure of our synthetic consortia composed of two strains degrading salicylate through MDOL. Individual-based modelling and colony pattern formation assays further indicated that our rule is also applicable to estimating community structure in spatially structured environments. Our results demonstrate that the structure of the microbial communities can be quantitatively predicted from simple environmental factors, such as substrate concentration and its toxicity, which provides novel perspectives on understanding the assembly of natural communities, as well as insights into how to manage artificial microbial systems.
High resolution 3D imaging of liver reveals a central role for subcellular architectural organization in metabolism
Authors: Parlakgül Güneş , Arruda Ana Paula , Cagampan Erika , Pang Song , Güney Ekin , Lee Yankun , Hess Harald F. , Xu C. Shan , Hotamışlıgil Gökhan S
Published: November 2020

Cells display complex intracellular organization through compartmentalization of metabolic processes into organelles, yet neither the resolution of these structures in the native tissue context nor its functional consequences are well understood. Here, we resolved the 3-dimensional organelle structural organization in large (>2.8×105μm3) volumes of intact liver tissue (15 partial or full hepatocytes per condition) in high resolution (8nm isotropic pixel size) by utilizing enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging, followed by deep-learning-based image segmentation and 3D reconstruction. We also performed a comparative analysis of subcellular structures in liver tissue of lean and obese animals and found marked alterations particularly in hepatic endoplasmic reticulum (ER), which undergoes massive structural re-organization in obesity characterized by marked disorganization of stacks of ER sheets and predominance of ER tubules. Finally, we demonstrated the functional importance of these structural changes upon experimental recovery of the subcellular organization and its marked impact on cellular and systemic metabolism. We conclude that hepatic subcellular organization and ER’s architecture is highly dynamic, integrated with the metabolic state, and critical for adaptive homeostasis and tissue health.
In ovoelectroporation of chicken limb bud ectoderm
Authors: Tomizawa Reiko R. , Tabin Clifford J. , Atsuta Yuji
Published: November 2020

Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. Here we describe have an easy method for electroporation of chick embryo limb bud ectoderm, enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region byin ovoelectroporation. This approach results in efficient transgenesis of the limb ectodermal cells. Further, using a previously describedMsx2promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. Our method is simple and cheap, and permits high throughput tests for gene function during limb ectodermal development and wound healing.
Reducing error and increasing reliability of wildlife counts from citizen science surveys: counting Weddell Seals in the Ross Sea from satellite images
Authors: Salas Leo A. , LaRue Michelle , Nur Nadav , Ainley David G. , Stammerjohn Sharon E. , Pennycook Jean , Rotella Jay , Paterson John Terrill , Siniff Don , Stamatiou Kostas , Dozier Melissa , Saints Jon , Barrington Luke
Published: November 2020

Citizen science programs can be effective at collecting information at large temporal and spatial scales. However, sampling bias is a concern in citizen science datasets and can lead to unreliable estimates. We address this issue with a novel approach in a first-of-its-kind citizen science survey of Weddell seals for the entire coast of Antarctica. Our citizen scientists inspected very high-resolution satellite images to tag any presumptive seals hauled out on the fast ice during the pupping period. To assess and reduce the error in counts in term of bias and other factors, we ranked surveyors on how well they agreed with each other in tagging a particular feature (presumptive seal), and then ranked these features based on the ranking of surveyors placing tags on them. We assumed that features with higher rankings, as determined by “the crowd wisdom,” were likely to be seals. By comparing citizen science feature ranks with an expert’s determination, we found that non-seal features were often highly ranked. Conversely, some seals were ranked low or not tagged at all. Ranking surveyors relative to their peers was not a reliable means to filter out erroneous or missed tags; therefore, we developed an effective correction factor for both sources of error by comparing surveyors’ tags to those by the expert. Furthermore, counts may underestimate true abundance due to seals not being present on the ice when the image was taken. Based on available on-the-ground haul-out location counts in Erebus Bay, the Ross Sea, we were able to correct for the proportion of seals not detected through satellite images after accounting for year, time-of-day, location (islet vs. mainland locations), and satellite sensor effects. We show that a prospective model performed well at estimating seal abundances at appropriate spatial scales, providing a suitable methodology for continent-wide Weddell Seal population estimates.
Shape and size complementarity induced formation of supramolecular protein assemblies with metal-oxo clusters
Authors: Vandebroek Laurens , Noguchi Hiroki , Kamata Kenichi , Tame Jeremy R. H. , Van Meervelt Luc , Parac-Vogt Tatjana N. , Voet Arnout R. D.
Published: November 2020

The controlled formation of protein supramolecular assemblies is challenging but it could provide an important route for the development of hybrid biomaterials. In this work, we demonstrate formation of well-defined complexes formed between the 8-fold symmetrical designer protein Tako8 and soluble metal-oxo clusters from the family of Anderson-Evans, Keggin and ZrIV- substituted Wells-Dawson polyoxometalates. A combination of x-ray crystallography and solution studies showed that metal-oxo clusters are able to serve as linker nodes for the bottom-up creation of protein based supramolecular assemblies. Our findings indicate that clusters with larger size and negative charge are capable of modulating the crystal packing of the protein, highlighting the need for a size and shape complementarity with the protein node for optimal alteration of the crystalline self-assembly.
Subtractive CRISPR screen identifies factors involved in non-canonical LC3 lipidation
Authors: Ulferts Rachel , Marcassa Elena , Timimi Lewis , Lee Liam C , Daley Andrew , Montaner Beatriz , Turner Suzanne D. , Florey Oliver , Baillie J. Kenneth , Beale Rupert
Published: November 2020

Although commonly associated with autophagosomes, LC3 can also be recruited to membranes in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. LC3 is attached to membranes by covalent lipidation that depends on recruitment of the ATG5-12-16L1 complex. Non-canonical LC3 lipidation requires the C-terminal WD40 domain of ATG16L1 that is dispensable for canonical autophagy. We devised a subtractive CRISPR knock-out screening strategy to investigate the requirements for non-canonical LC3-lipidation. This correctly identified the enzyme complexes directly responsible for LC3-lipidation. We additionally identified the RALGAP complex as important for M2-induced, but not ionophore drug induced LC3 lipidation. In contrast, we identified ATG4D as responsible for LC3 recycling in M2-induced and basal LC3-lipidation. Identification of a vacuolar ATPase subunit in the screen suggested a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug induced LC3-lipidation leads to association of the vacuolar ATPase and ATG16L1 and can be antagonised by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.
Long-chain polyphosphates impair SARS-CoV-2 infection and replication: a route for therapy in man
Authors: Ferrucci Veronica , Kong Dae-Young , Asadzadeh Fatemeh , Marrone Laura , Siciliano Roberto , Cerino Pellegrino , Criscuolo Giuseppina , Pisano Ida , Quarantelli Fabrizio , Izzo Barbara , Fusco Giovanna , Comegna Marika , Boccia Angelo , Viscardi Maurizio , Borriello Giorgia , Brandi Sergio , Pierri Bianca Maria , Tiberio Claudia , Atripaldi Luigi , Paolella Giovanni , Castaldo Giuseppe , Pascarella Stefano , Bianchi Martina , Monica Rosa Della , Chiariotti Lorenzo , Yun Kyong-Seop , Cheong Jae-Ho , Kim Hong-Yeoul , Zollo Massimo
Published: November 2020

Anti-viral activities of long-chain inorganic polyphosphates (PolyPs) against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection were investigated. In molecular docking analyses, PolyPs interacted with several conserved angiotensin-converting enzyme (ACE)2 and RNA-dependent RNA polymerase (RdRp) amino acids. We thus tested PolyPs for functional interactionsin vitroin SARS-CoV-2–infected Vero E6, Caco2 and human primary nasal epithelial cells. Immunofluorescence, qPCR, direct RNA sequencing, FISH and Immunoblotting were used to determine virus loads and transcription levels of genomic(g)RNAs and sub-genomic(sg)RNAs. We show that PolyP120 binds to ACE2 and enhances its proteasomal degradation. PolyP120 shows steric hindrance of the genomic Sars-CoV-2-RNA/RdRP complex, to impair synthesis of positive-sense gRNAs, viral subgenomic transcripts and structural proteins needed for viral replication. Thus, PolyP120 impairs infection and replication of Korean and European (containing non-synonymous variants) SARS-CoV-2 strains. As PolyPs have no toxic activities, we envision their use as a nebulised formula for oropharyngeal delivery to prevent infections of SARS-CoV-2 and during early phases of antiviral therapy.
Bivariate confidence probability plots as a method to test the accuracy and variability of microbiological measures
Authors: Monleon-Getino Antonio ,   AC Marca Home Care
Published: November 2020

Introduction

In an interlaboratory calibration analysis to validate a methodology that will be proposed as a European standard for domestic laundry disinfection, tests were carried out to detect if there are different behaviors in the measurements regarding accuracies and variabilities. Interlaboratory tests using different doses of disinfectant and microorganisms were carried out. ISO 5725-2 and ISO 13528 form the basis of validations of quantitative methods, providing validation specifications for interlaboratory studies. However, a need for a simple graphical method to detect interlaboratory differences in accuracy and variability was observed.

Objectives

The general goal of this work is to present a new exploratory methodology, graphical and easy to interpret, that can determine the accuracy and variability (precision) of a variable, and compare it to the methodology applied in ISO 5725-2 and ISO 13528.

Methods

We used confidence probability plots of the multivariate Student’s t-distribution to observe the accuracy and variability of microbiological measures carried out by different laboratories during a ring trial exercise. A function in R was built for this purpose: Miriam.analysis.ellipse(Y, factor_a, eel.plot = “ t-Student”). The different observations of accuracy and variability are represented in the ellipses. If any of the points are outside the ellipse with 95% confidence, we can assume a deviation in accuracy and / or variability.

Results

Two examples are provided with real microbiological data (logarithmic unit reductions (LR) forPseudomonas aeruginosa, Escherichia coli, Staphilococcus aureus, Enterococcus hirae, Candida albicansand microbial counts in water (WW)). The proposed new method allowed us to detect possible deviations in the WWMEA variable and we believe it has future application for the rapid control of microbiological measures.
Cell-to-cell heterogeneity in Sox2 and Brachyury expression ratios guides progenitor destiny by controlling their motility.
Authors: Romanos Michèle , Allio Guillaume , Combres Léa , Médevielle Francois , Escalas Nathalie , Soula Cathy , Steventon Ben , Trescases Ariane , Bénazéraf Bertrand
Published: November 2020

Although cell-to-cell heterogeneity in gene and protein expression within cell populations has been widely documented, we know little about its potential biological functions. We addressed this issue by studying progenitors that populate the posterior region of the vertebrate embryos, a cell population known for its capacity to self-renew or to contribute to the formation of the neural tube and paraxial mesoderm tissues. Posterior progenitors are characterized by the co-expression of Sox2 and Brachyury (Bra), two transcription factors related to neural and mesodermal lineages, respectively. In this study, we show that the respective levels of Sox2 and Bra proteins display a high degree of variability among posterior progenitors of the quail embryo. By developing forced expression and downregulation approaches, we further provide evidence that the value of the Sox2-to-Bra ratio in a given progenitor directs its choice of staying in place or exit the progenitor zone to generate neural or mesodermal cells. Time-lapse imaging together with mathematical modeling then reveal that variations of the Sox2-to-Bra ratio confer these cells heterogeneous motile behaviors. While high Bra levels display high motile properties that push cells to join the mesoderm, high levels of Sox2 tend to inhibit cell movement making cells get integrated into the neural tube. Our work thus provides evidence that the spatial heterogeneity of posterior progenitors, with regards to their relative contents of Sox2 and Bra and thus to their motile properties, is fundamental to maintain a pool of resident progenitors while others segregate to contribute to tissue formation. More broadly, our work reveals that heterogeneity among a population of progenitor cells is critical to ensure robust multi-tissue morphogenesis.
Formulation of a composite nasal spray enabling enhanced surface coverage and prophylaxis of SARS-COV-2
Authors: Moakes R. J. A. , Davies S. P. , Stamataki Z. , Grover L. M.
Published: November 2020

Airborne pathogens pose high risks in terms of both contraction and transmission within the respiratory pathways, in particular the nasal region. Although knowledge of airborne transmission has long been known, there is little in the way of adequate intervention that can protect the individual, or even prevent further spread. This study focuses on a nasal applicant with the capacity to combat such issues, by focussing on the SARS-CoV-2 virus. Formulation of a spray containing polysaccharides known for their mucoadhesive properties was undertaken and characterised for their mechanical, spray patterns and antiviral properties. The ability to engineer key behaviours such as yielding have been shown, through systematic understanding of a composite mixture containing two polymers: gellan and λcarrageenan. Furthermore, spray systems demonstrated highly potent antiviral capacities, resulting in complete inhibition of the virus when studied for both prophylaxis and prevention of spread. Finally, a mechanism has been proposed to explain such findings. Therefore, demonstrating the first fully preventative device, targeted to protect the lining of the upper respiratory pathways.
The centriolar satellite protein Cfap53/Ccdc11 facilitates the formation of the first zygotic microtubule organizing center in the zebrafish embryo
Authors: Willekers Sven , Tessadori Federico , van der Vaart Babet , Henning Heiko , Stucchi Riccardo , Altelaar Maarten , Roelen Bernard A.J. , Akhmanova Anna , Bakkers Jeroen
Published: November 2020

In embryos from most animal species a zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle in the first and all subsequent cell divisions. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the first zygotic MTOC. Here we find that zebrafish (Danio rerio) embryos lacking maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle due to a failure in proper formation of the mitotic spindle. During the first cell cycle Cfap53 co-localizes with γ-tubulin and other centrosomal and centriolar satellite proteins to the very large MTOC. Furthermore, we find that γ-tubulin localization to the MTOC is impaired in the absence of Cfap53 or when the microtubule network is disrupted. Based on these results we propose a model in which maternal and paternal Cfap53 participates in the organization of the first zygotic MTOC of the embryo. Once the zygotic MTOC is formed, Cfap53 is dispensable for MTOC formation and integrity in subsequent cell divisions.
Universally available herbal teas based on sage and perilla elicit potent antiviral activity against SARS-CoV-2 variants of concern by HMOX-1 upregulation in human cells
Authors: Le-Trilling Vu Thuy Khanh , Mennerich Denise , Schuler Corinna , Sakson Roman , Lill Julia K. , Kopczynski Dominik , Loroch Stefan , Flores-Martinez Yulia , Katschinski Benjamin , Wohlgemuth Kerstin , Gunzer Matthias , Meyer Folker , Dittmer Ulf , Sickmann Albert , Trilling Mirko
Published: November 2020

The current SARS-CoV-2/COVID-19 pandemic wreaks medical and socioeconomic havoc. Despite the availability of vaccines, cost-effective acute treatment options preventing morbidity and mortality are urgently needed. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2. Aqueous infusions prepared by boiling leaves of theLamiaceaeperilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 in therapeutic as well as prophylactic regimens. The herbal infusions exerted antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin. In conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron.
The zebrafish mutantdreammistimplicates sodium homeostasis in sleep regulation
Authors: Barlow Ida L. , Mackay Eirinn , Wheater Emily , Goel Aimee , Lim Sumi , Zimmerman Steve , Woods Ian , Prober David A. , Rihel Jason
Published: November 2020

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene,dreammist(dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expresseddmistgene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of eitherfxyd1oratp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Sinceatpa1a3aanddmistmutants have elevated intracellular Na+levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+pump function modulates neuronal excitability to maintain normal sleep behaviour.

Significance statement

Sleep is an essential behavioral state, but the genes that regulate sleep and wake states are still being uncovered. A viral insertion screen in zebrafish identified a novel sleep mutant calleddreammist, in which a small, highly-conserved transmembrane protein is disrupted. The discovery ofdreammisthighlights the importance of a class of small transmembrane-protein modulators of the sodium pump in setting appropriate sleep duration.
Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays
Authors: Mehalko Jennifer , Drew Matthew , Snead Kelly , Denson John-Paul , Wall Vanessa , Taylor Troy , Sadtler Kaitlyn , Messing Simon , Gillette William , Esposito Dominic
Published: November 2020

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.

Highlights:

Improved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parameters

Two versions of RBD show different sensitivity in serology assays

Yields of greater than 50 mg/l obtained under optimal conditions

Magnetic bead purification technology improves throughput of protein production
Heme is crucial for medium-dependent metronidazole resistance in clinical isolates ofC. difficile
Authors: Boekhoud Ilse M. , Sidorov Igor , Nooij Sam , Harmanus Céline , Bos-Sanders Ingrid M.J.G. , Viprey Virginie , Spittal Bill , Clark Emma , Davies Kerrie , Freeman Jane , Kuijper Ed J. , Smits Wiep Klaas
Published: November 2020

Until recently, metronidazole was the first-line treatment forClostridioides difficileinfection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. Here, we investigate resistance to metronidazole in a collection of clinical isolates ofC. difficile. We find that nearly all isolates demonstrate a heme-dependent increase in the minimal inhibitory concentration for metronidazole, which in some cases leads to isolates being qualified as resistant (MIC > 2 mg/L). Moreover, whole genome sequence analysis reveals a single nucleotide polymorphism in the heme responsive genehsmA, which defines a metronidazole resistant lineage of PCR ribotype 010 / multilocus sequence type 15 isolates that also includes pCD-METRO containing strains. Together our data demonstrate that heme is crucial for medium-dependent metronidazole resistance inC. difficile.
The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility
Authors: Leung Miguel Ricardo , Roelofs Marc C. , Ravi Ravi Teja , Maitan Paula , Zhang Min , Henning Heiko , Bromfield Elizabeth G. , Howes Stuart C. , Gadella Bart M. , Bloomfield-Gadêlha Hermes , Zeev-Ben-Mordehai Tzviya
Published: November 2020

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.
Production of anti-SARS-CoV-2 hyperimmune globulin from convalescent plasma
Authors: Vandeberg Peter , Cruz Maria , María Diez José , Merritt W. Keither , Santos Benjamin , Trukawinski Susan , Wellhouse Andrea , José Marta , Willis Todd
Published: November 2020

BACKGROUND

In late 2019, the SARS-CoV-2 virus emerged in China and quickly spread into a world-wide pandemic. Prior to the development of specific drug therapies or a vaccine, more immediately available treatments were sought including convalescent plasma. A potential improvement from convalescent plasma could be the preparation of anti-SARS-CoV-2 hyperimmune globulin (hIVIG).

STUDY DESIGN AND METHODS

Convalescent plasma was collected from an existing network of plasma donation centers. A caprylate/chromatography purification process was used to manufacture hIVIG. Initial batches of hIVIG were manufactured in a versatile, small-scale facility designed and built to rapidly address emerging infectious diseases.

RESULTS

Processing convalescent plasma into hIVIG resulted in a highly purified IgG product with more concentrated neutralizing antibody activity. hIVIG will allow for the administration of greater antibody activity per unit of volume with decreased potential for several adverse events associated with plasma administration. IgG concentration and IgG antibody specific to SARS-CoV-2 were increased over 10-fold from convalescent plasma to the final product. Normalized ELISA activity (per mg/mL IgG) was maintained throughout the process. Protein content in these final product batches was 100% IgG, consisting of 98% monomer and dimer forms. Potentially hazardous proteins (IgM, IgA, and anti-A, anti-B and anti-D antibodies) were reduced to minimal levels.

CONCLUSIONS

Multiple batches of anti-SARS-CoV-2 hyperimmune globulin (hIVIG) that met regulatory requirements were manufactured from human convalescent plasma. The first clinical study in which the hIVIG will be evaluated will be Inpatient Treatment with Anti-Coronavirus Immunoglobulin (ITAC) [NCT04546581].
Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse neocortex
Authors: Liang Lindsay , Darbandi Siavash Fazel , Pochareddy Sirisha , Gulden Forrest O. , Gilson Michael C. , Sheppard Brooke K. , Sahagun Atehsa , An Joon-Yong , Werling Donna M. , Rubenstein John L.R. , Šestan Nenad , Bender Kevin J. , Sanders Stephan J.
Published: November 2020

Objective

Genetic variants in the voltage-gated sodium channelsSCN1A, SCN2A, SCN3A,andSCN8Aare leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported inSCN8Aonly (18N and 18A). We aimed to quantify the expression of individual exons in the developing human neocortex.

Methods

RNA-seq data from 176 human dorsolateral prefrontal cortex samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples.

Results

In the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. ForSCN2A, SCN3A,andSCN8Aa synchronized 5N/5A transition occurs between 24 post-conceptual weeks (2ndtrimester) and six years of age. In mice, the equivalent 5N/5A transition begins at or before embryonic day 15.5. InSCN8A,over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories.

Significance

Splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.
Dorsal Periaqueductal gray ensembles represent approach and avoidance states
Authors: Reis Fernando MCV , Lee Johannes Y , Maesta-Pereira Sandra , Schuette Peter J , Chakerian Meghmik , Liu Jinhan , La-Vu Mimi Q , Tobias Brooke C , Canteras Newton S , Kao Jonathan C , Adhikari Avishek
Published: November 2020

Animals must balance needs to approach threats for risk-assessment and to avoid danger. The dorsal periaqueductal gray (dPAG) controls defensive behaviors, but it is unknown how it represents states associated with threat approach and avoidance. We identified a dPAG threat-avoidance ensemble in mice that showed higher activity far from threats such as the open arms of the elevated plus maze and a live predator. These cells were also more active during threat-avoidance behaviors such as escape and freezing, even though these behaviors have antagonistic motor output. Conversely, the threat-approach ensemble was more active during risk-assessment behaviors and near threats. Furthermore, unsupervised methods showed approach/avoidance states were encoded with shared activity patterns across threats. Lastly, the relative number of cells in each ensemble predicted threat-avoidance across mice. Thus, dPAG ensembles dynamically encode threat approach and avoidance states, providing a flexible mechanism to balance risk-assessment and danger avoidance.
Real-space quantum-based refinement for cryo-EM: Q|R#3
Authors: Wang Lum , Kruse Holger , Sobolev Oleg V. , Moriarty Nigel W. , Waller Mark P. , Afonine Pavel V. , Biczysko Malgorzata
Published: November 2020

Electron cryo-microscopy (cryo-EM) is rapidly becoming a major competitor to X-ray crystallography, especially for large structures that are difficult or impossible to crystallize. While recent spectacular technological improvements have led to significantly higher resolution three-dimensional reconstructions, the average quality of cryo-EM maps is still at the low-resolution end of the range compared with crystallography. A long-standing challenge for atomic model refinement has been the production of stereochemically meaningful models for this resolution regime. Here, it is demonstrated that including accurate model geometry restraints derived from ab initio quantum-chemical calculations (HF-D3/6-31G) can improve the refinement of an example structure (chain A of PDB entry 3j63). The robustness of the procedure is tested for additional structures with up to 7000 atoms (PDB entry 3a5x and chain C of PDB entry 5fn5) using the less expensive semi-empirical (GFN1-xTB) model. The necessary algorithms enabling real-space quantum refinement have been implemented in the latest version of qr.refine and are described here.
The crystal structure of AjiA1 reveals a novel structural motion mechanism in the adenylate-forming enzyme family
Authors: de Paiva Fernanda C. R. , Chan Karen , Samborskyy Markiyan , Silber Ariel M. , Leadlay Peter F. , Dias Marcio V. B.
Published: November 2020

Adenylate-forming enzymes (AFEs) are a mechanistic superfamily of proteins that are involved in many cellular roles. In the biosynthesis of benzoxazole antibiotics, an AFE has been reported to play a key role in the condensation of cyclic molecules. In the biosynthetic gene cluster for the benzoxazole AJI9561, AjiA1 catalyzes the condensation of two 3-hydroxyanthranilic acid (3-HAA) molecules using ATP as a co-substrate. Here, the enzymatic activity of AjiA1 is reported together with a structural analysis of its apo form. The structure of AjiA1 was solved at 2.0 Å resolution and shows a conserved fold with other AFE family members. AjiA1 exhibits activity in the presence of 3-HAA (K
m = 77.86 ± 28.36, k
cat = 0.04 ± 0.004) and also with the alternative substrate 3-hydroxybenzoic acid (3-HBA; K
m = 22.12 ± 31.35, k
cat = 0.08 ± 0.005). The structure of AjiA1 in the apo form also reveals crucial conformational changes that occur during the catalytic cycle of this enzyme which have not been described for any other AFE member. Consequently, the results shown here provide insights into this protein family and a new subgroup is proposed for enzymes that are involved in benzoxazole-ring formation.
Influence of the presence of the heme cofactor on the JK-loop structure in indoleamine 2,3-dioxygenase 1
Authors: Mirgaux Manon , Leherte Laurence , Wouters Johan
Published: November 2020

Indoleamine 2,3-dioxygenase 1 has sparked interest as an immunotherapeutic target in cancer research. Its structure includes a loop, named the JK-loop, that controls the orientation of the substrate or inhibitor within the active site. However, little has been reported about the crystal structure of this loop. In the present work, the conformation of the JK-loop is determined for the first time in the presence of the heme cofactor in the active site through X-ray diffraction experiments (2.44 Å resolution). Molecular-dynamics trajectories were also obtained to provide dynamic information about the loop according to the presence of cofactor. This new structural and dynamic information highlights the importance of the JK-loop in confining the labile heme cofactor to the active site.
The copper(II)-binding tripeptide GHK, a valuable crystallization and phasing tag for macromolecular crystallography
Authors: Mehr Alexander , Henneberg Fabian , Chari Ashwin , Görlich Dirk , Huyton Trevor
Published: November 2020

The growth of diffraction-quality crystals and experimental phasing remain two of the main bottlenecks in protein crystallography. Here, the high-affinity copper(II)-binding tripeptide GHK was fused to the N-terminus of a GFP variant and an MBP-FG peptide fusion. The GHK tag promoted crystallization, with various residues (His, Asp, His/Pro) from symmetry molecules completing the copper(II) square-pyramidal coordination sphere. Rapid structure determination by copper SAD phasing could be achieved, even at a very low Bijvoet ratio or after significant radiation damage. When collecting highly redundant data at a wavelength close to the copper absorption edge, residual S-atom positions could also be located in log-likelihood-gradient maps and used to improve the phases. The GHK copper SAD method provides a convenient way of both crystallizing and phasing macromolecular structures, and will complement the current trend towards native sulfur SAD and MR-SAD phasing.
The influence of the shoulder depth on the properties of the thin sheet joint made by FSW technology
Authors: Burek Rafał , Wydrzyński Dawid , Kubit Andrzej , Łogin Waldemar
Published: November 2020

Purpose

This paper aims to experimentally determine the influence of the tool shoulder depth value on the structural and strength properties of the single lap joints made of 7075-T6 aluminium alloy made with friction stir welding (FSW) technology. The aim of the preliminary tests is to optimize the parameters of joining process of thin-walled structures such as the skin-stringer joint or skin-frame joint of the aircraft fuselage. The tests were carried out for materials commonly used in such structures, i.e. 1.6 mm thick sheet 7075–T6 aluminium alloy with cladding on both sides (cladding thickness 4% per each side). The layer of clad protects plates from corrosion.

Design/methodology/approach

This study shows the results of the investigation for the joining of 7075–T6 ALCLAD aluminium alloy sheets. The welding process was carried out on a computer numerical control milling machine SOLARUCE TA–20A. Linear FSW welding was performed using a commercial tool from RSS SCHILLING with the symbol 10–K–4–Z–M–O, which is fabricated of hot work tool steel. Constant parameters of the technological process were applied. The welding process was executed for different values of the shoulder depth ZS.

Findings

This paper investigated the dependence between the thinning of the welded material and the depth value of the tool shoulder during the FSW process. The influence of the depth value of tool shoulder on joint strength in the static tensile/shear test was also performed. With the increase of the depth of the tool, the size of flash and structures of the face of the joint changes as well (its annular surface resulting from the tool’s work and the accompanying process of material flow on the run-off side). Such conditions in the process require a proper tool depression to reduce the occurrence of flash and minimize material thinning to achieve high joint strength and maintain the conditions for plasticizing the material.

Practical implications

Based on the experimental tests carried out, a number of guidelines for the correct conduct of the welding process can be outlined.

Originality/value

Taking into account the various aspects of the process, the optimal range of the tool depth into the material is a value of approximately 0.06 mm. At this value, the face of the weld is not porous; the flash is easily removed; and the strength of the joint and the deformation of contact line are at an acceptable quality level.
Mobile money system and market risk mitigation: an econometric case study of Kenya's farm business
Authors: Gopane Thabo J.
Published: November 2020

Purpose

The study presents and evaluates a proposition that there is market information bundled with the mobile money (m-money) system. Also, that such information is disseminated to farmers in the process of their normal financial transactions and business interactions. This market information is important to mitigate the price uncertainty of farm products.

Design/methodology/approach

The empirical analysis is conducted with a two-stage econometric procedure. First, a probit model is used to estimate the likelihood of m-money take-up by farm entrepreneurs. Second, the ordinary least squares model is used to regress the farming price uncertainty index on the m-money take-up, which is quantified with a set of predicted values from the probit model in the first stage. The data used in econometric estimation come from Kenya's FinAccess Household Survey published in 2016.

Findings

The results of this paper show a negative effect of m-money service take-up on the price uncertainty index. A one unit increase in the m-money system is associated with a 12% reduction in the price uncertainty index. The authors interpret this as saying that access to the m-money system by farm business has a beneficial spillover effect on product risk mitigation, ceteris paribus.

Research limitations/implications

The implication of this research is that policy initiatives which support farm entrepreneurs to access the m-money system should be encouraged not only for their financial inclusion advantage but also for their positive externality on product risk amelioration for rural farm businesses.

Originality/value

The paper introduces an idea that the m-money system is bundled with relevant information for mitigating price risk to the benefit of rural farm businesses.
Attitude and labelling preferences towards gene-edited food: a consumer study amongst millennials and Generation Z
Authors: Ferrari Linda , Baum Chad M. , Banterle Alessandro , De Steur Hans
Published: November 2020

Purpose

This study jointly examines consumer attitudes towards gene-edited (GE) food and their preferences for labelling such products. Thus, it contributes to understanding the role of educational background, objective/subjective knowledge, environmental concern and socio-demographics in the context of GE food.

Design/methodology/approach

An online survey was administered to two generations of young individuals (millennials and Generation Z; n = 234) from two neighbouring European Union (EU) regions (Belgium and The Netherlands), which have a stringent policy on (labelling) genetically modified (GM) food. Ordinary least squares (OLS) and ordered logit models (OLMs) were employed to identify key determinants of attitudes towards GE food and GE labelling preferences, respectively.

Findings

Attitudes towards GE food were determined by environmental concern (negative) and objective knowledge (positive). Key factors influencing preferences for GE labelling were a non-hard-scientific background, knowledge about relevant policies and a negative attitude towards GE food. Preference for applying a similar labelling policy to both GM and GE was itself linked to having low, objective EU policy-related GM food knowledge and one's nationality.

Originality/value

This is one of the first studies to examine consumer attitudes towards GE food products, while also addressing a lack of research on GE food labelling preferences. By highlighting the preferences of young generations for a revised policy approach, this study sheds new light on the current GE debate, notably, by promoting a deeper understanding of a group which has so far received limited attention in the discourse on the acceptance of novel plant-breeding technologies.