bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain
Authors: Wolff Ian D. , Hollis Jeremy A. , Wignall Sarah M. 
Published: June 2021

SUMMARYDuring the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes [1]. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity [2–7]. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force [8]. Instead, the kinesin-12 family motor KLP-18 performs this function [9–12]. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles [12]. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.
Exosomal TAR DNA binding protein 43 profile in canine model of amyotrophic lateral sclerosis: A preliminary study in developing blood-based biomarker for neurodegenerative diseases
Authors: Pfeiffer Penelope , Coates Joan R. , Esqueda Yajaira M. , Kennedy Andrew , Getchell Kyleigh , McLenon Myra , Kosa Edina , Agbas Abdulbaki 
Published: June 2021

AbstractObjectiveBlood-based biomarkers provide a crucial information in progress of neurodegenerative diseases with minimally invasive sampling method. Validated blood-based biomarker application in people with amyotrophic lateral sclerosis would derive numerous benefits. Canine degenerative myelopathy is a naturally occurring animal disease model to study the biology of human amyotrophic lateral sclerosis. Serum derived exosomes are potential carriers for cell-specific cargoes making them ideal venue to study biomarkers for a variety of diseases and biological processes. This study assessed the exosomal proteins that may be assigned as surrogate biomarker that may reflect biochemical changes in central nervous system.MethodsExosomes were isolated from canine serum using commercial exosome isolation reagents. Exosomes target proteins contents were analysed by Western blotting method.ResultsThe profiles of potential biomarker candidates in spinal cord homogenate and that of serum-derived exosomes were found elevated in dogs with degenerative myelopathy as compare to control subjects.ConclusionsSerum-derived exosomal biomolecules can serve as surrogate biomarkers in neuro degenerative diseases.Key MessagesA canine with degenerative myelopathy can serve as a model animal to study human amyotrophic lateral sclerosis.Serum-derived exosomes contains Transactive Response DNA Binding Protein 43 (TDP-43), potential biomarker candidate.The levels of spinal cord TDP-43 proteins and that of serum-derived exosomes exhibited a similar profiling. Therefore, serum derived exosomes may be used as a venue for establishing blood-based biomarkers for neurodegenerative diseases.
Predicting microbiome compositions from species assemblages through deep learning
Authors: Michel-Mata Sebastian , Wang Xu-Wen , Liu Yang-Yu , Angulo Marco Tulio 
Published: June 2021

AbstractMicrobes can form complex communities that perform critical functions in maintaining the integrity of their environment or their hosts’ well-being. Rationally managing these microbial communities requires improving our ability to predict how different species assemblages affect the final species composition of the community. However, making such a prediction remains challenging because of our limited knowledge of the diverse physical, biochemical, and ecological processes governing microbial dynamics. To overcome this challenge, here we present a deep learning framework that automatically learns the map between species assemblages and community compositions from training data only, without knowledge of any of the above processes. First, we systematically validate our framework using synthetic data generated by classical population dynamics models. Then, we apply it to experimental data of both in vitro and in vivo communities, including ocean and soil microbial communities, Drosophila melanogaster gut microbiota, and human gut and oral microbiota. In particular, we show how our framework learns to perform accurate out-of-sample predictions of complex community compositions from a small number of training samples. Our results demonstrate how deep learning can enable us to understand better and potentially manage complex microbial communities.
Light-induction of endocannabinoids and activation of Drosophila TRPC channels
Authors: Sokabe Takaaki , Bradshaw Heather B. , Tominaga Makoto , Leishman Emma , Montell Craig 
Published: June 2021

AbstractDrosophila phototransduction represents a classical model for signaling cascades that culminate with activation of TRP channels. TRP and TRPL are the canonical TRP (TRPC) channels, which are gated by light stimulation of rhodopsin and engagement of Gq and phospholipase Cβ (PLC). Despite decades of investigation, the mechanism of TRP activation in photoreceptor cells is unresolved. Here, using a combination of genetics, lipidomics and Ca2+ imaging, we found that light increased the levels of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG) in vivo. The elevation in 2-LG strictly depended on the PLC encoded by norpA. Moreover, this endocannabinoid upregulated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.
Structural basis for the context-specific action of classic peptidyl transferase inhibitors
Authors: Syroegin Egor A. , Flemmich Laurin , Klepacki Dorota , Vazquez-Laslop Nora , Micura Ronald , Polikanov Yury S. 
Published: June 2021

ABSTRACTRibosome-targeting antibiotics serve both as powerful antimicrobials and as tools for studying the ribosome. The ribosomal catalytic site, the peptidyl transferase center (PTC), is targeted by a large number of various drugs. The classical and best-studied PTC-acting antibiotic chloramphenicol, as well as the newest clinically significant linezolid, were considered indiscriminate inhibitors of every round of peptide bond formation, presumably inhibiting protein synthesis by stalling ribosomes at every codon of every gene being translated. However, it was recently discovered that chloramphenicol or linezolid, and many other PTC-targeting drugs, preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms and structural bases that underlie this phenomenon of context-specific action of even the most basic ribosomal antibiotics, such as chloramphenicol, are unknown. Here we present high-resolution structures of ribosomal complexes, with or without chloramphenicol, carrying specific nascent peptides that support or negate the drug action. Our data suggest that specific amino acids in the nascent chains directly modulate the antibiotic affinity to the ribosome by either establishing specific interactions with the drug molecule or obstructing its placement in the binding site. The model that emerged from our studies rationalizes the critical importance of the penultimate residue of a growing peptide for the ability of the drug to stall translation and provides the first atomic-level understanding of context specificity of antibiotics that inhibit protein synthesis by acting upon the PTC.
Electrodetection of small molecules by conformation-mediated signal enhancement
Authors: Murugappan Krishnan , Sundaramoorthy Uthayasuriya , Damry Adam M. , Nisbet David R. , Jackson Colin J. , Tricoli Antonio 
Published: June 2021

AbstractElectrochemical biosensors allow the rapid, selective, and sensitive transduction of critical biological parameters into measurable signals. However, current electrochemical biosensors often fail to selectively and sensitively detect small molecules due to their small size and low molecular complexity. We have developed an electrochemical biosensing platform that harnesses the analyte-dependent conformational change of highly selective solute-binding proteins to amplify the signal generated by analyte binding. Using this platform, we constructed and characterized two biosensors that can sense leucine and glycine, respectively. We show that these biosensors can selectively and sensitively detect their targets over a wide range of concentrations – up to seven orders of magnitude – and that the selectivity of these sensors can be readily altered by switching the bioreceptor’s binding domain. Our work represents a new paradigm for the design of a family of modular electrochemical biosensors, where access to electrode surfaces can be controlled by protein conformational change.
Generation and transmission of inter-lineage recombinants in the SARS-CoV-2 pandemic
Authors: Jackson Ben , Boni Maciej F , Bull Matthew J , Colleran Amy , Colquhoun Rachel M , Darby Alistair , Haldenby Sam , Hill Verity , Lucaci Anita , McCrone John T , Nicholls Samuel , O’Toole Áine , Pacchiarini Nicole , Poplawski Radoslaw , Scher Emily , Todd Flora , Webster Hermione , Whitehead Mark , Wierzbicki Claudia ,   The COVID-19 Genomics UK (COG-UK) consortium, Loman Nicholas J , Connor Thomas R , Robertson David L , Pybus Oliver G , Rambaut Andrew 
Published: June 2021

SummaryWe present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern, but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of two months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7’s set of mutations.
Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis
Authors: Živković Dušan , Sanchez Dafun Angelique , Menneteau Thomas , Schahl Adrien , Lise Sandrine , Kervarrec Christine , Toste Rêgo Ana , da Fonseca Paula C. A. , Chavent Matthieu , Pineau Charles , Burlet-Schiltz Odile , Marcoux Julien , Bousquet Marie-Pierre 
Published: June 2021

AbstractDuring spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82-92%) during differentiation, presumably due to the shift from α4 to α4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S, and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners, and dictate its role in germ cell differentiation.
Clathrin-independent endocytosis of Human Papillomaviruses is facilitated by actin nucleation promoting factor WASH
Authors: Brinkert Pia , Krebs Lena , Samperio Ventayol Pilar , Greune Lilo , Bannach Carina , Bucher Delia , Kollasser Jana , Dersch Petra , Boulant Steeve , Stradal Theresia E.B. , Schelhaas Mario 
Published: June 2021

SummaryEndocytosis is a fundamental cellular process facilitated by diverse mechanisms. Remarkably, several distinct clathrin-independent endocytic processes were identified and characterized following virus uptake into cells. For some, however, mechanistic execution and biological function remain largely unclear. This includes an endocytic process exploited by human papillomavirus type 16 (HPV16). Using HPV16, we examined how vesicles are formed by combining systematic cellular perturbations with electron and video microscopy. Cargo uptake occurred by uncoated, inward-budding pits. Mechanistically, vesicle scission was facilitated by actin polymerization controlled through the actin nucleation promoting factor WASH. While WASH typically functions in conjunction with the retromer complex on endosomes during retrograde trafficking, endocytic vesicle formation was largely independent of retromer itself and the heterodimeric membrane-bending SNX-BAR retromer adaptor, thereby uncovering a role of WASH in endocytosis in addition to its canonical role in intracellular membrane trafficking.
The widespread nature of Pack-TYPE transposons reveals their importance for plant genome evolution
Authors: Gisby Jack S. , Catoni Marco 
Published: June 2021

AbstractPack-TYPE transposable elements (TEs) are a group of non-autonomous DNA transposons found in plants. These elements can efficiently capture and shuffle coding DNA across the host genome, accelerating the evolution of genes. Despite their relevance for plant genome plasticity, the detection and study of Pack-TYPE TEs are challenging due to the high similarity these elements have with genes. Here, we produced an automated annotation procedure designed to study Pack-TYPE elements and used it to successfully annotate and analyse more than 4000 new Pack-TYPE TEs in the rice and maize genomes. Our analysis indicates that Pack-TYPE TEs are an abundant and heterogeneous group of elements. We found that these elements are associated with all main superfamilies of Class II DNA transposons in plants and likely share a similar mechanism to capture new chromosomal DNA sequences. Furthermore, we report examples of the direct contribution of these TEs to coding genes, suggesting a generalised and extensive role of Pack-TYPE TEs in plant genome evolution.
A mutational gradient drives somatic mutation accumulation in mitochondrial DNA and influences germline polymorphisms and genome composition
Authors: Sanchez-Contreras Monica , Sweetwyne Mariya T. , Kohrn Brendan F. , Tsantilas Kristine A. , Hipp Michael J , Schmidt Elizabeth K. , Fredrickson Jeanne , Whitson Jeremy A. , Campbell Matthew D. , Rabinovitch Peter S. , Marcinek David J. , Kennedy Scott R. 
Published: June 2021

AbstractBackgroundMutations in the mitochondrial genome (mtDNA) can cause devastating maternally inherited diseases, while the accumulation of somatic mtDNA mutations is linked to common diseases of aging. Although mtDNA mutations impact human health, the process(es) that give rise to these mutations are unclear and are under considerable debate. We analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing to provide clues to the mechanism by which de novo mutations arise as well as how the genome is replicated.ResultsWe observe two distinct mutational gradients in G→A and T→C transitions, but not their complements, that are delimited by the light-strand origin and the control region (CR). The gradients increase with age and are lost in the absence of DNA polymerase γ proofreading activity. A nearly identical pattern is present in human mtDNA somatic mutations. The distribution of mtDNA single nucleotide polymorphisms (SNPs) in the human population and genome base composition across >3,000 vertebrate species mirror this gradient pattern, pointing to evolutionary conservation of this phenomenon. Lastly, high-resolution analysis of the mtDNA control region highlights mutational ‘hotspots’ and ‘cold-spots’ that strongly align with important regulatory regions.ConclusionsCollectively, these patterns support an asymmetric strand-displacement mechanism with key regulatory structures in the CR and argue against alternative replication models. The mutational gradient is a fundamental consequence of mtDNA replication that drives somatic mutation accumulation and influences inherited polymorphisms and, over evolutionary timescales, genome composition.
Detection of a novel enterotropic Mycoplasma gallisepticum-like in European Starling (Sturnus vulgaris) around poultry farms in France
Authors: Gall-Ladevèze Chloé Le , Nouvel Laurent-Xavier , Souvestre Marie , Croville Guillaume , Hygonenq Marie-Claude , Guérin Jean-Luc , Le Loc’h Guillaume 
Published: June 2021

SUMMARYRecent outbreaks of highly pathogenic avian influenza in southwest France have raised questions regarding the role of commensal wild birds in the introduction and dissemination of pathogens between poultry farms. To assess possible infectious contacts at the wild-domestic bird interface, the presence of Mycoplasma gallisepticum (MG) was studied in the two sympatric compartments in southwest France. Among various peridomestic wild birds (n=385), standard PCR primers targeting the 16S rRNA of MG showed a high apparent prevalence (up to 45%) in cloacal swabs of European starlings (Sturnus vulgaris, n=108), while the MG-specific mgc2 gene was not detected. No tracheal swab of these birds tested positive, and no clinical sign was observed in positive birds, suggesting commensalism in the digestive tract of starlings. A mycoplasma strain was then isolated from a starling swab and its whole genome was sequenced using both Illumina and Nanopore technologies. Phylogenetic analysis showed that it was closely related to MG and M. tullyi, although it was a distinct species.A pair of specific PCR primers targeting the mgc2-like gene of this MG-like strain was designed and used to screen again the same avian populations and a wintering urban population of starlings (n=50). Previous PCR results obtained in starlings were confirmed to be mostly due to this strain (20/22 positive pools). In contrast, the strain was not detected in fresh faeces of urban starlings. Furthermore, it was detected in one tracheal pool of cattle egrets and one cloacal pool of white wagtails, suggesting infectious transmissions between synanthropic birds with similar feeding behaviour. As the new starling mycoplasma was not detected in free-range ducks (n=80) in close contact with positive starlings, nor in backyard (n=320) and free-range commercial (n=720) chickens of the area, it might not infect poultry. However, it could be involved in mycoplasma gene transfer in such multi-species contexts.
SARS-CoV-2 spike RBD and nucleocapsid encoding DNA vaccine elicits T cell and neutralising antibody responses that cross react with variants
Authors: Brentville VA , Vankemmelbeke M , Metheringham RL , Symonds P , Cook KW , Urbanowicz RA , Tsoleridis T , Coleman CM , Chang K-C , Skinner A , Dubinina E , Daniels I , Shah S , Argonza M , Delgado J , Dwivedi V , Kulkarni V , Dixon JE , Pockley AG , Adams SE , Paston SJ , Daly JM , Ball JK , Durrant LG 
Published: June 2021

AbstractAlthough the efficacy of vaccines targeting SARS-CoV-2 is apparent now that the approved mRNA and adenovirus vector vaccines are in widespread use, the longevity of the protective immune response and its efficacy against emerging variants remains to be determined. We have therefore designed a DNA vaccine encoding both the SARS-CoV-2 spike receptorbinding domain (‘RBD’) and nucleocapsid proteins, the latter of which is highly conserved amongst beta coronaviruses. The vaccine elicits strong pro-inflammatory CD4+Th1 and CD8+T-cell responses to both proteins in mice and rats, with responses being significantly enhanced by fusing the nucleocapsid sequence to a modified Fc domain. We have shown that the vaccine also stimulates high titre antibody responses to RBD in mice that efficiently neutralise in pseudotype and live virus neutralisation assays and show cross reactivity with spike proteins from the variants B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). The vaccine also showed good protection in a viral challenge model in ACE2 receptor transgenic mice. This DNA platform can be easily adapted to target variant proteins and we show that a vaccine variant encoding the Beta variant sequence stimulates cross-reactive humoral and T cell responses. These data support the translation of this DNA vaccine platform into the clinic, thereby offering a particular advantage for rapidly targeting emerging SARS-CoV-2 variants.
Nanodiscs-based proteomics identify Caj1 as an Hsp40 with affinity for phosphatidic acid lipids
Authors: Zhang Xiao X. , Young John William , Foster Leonard J. , Duong Franck 
Published: June 2021

ABSTRACTMany soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids’ poor solubility in aqueous solution. Here, we employ nanodiscs – small, water-soluble patches of lipid bilayer encircled with amphipathic scaffold proteins – along with quantitative proteomics to identify lipid-binding proteins in S. cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids, and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of the nanodisc system to identify and characterize protein-lipid interactions that may not be evident using other methods.