bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fibrinogen anchors for micropatterning of active proteins and subcellular receptor relocalisation
Authors: Watson Joseph L. , Aich Samya , Salvia Benjamí Oller , Drabek Andrew A. , Blacklow Stephen C. , Chin Jason , Derivery Emmanuel 
Published: September 2020

AbstractProtein micropatterning allows proteins to be precisely deposited onto a substrate of choice, and is now routinely used in cell biology and in vitro reconstitution. However, a drawback of current technology is that micropatterning efficiency can be variable between proteins, and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, Fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard to micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.
Mutagenic mechanisms of cancer-associated DNA polymerase ε alleles
Authors: Herzog Mareike , Alonso-Perez Elisa , Salguero Israel , Warringer Jonas , Adams David J. , Jackson Stephen P. , Puddu Fabio 
Published: September 2020

ABSTRACTA single amino acid residue change in the exonuclease domain of human DNA polymerase ε, P286R, is associated with the development of colorectal cancers, and has been shown to impart a mutagenic phenotype. Perhaps unexpectedly, the corresponding Pol ε allele in the yeastSaccharomyces cerevisiae(pol2-P301R), was found to drive greater mutagenesis than exonuclease-deficient Pol ε (pol2-4), a phenotype sometimes termedultra-mutagenesis. By studying the impact on mutation frequency, type, replication-strand bias, and sequence context, we show thatultra-mutagenesis is commonly observed in cells carrying a range of cancer-associated Pol ε exonuclease domain alleles. Similarities between mutations generated by these alleles and those generated inpol2-4cells indicate a shared mechanism of mutagenesis that yields a mutation pattern similar to cancer Signature 14. Comparison ofPOL2 ultra-mutator withpol2-M644G, a mutant in the polymerase domain decreasing Pol ε fidelity, revealed unexpected analogies in the sequence context and strand bias of mutations. Analysis of mutational patterns unique to exonuclease domain mutant cells suggests that backtracking of the polymerase, when the mismatched primer end cannot be accommodated in the proofreading domain, results in the observed increase in insertions and T>A mutations in specific sequence contexts.
Ratio of hydrophobic-hydrophilic and positive-negative residues at lipid-water-interface influences surface expression and channel-gating of TRPV1
Authors: Saha Somdatta , Das Rashmita , Divyanshi Divyanshi , Tiwari Nikhil , Tiwari Ankit , Dalai Ritesh , Kumar Abhishek , Goswami Chandan 
Published: September 2020

AbstractDuring evolution, TRPV1 has lost, retained or selected certain residues at Lipid-Water-Interface (LWI) and formed specific patterns there. The ratio of “hydrophobic-hydrophilic” and “positive-negative charged” residues at the inner LWI remains conserved throughout vertebrate evolution and play important role in regulating TRPV1 trafficking, localization and functions. Arg575 is an important residue as Arg575Asp mutant has reduced Capsaicin-sensitivity, surface expression, colocalization with lipid-raft markers, cell area, and increased cell lethality. This lethality is due to the disruption of the ratio between positive-negative charges there. Such lethality can be rescued by either using TRPV1-specfic inhibitor 5’-IRTX or by restoring the positive-negative charge ratio at that position, i.e. by introducing Asp576Arg mutation in Arg575Asp backbone. We propose that Arg575Asp mutant confers TRPV1 in a “constitutive-open-like” condition. These findings have broader implication in understanding the molecular basis of thermo-gating and channel-gating and the microenvironments involved in such process that goes erratic in different diseases.
Monoclonal Antibodies Capable of Binding SARS-CoV-2 Spike Protein Receptor Binding Motif Specifically Prevent GM-CSF Induction
Authors: Qiang Xiaoling , Zhu Shu , Li Jianhua , Wang Ping , Tracey Kevin J. , Wang Haichao 
Published: September 2020

AbstractA severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has recently caused a pandemic COVID-19 disease that infected more than 25.6 million and killed 852,000 people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its envelope spike protein for binding the host angiotensin-converting enzyme 2 (ACE2) to gain viral entry. Currently, extensive efforts are being made to produce vaccines against a surface fragment of a SARS-CoV-2, such as the spike protein, in order to boost protective antibody responses. It was previously unknown how spike protein-targeting antibodies would affect innate inflammatory responses to SARS-CoV-2 infections. Here we generated a highly purified recombinant protein corresponding to the RBM of SARS-CoV-2, and used it to screen for cross-reactive monoclonal antibodies (mAbs). We found two RBM-binding mAbs that competitively inhibited its interaction with human ACE2, and specifically blocked the RBM-induced GM-CSF secretion in both human monocyte and murine macrophage cultures. Our findings have suggested a possible strategy to prevent SARS-CoV-2-elicited “cytokine storm”, and provided a potentially useful criteria for future assessment of innate immune-modulating properties of various SARS-CoV-2 vaccines.One Sentence SummaryRBM-binding Antibodies Inhibit GM-CSF Induction.
Chemical validation ofMycobacterium tuberculosisphosphopantetheine adenylyltransferase using fragment linking and CRISPR interference
Authors: El Bakali Jamal , Blaszczyk Michal , Evans Joanna C. , Boland Jennifer A. , McCarthy William J. , Dias Marcio V. B. , Coyne Anthony G. , Mizrahi Valerie , Blundell Tom L. , Abell Chris , Spry Christina 
Published: September 2020

AbstractThe coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused byMycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors ofMtbphosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of theMtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD< 20 μM and on-target anti-Mtbactivity, as demonstrated using CRISPR interference. This study represents a big step toward validatingMtbPPAT as a potential drug target and designing aMtbPPAT-targeting anti-TB drug.Abstract Figure
The RNA-binding protein SFPQ preserves long-intron splicing and regulates circRNA biogenesis
Authors: Stagsted Lotte Victoria Winther , O’Leary Eoghan Thomas , Hansen Thomas Birkballe 
Published: September 2020

AbstractCircular RNAs (circRNAs) represent an abundant and conserved entity of non-coding RNAs, however the principles of biogenesis are currently not fully understood. To elucidate features important for circRNA production, we performed global analyses of RNA-binding proteins associating with the flanking introns of circRNAs, and we identified two factors, SFPQ and NONO, to be highly enriched with circRNAs. Using transient knockdown of both proteins in two human cell lines followed by total RNAseq, we found a subclass of circRNAs with distal invertedAluelements and long introns to be highly deregulated upon SFPQ knockdown. In addition, SFPQ depletion leads to increased intron retention with concomitant induction of cryptic splicing prevalent for long introns causing in some cases premature transcription termination and polyadenylation. While SFPQ depletion has an overall negative effect on circRNA production, premature termination is not the main causative explanation. Instead, data suggests that aberrant splicing in the upstream and downstream regions of circRNA producing exons are critical for shaping the circRNAome, and specifically, we observe a conserved impact of missplicing in the immediate upstream region to drive circRNA biogenesis. Collectively, our data show that SFPQ plays an important role in maintaining intron integrity by ensuring accurate splicing of long introns, and disclose novel features governingAlu-independent circRNA production.
Intensity coded octopaminergic modulation of aversive crawling behavior inDrosophila melanogasterlarvae
Authors: Bilz Florian , Gilles Madeleine-Marie , Schatton Adriana , Pflüger Hans-Joachim , Schubert Marco 
Published: September 2020

AbstractActivation and modulation of sensory-guided behaviors by biogenic amines assure appropriate adaptations to changes in an insect’s environment. Given its genetic tool kitDrosophila melanogasterrepresents an excellent model organism to study larger networks of neurons by optophysiological methods. Here, we studied stationary crawling movements of 3rdinstar larvae and revealed how the octopaminergic VUM neuron system reacts during crawling behavior and tactile stimulations. We conducted calcium imaging experiments on dissections of the isolated nervous system (missing all sensory input) and found spontaneous rhythmic wave pattern of neuronal activity in VUM neuron clusters over the range of thoracic and abdominal neuromeres in the VNC. In contrast,in vivopreparations (semi-intact animals, receiving sensory input) did not reveal such spontaneous rhythmic pattern. However, tactile stimulations activated different clusters of the VUM neuron system simultaneously in these preparations. The activation intensity of VUM neurons in the VNC was correlated with the location and degree of body wall stimulation. While VUM neuron cluster near the respective location of body wall stimulation were less activated more distant cluster showed stronger activation. Repeated gentle touch stimulations led to decreased response intensities, repeated harsh stimulations resulted in increasing intensities over trials. Optophysiological signals correlated highly with crawling behavior in freely moving larvae stimulated similarly. We conclude that the octopaminergic system is strongly coupled to the neuronal pattern generator of crawling movements and that it is simultaneously activated by physical stimulation, rather intensity than sequential coded. We hope that our work raises the interest in whole biogenic network activity and shows that octopamine release does not only underlie “the more the better” principle but instead has a more complex function in control and modulation of insect’s locomotion.
Fruitless decommissions regulatory elements to implement cell-type-specific neuronal masculinization
Authors: Brovkina Margarita V. , Duffié Rachel , Burtis Abbigayl E. C. , Clowney E. Josephine 
Published: September 2020

AbstractIn the fruit flyDrosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruMin sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. We identify 436 high-confidence genomic regions differentially accessible in malefruitlessneurons, validate candidate regions as bona-fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specificfru+cell types. Aside fromfruitself, genes are not dedicated to or common across thefrucircuit; rather, FruMappears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find FruMmotifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruMacts cell-type-specifically to decommission regulatory elements in malefruitlessneurons.
A conserved choreography of mRNAs at centrosomes reveals a localization mechanism involving active polysome transport
Authors: Safieddine Adham , Coleno Emeline , Traboulsi Abdel-Meneem , Kwon Oh Sung , Lionneton Frederic , Georget Virginie , Robert Marie-Cécile , Gostan Thierry , Lecellier Charles , Salloum Soha , Chouaib Racha , Pichon Xavier , Le Hir Hervé , Zibara Kazem , Peter Marion , Bertrand Edouard 
Published: September 2020

AbstractLocal translation allows for a spatial control of gene expression. Here, we used high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses revealed a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging revealed active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, theDrosophilaorthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysomes transport mediated by nascent proteins.
Mitochondrial, cell cycle control and neuritogenesis alterations in an iPSC-based neurodevelopmental model for schizophrenia
Authors: Zuccoli Giuliana S. , Nascimento Juliana M. , Codo Ana Campos , Moraes-Vieira Pedro M. , Rehen Stevens K. , Martins-de-Souza Daniel 
Published: September 2020

AbstractSchizophrenia is a severe psychiatric disorder of neurodevelopmental origin that affects around 1% of the world’s population. Proteomic studies and other approaches have provided evidence of compromised cellular processes in the disorder, including mitochondrial function. Most of the studies so far have been conducted on postmortem brain tissue from patients and do not allow the evaluation of the neurodevelopmental aspect of the disorder. To circumvent that, we studied the mitochondrial and nuclear proteomes of neural stem cells (NSCs) and neurons derived from induced pluripotent stem cells (iPSCs) from schizophrenia patients versus healthy controls. Our results revealed differentially regulated proteins in pathways related to mitochondrial function, oxidative phosphorylation, cell cycle control, DNA repair, and neuritogenesis. Moreover, metabolic analysis of NSCs revealed alterations in mitochondrial function in schizophrenia-derived cells. Hence, this study shows that changes in important cellular processes are present during neurodevelopment and could be involved with the establishment of schizophrenia, as well as the phenotypic traits observed in adult patients.
Cholinergic neuromodulation of inhibitory interneurons facilitates functional integration in whole-brain models
Authors: Coronel-Oliveros Carlos , Cofré Rodrigo , Orio Patricio 
Published: September 2020

AbstractSegregation and integration are two fundamental principles of brain structural and functional organization. Neuroimaging studies have shown that the brain transits between different functionally segregated and integrated states, and neuromodulatory systems have been proposed as key to facilitate these transitions. Although computational models have reproduced the effect of neuromodulation at the whole-brain level, the role of local inhibitory circuits and their cholinergic modulation has not been studied. In this article, we consider a Jansen & Rit whole-brain model in a network interconnected using a human connectome, and study the influence of the cholinergic and noradrenergic neuromodulatory systems on the segregation/integration balance. In our model, a newly introduced local inhibitory feedback enables the integration of whole-brain activity, and its modulation interacts with the other neuromodulatory influences to facilitate the transit between different functional states. Moreover, the new proposed model is able to reproduce an inverted-U relationship between noradrenergic modulation and network integration. Our work proposes a new possible mechanism behind segregation and integration in the brain.
Non-permissive human conventional CD1c+dendritic cells enabletrans-infection of human primary renal tubular epithelial cells and protect BK polyomavirus from neutralization
Authors: Sikorski Mathieu , Coulon Flora , Peltier Cécile , Braudeau Cécile , Garcia Alexandra , Giraud Matthieu , Renaudin Karine , Kandel-Aznar Christine , Nedellec Steven , Hulin Philipe , Branchereau Julien , Véziers Joëlle , Gaboriaud Pauline , Touzé Antoine , Burlaud-Gaillard Julien , Josien Régis , McIlroy Dorian , Bressollette-Bodin Céline , Halary Franck 
Published: September 2020

AbstractThe BK polyomavirus (BKPyV) is a ubiquitous human virus that persists in the renourinary epithelium. Immunosuppression can lead to BKPyV reactivation in the first year post-transplantation in kidney (KTR) and hematopoietic stem cell transplant recipients. In KTR, persistent DNAemia has been correlated to the occurrence of polyomavirus-associated nephropathy (PVAN) that can lead to graft loss if not properly controlled. Based on recent observations that conventional dendritic cells (cDC) specifically infiltrate PVAN lesions, we hypothesized that those cells could play a role in BKPyV infection. We first demonstrated that monocyte-derived DC (MDDC), anin vitromodel for mDC, captured BKPyV particles through an unconventional GRAF-1 endocytic pathway. Neither BKPyV particles nor BKPyV-infected cells were shown to activate MDDC. Endocytosed virions were efficiently transmitted to permissive cells and shown to be protected from the antibody-mediated neutralization. Finally, we demonstrated that freshly isolated CD1c+mDC from the blood and kidney parenchyma behaved similarly to MDDC thus extending our results to cells of clinical relevance. This study sheds light on a potential unprecedented CD1c+mDC involvement in the BKPyV infection as a promoter of viral spreading.
fruitlessmutant male mosquitoes gain attraction to human odor
Authors: Basrur Nipun S. , De Obaldia Maria Elena , Morita Takeshi , Herre Margaret , von Heynitz Ricarda K. , Tsitohay Yael N. , Vosshall Leslie B. 
Published: September 2020

SUMMARYWhile sexual dimorphism in courtship and copulation behavior is common in the animal kingdom, sexual dimorphism in feeding behavior is rare. TheAedes aegyptimosquito provides an example of extreme sexual dimorphism in feeding, because only the females show strong attraction to humans, and bite them to obtain a blood-meal necessary to stimulate egg production1-8. The genetic basis of this complex, modular, and sexually dimorphic feeding behavior is unknown. Thefruitlessgene is sex-specifically spliced in the brain of multiple insect species including mosquitoes9-11and encodes a BTB zinc-finger transcription factor that has been proposed to be a master regulator of male courtship and mating behavior across insects12-17. Here we use CRISPR-Cas9 to mutate thefruitlessgene in male mosquitoes.fruitlessmutant males fail to mate, confirming the ancestral function of this gene in male sexual behavior. Remarkably,fruitlessmutant males also gain strong attraction to a live human host, a behavior that wild-type males never display. Humans produce multiple sensory cues that attract mosquitoes and we show thatfruitlessspecifically controls hostseeking in response to human odor. These results suggest that male mosquitoes possess the neural circuits required to host-seek and that removingfruitlessreveals this latent behavior in males. Our results highlight an unexpected repurposing of a master regulator of male-specific sexual behavior to control one module of female-specific blood-feeding behavior in a deadly vector of infectious diseases.
Whole-Genome Enrichment and Sequencing ofChlamydia trachomatisDirectly from Patient Clinical Vaginal and Rectal Swabs
Authors: Bowden Katherine E. , Joseph Sandeep J. , Cartee John , Ziklo Noa , Danavall Damien , Raphael Brian H. , Read Timothy D. , Dean Deborah 
Published: September 2020

AbstractChlamydia trachomatisis the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. U.S. cases have been steadily increasing for more than a decade in both the urogenital tract and rectum.C. trachomatisis an obligate intracellular bacterium that is not easily cultured, limiting the capacity for genome studies to understand strain diversity and emergence among various patient populations globally. While Agilent SureSelectXTtarget-enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing ofC. trachomatisdirectly from clinical urine, vaginal, conjunctival and rectal samples, efficiencies are only 60-80% for ≥95-100% genome coverage. We therefore re-designed and expanded the RNA bait library to augment enrichment of the organism from clinical samples to improve efficiency. We describe the expanded library, the limit of detection forC. trachomatisgenome copy input, and the 100% efficiency and high-resolution of generated genomes where genomic recombination among paired vaginal and rectal specimens from four patients was identified. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporaryC. trachomatisSTIs across sample types, among geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.ImportanceChlamydia trachomatisis an obligate intracellular bacterium that is not easily cultured, and there is limited information on rectalC. trachomatistransmission and its impact on morbidity. To improve efficiency of previous studies involving whole genome target enrichment and sequencing ofC. trachomatisdirectly from clinical urine, vaginal, conjunctival, and rectal specimens, we expanded the RNA bait library to augment enrichment of the organism from clinical samples. We demonstrate an increased efficiency in the percentage of reads mapping toC. trachomatis. We show the new system is sensitive for near identical genomes ofC. trachomatisfrom two body sites in four women. Further, we provide a robust genomic epidemiologic approach to advance our understanding ofC. trachomatisstrains causing ocular, urogenital and rectal infections, and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.