bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the complete dimeric human GDAP1 core domain provides insights into ligand binding and clustering of disease mutations
Authors: Tuyet Nguyen Giang Thi , Sutinen Aleksi , Raasakka Arne , Muruganandam Gopinath , Loris Remy , Kursula Petri
Published: November 2020

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive towards classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca2+homeostasis. The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point towards allosteric mechanisms in regulating GDAP1 oligomeric state and function.
DHS-Crystallize: Deep-Hybrid-Sequence based method for predicting protein Crystallization
Authors: Alavi Azadeh , Ascher David B.
Published: November 2020

The key method for determining the structure of a protein to date is X-ray crystallography, which is a very expensive technique that suffers from high attrition rate. On the contrary, a sequence-based predictor that is capable of accurately determining protein crystallization property, would not only overcome such limitations, but also would reduce the trial-and-error settings required to perform crystallization. In this work, to predict protein crystallizability, we have developed a novel sequence-based hybrid method that employs two separate, yet fully automated, concepts for extracting features from protein sequences. Specifically, we use a deep convolutional neural network on a publicly available dataset to extract descriptive features directly from the sequences, then fuse such feature with structural-and-physio-chemical driven features (such as amino-acid composition or AAIndex-based physicochemical properties). Dimentionality reduction is then performed on the resulting features and the output vectors are applied to train optimized gradient boosting machine (XGBoostt). We evaluate our method through three publicly available test sets, and show that our proposed DHS-Crystallize algorithm outperforms state-of-the-art methods, and achieves higher performance compared to using DCNN-deriven features, or structural-and-physio-chemical driven features alone.
Plasma irradiation efficiently inactivates the coronaviruses mouse hepatitis virus and SARS-CoV-2
Authors: Morikawa Shigeru , Watanabe Shunpei , Fujii Hikaru , Tanaka Toshio , Arai Junichirou , Kyuwa Shigeru
Published: November 2020

Many inactivation methods have been shown to inactivate SARS-CoV-2 for safe and efficient diagnostic methods. COVID-19 is caused by airborne infection of SARS-CoV-2, and therefore, methods of inactivating the virus efficiently and safely are crucial for reducing the risk of airborne infection. In this regard, the effect of plasma discharge on the infectivity of the coronaviruses mouse hepatitis virus (MHV) and SARS-CoV-2 was tested. Plasma discharge efficiently reduced the infectivity of both coronaviruses. Treatment of SARS-CoV-2 in culture medium with a plasma discharge resulted in 95.17% viral inactivation after plasma irradiation after 1 hour (hr), 99.54% inactivation after 2 hrs and 99.93% inactivation after 3 hrs. Similar results were obtained for MHV. The results indicated that plasma discharge effectively and safely inactivated the airborne coronaviruses and may be useful in minimizing the risk of airborne infection of SARS-CoV-2.
Cloning and Functional characterization of seed-specificLEC1Apromoter from peanut (Arachis hypogaeaL.)
Authors: Tang Guiying , Xu Pingli , Li Pengxiang , Zhu Jieqiong , Chen Guangxia , Shan Lei , Wan Shubo
Published: November 2020

LEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor, which controls several aspects of embryo and postembryo development, including embryo morphogenesis, storage reserve accumulation and skotomorphogenesis. Herein, using the method of chromosomal walking, a 2707bp upstream sequence from the ATG initiation codon site ofAhLEC1Awhich is a homolog of ArabidopsisLEC1was isolated in peanut. Its transcriptional start site confirmed by 5’ RACE was located at 82 nt from 5’ upstream of ATG. The bioinformatics analysis revealed that there existed many tissue-specific elements and light responsive motifs in its promoter. To identify the functional region of theAhLEC1Apromoter, seven plant expression vectors expressing theGUS(β-glucuronidase) gene, driven by 5’ terminal series deleted fragments ofAhLEC1Apromoter, were constructed and transformed into Arabidopsis. Results of GUS histochemical staining showed that the regulatory region containing 82bp of 5’ UTR and 2228bp promoter could facilitate GUS to express preferentially in the embryos at different development periods of Arabidopsis. Taken together, it was inferred that the expression ofAhLEC1Aduring seed development of peanut might be controlled positively by several seed-specific regulatory elements, as well as negatively by some other regulatory elements inhibiting its expression in other organs. Moreover, theGUSexpression pattern of transgenic seedlings in darkness and in light was relevant to the light-responsive elements scattered inAhLEC1Apromoter segment, implying that these light-responsive elements harbored in theAhLEC1Apromoter regulate skotomorphogenesis of peanut seeds, andAhLEC1Aexpression was inhibited after the germinated seedlings were transferred from darkness to light.
A whole virion vaccine for COVID-19 produced via a novel inactivation method: results from animal challenge model studies
Authors: Ragan Izabela K , Hartson Lindsay M , Dutt Taru S , Obregon-Henao Andres , Maison Rachel M , Gordy Paul , Fox Amy , Karger Burton R , Cross Shaun T , Kapuscinski Marylee L , Cooper Sarah K , Podell Brendan K , Stenglein Mark D , Bowen Richard A , Henao-Tamayo Marcela , Goodrich Raymond P
Published: November 2020

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX™) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this study provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have utility in the preparation of one such vaccine candidate.

Author Summary

We have developed a vaccine for COVID-19 which is prepared by a novel method for inactivation of a whole virion particle and tested it in a hamster animal model for its ability to protect against SARS-CoV-2 infection.
ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2
Authors: Tribolet Leon , Alexander Marina R. , Brice Aaron M. , van Vuren Petrus Jansen , Rootes Christina L. , Mara Kostlend , McDonald Meg , Bruce Kerri L. , Gough Tamara J. , Shi Shuning , Cowled Christopher , Bean Andrew G. D. , Stewart Cameron R.
Published: November 2020

The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domain, previously C6orf106) is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we have utilised RNA-seq to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) infection. We find that inhibition of ILRUN expression increases cellular expression of several members of the renin-angiotensin aldosterone system (RAAS), including the SARS-CoV-2 entry receptor angiotensin converting enzyme 2 (ACE2). Furthermore, inhibition of ILRUN results in increased SARS-CoV-2 replication. These data identify ILRUN as a novel inhibitor of SARS-CoV-2 replication and represents, to our knowledge, the first report of ILRUN as a regulator of the RAAS.

SIGNIFICANCE STATEMENT

There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions and the development of innovative and exciting therapeutic strategies and new knowledge and tools to better protect against the impacts of disease. The human protein-coding gene ILRUN is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we present the first evidence that ILRUN modulation has implications for SARS-CoV-2 infections. Virus infectivity assays confirmed that gene silencing of ILRUN had a proviral effect and increased SARS-CoV-2 replication, whilst over-expression of ILRUN inhibited SARS-CoV-2 production. Additionally, we observed that ILRUN also regulates the expression of key elements of the RAAS. These data have important implications for the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.
Lipid-specific protein oligomerization is regulated by two interfaces in Marburg virus matrix protein VP40
Authors: Amiar Souad , Husby Monica L. , Wijesinghe Kaveesha J. , Angel Stephanie , Bhattarai Nisha , Gerstman Bernard S. , Chapagain Prem P. , Li Sheng , Stahelin Robert V.
Published: November 2020

Marburg virus major matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane and undergo a dynamic and extensive self-oligomerization into the structural matrix layer which confers the virion shape and stability. Using a myriad ofin vitroand cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces (N-terminal domain (NTD) and C-terminal domain (CTD)) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrated the importance of each interface in the mVP40 matrix assembly through protein trafficking to the plasma membrane and homo-multimerization that induced protein enrichment, plasma membrane fluidity changes and elongations at the plasma membrane. A novel APEX-TEM method was employed to closely assess the ultrastructural localization of and formation of viral particles for wild type and mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly.
A metabolic coincidence mechanism controls winter photoperiodism in plants
Authors: Liu Wei , Feke Ann , Leung Chun Chung , Tarté Daniel A. , Yuan Wenxin , Vanderwall Morgan , Sager Garrett , Wu Xing , Schear Ariela , Clark Damon A. , Thines Bryan C. , Gendron Joshua M.
Published: November 2020

Plants have served as a preeminent study system for photoperiodism because of their propensity to flower in concordance with the seasons. A nearly singular focus on understanding seasonal flowering has been to the detriment of discovering other photoperiod measuring mechanisms that may be necessary for vegetative health. Here we use bioinformatics to identify a group of winter photoperiod-induced genes in Arabidopsis and show that one,PP2-A13, is critical for fitness and survival, exclusively in winter-like photoperiods. We create a real-time photoperiod reporter, using thePP2-A13promoter driving luciferase, and show that winter photoperiod genes are regulated independent of the canonical CO/FT mechanism for photoperiodic flowering. The reporter then allows us to identify the first genetic and cellular drivers of winter photoperiodism and reveal a mechanism that relies on coincidence between light capture through photosynthesis and rhythmic metabolism. This work demonstrates that plants have distinct photoperiod measuring mechanisms that enact critical biological and developmental processes in different seasons.
Stimulus-specific plasticity in human visual gamma-band activity and functional connectivity
Authors: Stauch Benjamin J. , Peter Alina , Schuler Heike , Fries Pascal
Published: November 2020

Under natural conditions, the visual system often sees a given input repeatedly. This provides an opportunity to optimize processing of the repeated stimuli. Stimulus repetition has been shown to strongly modulate neuronal-gamma band synchronization, yet crucial questions remained open. Here we used magnetoencephalography in 30 human subjects and find that gamma decreases across ~10 repetitions and then increases across further repetitions, revealing plastic changes of the activated neuronal circuits. Crucially, changes induced by one stimulus did not affect responses to other stimuli, demonstrating stimulus specificity. Changes partially persisted when the inducing stimulus was repeated after 25 minutes of intervening stimuli. They were strongest in early visual cortex and increased interareal feedforward influences. Our results suggest that early visual cortex gamma synchronization enables adaptive neuronal processing of recurring stimuli. These and previously reported changes might be due to an interaction of oscillatory dynamics with established synaptic plasticity mechanisms.
Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2
Authors: Clark Sarah A. , Clark Lars E. , Pan Junhua , Coscia Adrian , McKay Lindsay G.A. , Shankar Sundaresh , Johnson Rebecca I. , Griffiths Anthony , Abraham Jonathan
Published: November 2020

The SARS-CoV-2 viral spike (S) protein mediates attachment and entry into host cells and is a major target of vaccine and drug design. Potent SARS-CoV-2 neutralizing antibodies derived from closely related antibody heavy chain genes (IGHV3-53 or 3-66) have been isolated from multiple COVID-19 convalescent individuals. These usually contain minimal somatic mutations and bind the S receptor-binding domain (RBD) to interfere with attachment to the cellular receptor angiotensin-converting enzyme 2 (ACE2). We used antigen-specific single B cell sorting to isolate S-reactive monoclonal antibodies from the blood of a COVID-19 convalescent individual. The seven most potent neutralizing antibodies were somatic variants of the same IGHV3-53-derived antibody and bind the RBD with varying affinity. We report X-ray crystal structures of four Fab variants bound to the RBD and use the structures to explain the basis for changes in RBD affinity. We show that a germline revertant antibody binds tightly to the SARS-CoV-2 RBD and neutralizes virus, and that gains in affinity for the RBD do not necessarily correlate with increased neutralization potency, suggesting that somatic mutation is not required to exert robust antiviral effect. Our studies clarify the molecular basis for a heavily germline-biased human antibody response to SARS-CoV-2.
AUXIN RESPONSE FACTOR 6(ARF6)andARF8promote Gibberellin-mediated hypocotyl xylem expansion and cambium homeostasis
Authors: Ben-Targem Mehdi , Ripper Dagmar , Bayer Martin , Ragni Laura
Published: November 2020

During secondary growth, the thickening of plant organs, wood (xylem) and bast (phloem) are continuously produced by the vascular cambium. In Arabidopsis hypocotyl and root, we can distinguish two phases of secondary growth based on cell morphology and production rate. The first phase, in which xylem and phloem are equally produced, precedes the xylem expansion phase in which xylem formation is enhanced and xylem fibers differentiate. It is known that Gibberellins (GA) trigger this developmental transition via the degradation of DELLA proteins and that the cambium master regulator BREVIPEDICELLUS/KNAT1 (BP/KNAT1) and the receptor like kinases ERECTA and ERL1 regulate this process downstream of GA. However, our understandings on the regulatory network underlying GA-mediated secondary growth, are still limited.

Here, we demonstrate that DELLA-mediated xylem expansion is mainly achieved through RGA and GAI and that RGA and GAI promote cambium senescence. We further show that AUXIN RESPONSE FACTOR (ARF6) and ARF8, which physically interact with DELLAs, specifically repress phloem proliferation and induce cambium senescence during the xylem expansion phase. Moreover, the inactivation ofBPinarf6 arf8background revealed an essential role for ARF6 and ARF8 in cambium establishment and maintenance. Overall, our results shed light on a pivotal hormone cross-talk between GA and auxin in the context of plant secondary growth.
The two formyl peptide receptors differently regulate GPR84-mediated neutrophil NADPH-oxidase activity
Authors: Mårtensson Jonas , Sundqvist Martina , Manandhar Asmita , Ieremias Loukas , Zhang Linjie , Ulven Trond , Xie Xin , Björkman Lena , Forsman Huamei
Published: November 2020

Neutrophils express many G protein-coupled receptors (GPCRs) including the two formyl peptide receptors (FPR1 and FPR2) and the medium chain fatty acid receptor GPR84. The FPRs are known to define a hierarchy among neutrophil GPCRs, i.e., the GPCR-mediated response can be either suppressed or amplified by signals generated by FPRs. In this study, we investigated the position of GPR84 in the FPR-defined hierarchy regarding the activation of neutrophil NADPH-oxidase, an enzyme system designed to generate reactive oxygen species (ROS). When naïve neutrophils are activated by GPR84 agonists a modest ROS release was induced. However, vast amounts of ROS production was induced by these GPR84 agonists in FPR2-desensitized neutrophils, and the response is inhibited not only by a GPR84 antagonist but also by an FPR2 specific antagonist. This suggests that the amplified GPR84 agonist response is achieved through a reactivation of the desensitized FPR2. In addition, the GPR84-mediated FPR2 reactivation was independent of β-arrestin recruitment and sensitive to a protein phosphatase inhibitor. In contrast, the modest ROS production induced by GPR84 agonists was primarily suppressed in FPR1-desensitized neutrophils through hierarchical desensitization of GPR84 by FPR1 generated signals.

In summary, our data show that FPRs control the NADPH-oxidase activity mediated through GPR84 in human neutrophils. While an amplified ROS generation is achieved by GPR84 agonists through reactivation of desensitized FPR2, FPR1 heterologously desensitizes GPR84 and by that suppresses the release of ROS induced by GPR84 agonists.
DNA methylation during development and regeneration of the annelidPlatynereis dumerilii
Authors: Planques Anabelle , Kerner Pierre , Ferry Laure , Grunau Christoph , Gazave Eve , Vervoort Michel
Published: November 2020

Background

Methylation of cytosines in DNA (5mC methylation) is a major epigenetic modification that modulates gene expression and is important for embryonic development and cell reprogramming in vertebrates. In mammals, 5mC methylation in promoter regions is linked to transcriptional repression. Transcription regulation by 5mC methylation notably involves the Nucleosome Remodeling and Deacetylase complex (NuRD complex) which bridges DNA methylation and histone modifications. Less is known about roles and mechanisms of 5mC methylation in non-vertebrate animals. In this paper, we study 5mC methylation in the marine annelid wormPlatynereis dumerilii,an emerging evolutionary and developmental biology model capable of regenerating the posterior part of its body upon amputation. The regenerated region includes both differentiated structures and a growth zone consisting of stem cells required for the continuous growth of the worm.

Results

Usingin silicoand experimental approaches, we show thatP. dumeriliidisplays a high level of DNA methylation comparable to that of mammalian somatic cells. 5mC methylation inP. dumeriliiis dynamic along the life cycle of the animal and markedly decreases at the transition between larval to post-larval stages. We identify a full repertoire of mainly singlecopy genes encoding the machinery associated to 5mC methylation or members of the NuRD complex inP. dumeriliiand show, through phylogenetic analyses, that this repertoire is close to the one inferred for the last common ancestor of bilaterians. These genes are dynamically expressed duringP. dumeriliidevelopment, growth and regeneration. Treatment with the DNA hypomethylating agent Decitabine, impairsP. dumeriliilarval development and regeneration, and has long-term effects on post-regenerative growth by affecting the functionality of stem cells of the growth zone.

Conclusions

Our data indicate high-level of 5mC methylation in the annelidP. dumerilii,highlighting that this feature is not specific to vertebrates in the bilaterian clade. Analysis of DNA methylation levels and machinery gene expression during development and regeneration, as well as the use of a chemical inhibitor of DNA methylation, suggest an involvement of 5mC methylation inP. dumeriliidevelopment, regeneration and stem cell-based post-regenerative growth. We also present data indicating thatP. dumeriliiconstitutes a promising model to study biological roles and mechanisms of DNA methylation in non-vertebrate bilaterians and to provide new knowledge about evolution of the functions of this key epigenetic modification in bilaterian animals.
Properdin oligomers adopt rigid extended conformations supporting function
Authors: Pedersen Dennis V. , Pedersen Martin Nors , Mazarakis Sofia M.M. , Wang Yong , Lindorff-Larsen Kresten , Arleth Lise , Andersen Gregers R.
Published: November 2020

Properdin stabilizes convertases formed upon activation of the complement cascade within the immune system. The biological activity of properdin depends on the oligomerization state, but whether properdin oligomers are rigid and how their structure links to function remains unknown. We show by combining electron microscopy and solution scattering, that properdin oligomers adopt extended rigid and well-defined conformations that are well approximated by single models of apparent n-fold rotational symmetry with dimensions of 23-36 nm. Properdin monomers are pretzel shaped molecules with limited flexibility. In solution, properdin dimers are curved molecules whereas trimers and tetramers are close to being planar molecules. Structural analysis indicates that simultaneous binding through all binding sites to surface linked convertases is unlikely for properdin trimer and tetramers. We show that multivalency alone is insufficient for full activity in a cell lysis assay. Hence, the observed rigid extended oligomer structure is an integral component of properdin function.
Small RNAs couple embryonic developmental programs to gut microbes
Authors: Ohno Hayao , Bao Zhirong
Published: November 2020

Maternal exposure to microbes and other environmental factors is known to induce adaptive changes in the progeny, but little is understood about how development of the progeny is changed. We show thatCaenorhabditis elegansundergoes additional embryonic cell divisions in response to maternal gut microbes such as one producing the biopolymer γ-poly-DL-glutamic acid. The divisions coincide with anatomical changes including left-right asymmetric cell alignment, doubling the association between intestinal cells and primordial germ cells, and improved fecundity. The developmental changes are regulated by soma-to-germline transmission of endogenous RNAi and the miR-35 microRNA family, which targets the LIN-23/CDC-25 pathway. Our findings challenge the widespread assumption thatC. eleganshas an invariant cell lineage that consists of 959 somatic cells and provide insights into how organisms optimize embryogenesis to adapt to environmental changes through epigenetic controls.
Spatial and Temporal Localization of SPIRRIG and WAVE/SCAR Reveal Roles for These Proteins in Actin-Mediated Root Hair Development
Authors: Chin Sabrina , Kwon Taegun , Khan Bibi Rafeiza , Sparks J. Alan , Mallery Eileen L. , Szymanski Daniel B. , Blancaflor Elison B.
Published: November 2020

Root hairs are single cell protrusions that enable roots to optimize nutrient and water acquisition. They attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a BEACH domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations to root hairs at distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi vesicles and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced inspiand the position of root hair emergence was disrupted inbrk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development through pathways that might intersect with W/SC.
Natural genetic diversity in the potato resistance geneRBconfers suppression avoidance from Phytophthora effector IPI-O4
Authors: Karki Hari S. , Abdullah Sidrat , Chen Yu , Halterman Dennis A.
Published: November 2020

RBis a potato gene that provides resistance to a broad spectrum of genotypes of the late blight pathogenPhytophthora infestans.RBbelongs to the CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat) class of resistance (R) genes, a major component of the plant immune system. It directly interacts with Class I and II IPI-O effectors fromP. infestansto initiate a hypersensitive resistance response, but this activity is suppressed in the presence of the Class III effector IPI-O4. Using natural genetic variation of RB within potato wild relatives, we identified two amino acids in the CC domain that alter interactions needed for suppression of resistance by IPI-O4. We have found that separate modification of these amino acids in RB can diminish or enhance the resistance capability of this protein againstP. infestansin bothNicotiana benthamianaand potato. Our results demonstrate that increased knowledge of the molecular mechanisms that determine resistance activation and R protein suppression by effectors can be utilized to tailor-engineer genes with the potential to provide increased durability.
Global gene expression analysis ofStreptococcus agalactiaeat exponential growth phase
Authors: Silvestre Inês , Borges Vítor , Duarte Sílvia , Nunes Alexandra , Sobral Rita , Vieira Luís , Gomes João Paulo , Borrego Maria José
Published: November 2020

Streptococcus agalactiaeis a leading cause of neonatal infections and an increasing cause of infections in adults with underlying diseases. One of the firstS. agalactiaeisolates to be subjected to whole genome sequencing was NEM316, a strain responsible for a fatal case of septicemia that has been widely used as reference strain forin vitroassays. Whole transcriptome analyses may provide an essential contribute to the understanding of the molecular mechanisms responsible for bacteria adaptation and pathogenicity, still, so far, very few studies were dedicated to the analysis of global gene expression ofS. agalactiae. Here, we applied RNA-sequencing to perform a comparative overview of the global gene expression levels of theS. agalactiaereference strain NEM316 at the exponential growth phase. Genes were ranked by expression level and grouped by functional category and 46% of the top-100 expressed genes encode proteins involved in “Translation, ribosomal structure and biogenesis”. Among the group of highly expressed genes were also represented genes with no assigned functional category. Although this result warrants further investigation, most of them might be implicated in stress response. As very little is known about the molecular mechanisms behind the release of DNase’sin vitroandin vivo, we also performed preliminary assays to understand whether direct DNA exposure affects the gene expression of strain NEM316 at the exponential growth phase. No differentially expressed genes were detected, which indicates that follow-up studies are needed to disclose the complex molecular pathways (and stimuli) triggering the release of DNase’s. In general, we provide data on the global expression levels of NEM316 at exponential growth phase that may contribute to better understandS. agalactiaeadaptation and virulence.
alms1mutant zebrafish do not show hair cell phenotypes seen in other cilia mutants
Authors: Parkinson Lauren , Stawicki Tamara M.
Published: November 2020

Multiple cilia-associated genes have been shown to affect hair cells in zebrafish (Danio rerio),including the human deafness genedcdc2,the radial spoke genersph9,and multiple intraflagellar transport (IFT) and transition zone genes. Recently a zebrafishalms1mutant was generated. TheALMS1gene is the gene mutated in the ciliopathy Alström Syndrome a disease that causes hearing loss among other symptoms. The hearing loss seen in Alström Syndrome may be due in part to hair cell defects asAlms1mutant mice show stereocilia polarity defects and a loss of hair cells. Hair cell loss is also seen in postmortem analysis of Alström patients. The zebrafishalms1mutant has metabolic defects similar to those seen in Alström syndrome andAlms1mutant mice. We wished to investigate if it also had hair cell defects. We, however, failed to find any hair cell related phenotypes inalms1mutant zebrafish. They had normal lateral line hair cell numbers as both larvae and adults and normal kinocilia formation. They also showed grossly normal swimming behavior, response to vibrational stimuli, and FM1-43 loading. Mutants also showed a normal degree of sensitivity to both short-term neomycin and long-term gentamicin treatment. These results indicate that cilia-associated genes differentially affect different hair cell types.
Zika Virus Capsid Anchor Forms Cytotoxic Amyloid-like Fibrils
Authors: Saumya Kumar Udit , Gadhave Kundlik , Kumar Amit , Giri Rajanish
Published: November 2020

Capsid-anchor (CA) of Zika virus (ZIKV) is a small, single-pass transmembrane sequence that separates the capsid (C) protein from downstream pre-membrane (PrM) protein. During ZIKV polyprotein processing, CA is cleaved-off from C and PrM and left as a membrane-embedded peptide. CA plays an essential role in the assembly and maturation of the virus. However, its independent folding behavior is still unknown. Since misfolding and aggregation propensity of transmembrane proteins are now increasingly recognized and has been linked to several proteopathic disorders. Therefore, in this study, we investigated the amyloid-forming propensity of CA at physiological conditions. We observed aggregation behavior of CA peptide using dyebinding assays and ThT kinetics. The morphological analysis of CA aggregates explored by high-resolution microscopy (TEM and AFM) revealed characteristic amyloid-like fibrils. Further, the effect on mammalian cells exhibited the cytotoxic nature of the CA amyloid-fibrils. Our findings collectively shed light on the amyloidogenic phenomenon of flaviviral protein, which may contribute to their infection.

Graphical Abstract:

Schematic representation of Zika virus Capsid anchor forming amyloid aggregates with cytotoxic and hemolytic properties.
The solution structures of higher-order human telomere G-quadruplex multimers
Authors: Monsen Robert C. , Chakravarthy Srinivas , Dean William L. , Chaires Jonathan B. , Trent John O.
Published: November 2020

Human telomeres contain the repeat DNA sequence 5’(TTAGGG), with duplex regions that are several kilobases long terminating in a 3’ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate modes proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5’(TTAGGG)n, with n=8, 12, and 16, fold into multimeric structures containing the maximal number (2, 3, and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of about 34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.
The Nab2 RNA binding protein promotes sex-specific splicing ofSex lethalinDrosophilaneuronal tissue
Authors: Jalloh Binta , Rounds J. Christopher , Brown Brianna E. , Lancaster Carly L. , Leung Sara W. , Banerjee Ayan , Morton Derrick J. , Bienkowski Rick S. , Kremsky Isaac J. , Fasken Milo B. , Corbett Anita H. , Moberg Kenneth H.
Published: November 2020

TheDrosophilapolyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls axon projection, locomotion, and memory. Here we define an unexpectedly specific role for Nab2 in regulating splicing of ∼150 exons/introns in the head transcriptome and link the most prominent of these, female retention of a male-specific exon in the sex determination factorSex-lethal(Sxl), to a role in m6A-dependent mRNA splicing. Genetic evidence indicates that aberrantSxlsplicing underlies multiple phenotypes inNab2mutant females. At a molecular level, Nab2 associates withSxlpre-mRNA and ensures proper female-specific splicing by preventing m6A hypermethylation by Mettl3 methyltransferase. Consistent with these results, reducing Mettl3 expression rescues developmental, behavioral and neuroanatomical phenotypes inNab2mutants. Overall these data identify Nab2 as a required regulator of m6A-regulatedSxlsplicing and imply a broader link between Nab2 and Mettl3-regulated brain RNAs.
Single cell transcriptomics reveals the effect of PD-L1/TGF-β blockade on the tumor microenvironment
Authors: Lim Yoong Wearn , Coles Garry L. , Sandhu Savreet K. , Johnson David S. , Adler Adam S. , Stone Erica L.
Published: November 2020

Background

The anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-β has been shown to contribute to T cell exclusion and anti-TGF-β improves anti-PD-L1 efficacyin vivo. However, TGF-β inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-β blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-β blockade in the tumor microenvironment should be further elucidated.

Results

For the first time, we used single-cell RNA sequencing to characterize the transcriptomic effects of PD-L1 and/or TGF-β blockade on nearly 30,000 single cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in CD45+ cells, and down-regulation of extracellular matrix genes in CD45-cells. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation within vivomodels showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1.

Conclusions

Taken together, our data could be leveraged translationally to improve anti-PD-L1 plus anti-TGF-β combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.
Functional microstructure of CaV-mediated calcium signaling in the axon initial segment
Authors: Lipkin Anna M , Cunniff Margaret M , Spratt Perry WE , Lemke Stefan M , Bender Kevin J
Published: November 2020

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we utilize conventional 2-photon laser scanning and diffraction-limited, high-speed spot 2-photon imaging to resolve action potential-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-dependent stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function.
RBM39 alters phosphorylation of c-Jun and binds to viral RNA to promote PRRSV proliferation
Authors: Song Yinna , Guo Yanyu , Li Xiaoyang , Sun Ruiqi , Zhu Min , Shi Jingxuan , Zhang Lilin , Huang Jinhai
Published: November 2020

ABTRAST

As transcriptional co-activator of AP-1/Jun, estrogen receptors and NF-κB, nuclear protein RBM39 also involves in precursor mRNA (pre-mRNA) splicing. Porcine reproductive and respiratory syndrome virus (PRRSV) causes sow reproductive disorders and piglet respiratory diseases, which resulted in serious economic losses worldwide. In this study, the up-regulated expression of RBM39 and down-regulated of inflammatory cytokines (TNF, IL-1β) were determined in PRRSV-infected 3D4/21 cells, and accompanied with the PRRSV proliferation. The roles of RBM39 altering phosphorylation of c-Jun to inhibit the AP-1 pathway to promote PRRSV proliferation were further verified. In addition, the nucleocytoplasmic translocation of RBM39 and c-Jun from nucleus to cytoplasm were enhanced in PRRSV-infected cells. The three RRM domain of RBM39 are crucial to support the proliferation of PRRSV. several PRRSV RNA (nsp4, nsp5, nsp11 and N) binding with RBM39 were determined, which may also contribute to the PRRSV proliferation. Our results revealed a complex mechanism of RBM39 by altering c-Jun phosphorylation and nucleocytoplasmic translocation, and regulating binding of RBM39 with viral RNA to prompt PRRSV proliferation. The results provide new viewpoints to understand the immune escape mechanism of PRRSV infection.
Functional pathway redundancy in the metabolism of plant-derived phenolics byNovosphingobium aromaticivorans
Authors: Perez Jose M. , Kontur Wayne S. , Gehl Carson , Gille Derek M. , Ma Yanjun , Niles Alyssa V. , Umana German , Donohue Timothy J. , Noguera Daniel R.
Published: November 2020

Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds followed by biological funneling into a single product.Novosphingobium aromaticivoransDSM12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible forO-demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolics compounds byN. aromaticivorans. The results showed the involvement of DesA inO-demethylation of syringic and vanillic acids, LigM inO-demethylation of vanillic acid and 3-MGA, and a newO-demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring opening dioxygenase involved in 3-MGA, PCA, and GA metabolism, and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified inN. aromaticivoransthat involvesO-demethylation of 3-MGA to GA. We predict this pathway channels ∼15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC.

IMPORTANCE

For lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, andN. aromaticivoransis a promising chassis for this technology. The ability ofN. aromaticivoransto efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway inN. aromaticivoransand the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.
m6A RNA methylation-dependent APC translation is required for local translation of beta-actin and axon development
Authors: Broix Loic , Roy Rohini , Oomoto Ikumi , Umeshima Hiroki , Wang Dan Ohtan
Published: November 2020

Subcellular mRNA localization and local translation are crucial for spatially regulated gene expression in neurons. However, the precise mechanisms governing the selective transport and translation of mRNAs contributing to processes such as axonal growth and branching are only partially understood. Here, we present evidence of N6-methyladenosine (m6A)-mediated translational control of the RNA-binding protein, APC, influencing cytoskeletal dynamics at the growth cone. Our findings demonstrate that m6A modifications occur onApcmRNA, facilitating its recognition by YTHDF1 to promote APC translation in neuronal somata. Additionally, we observe that disrupting the m6A pathway impairs the transport and local translation of β-actin mRNA in the axon and growth cones, a deficiency that can be rescued by the exogenous expression of APC protein in cultured neurons. Furthermore, we establish the essential role of YTHDF1 in axon development, particularly in callosal projection neurons during cortical development. Our findings suggest a novel mechanism involving m6A-mediated regulation of APC protein translation, linking epitranscriptomics to axonal mRNA targeting, cytoskeletal dynamics, and axon development.
Amalgamation and Auckland city: A New Zealand success story?
Authors: Asquith Andy , McNeill Jeffrey , Stockley Elizabeth
Published: November 2020

The amalgamation of Auckland's local authorities in 2010 was remarkable for New Zealand. It created a city‐region to address perceived failures arising from trying to coordinate and align leadership and strategic planning while maintaining political accountability within a fractured local government system. The factors leading up to the Royal Commission on Auckland Governance in 2009 and its findings that led to amalgamation are examined and the Commission's report and its implementation are addressed. Finally, an assessment is made of the operation of the newly created Auckland Council during its first 9 years. Crucial to the amalgamation process was a change of national government, which had a very different political philosophy of the role of (local) government than its predecessor. Despite a difficult start, the Council achieved two of its three initial criteria: creating leadership for Auckland and providing strategic direction for New Zealand's biggest city.
Characterization of reproductive gene diversity in the endangered Tasmanian devil
Authors: Brandies Parice A. , Wright Belinda R. , Hogg Carolyn J. , Grueber Catherine E. , Belov Katherine
Published: November 2020

Interindividual variation at genes known to play a role in reproduction may impact reproductive fitness. The Tasmanian devil is an endangered Australian marsupial with low genetic diversity. Recent work has shown concerning declines in productivity in both wild and captive populations over time. Understanding whether functional diversity exists at reproductive genes in the Tasmanian devil is a key first step in identifying genes that may influence productivity. We characterized single nucleotide polymorphisms (SNPs) at 214 genes involved in reproduction in 37 Tasmanian devils. Twenty genes contained nonsynonymous substitutions, with genes involved in embryogenesis, fertilization and hormonal regulation of reproduction displaying greater numbers of nonsynonymous SNPs than synonymous SNPs. Two genes, ADAMTS9 and NANOG, showed putative signatures of balancing selection indicating that natural selection is maintaining diversity at these genes despite the species exhibiting low overall levels of genetic diversity. We will use this information in future to examine the interplay between reproductive gene variation and reproductive fitness in Tasmanian devil populations.