bluuebunny/crossref_metadata_2025 huggingface dataset:
Plk4 triggers autonomous de novo centriole biogenesis and maturation
Authors: Nabais Catarina , Pessoa Delphine , de-Carvalho Jorge , van Zanten Thomas , Duarte Paulo , Mayor Satyajit , Carneiro Jorge , Telley Ivo A. , Bettencourt-Dias Mónica
Published: May 2020

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell-types, centrioles assemble de novo, yet by poorly understood mechanisms. Here, we established a controlled system to investigate de novo centriole biogenesis, usingDrosophila melanogasteregg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at high Plk4 concentration, centrioles form de novo, mature and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the kinetics of centriole assembly. Moreover, our results suggest Plk4 operates in a switch-like manner to control the onset of de novo centriole formation, and that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and reveal that proteins of the pericentriolar matrix (PCM) promote biogenesis, likely by locally concentrating critical components.
Site-specific glycosylation mapping of Fc gamma receptor IIIb from neutrophils of health donors
Authors: Wojcik Iwona , Sénard Thomas , de Graaf Erik L , Janssen George MC , de Ru Arnoud H , Mohammed Yassene , van Veelen Peter A , Vidarsson Gestur , Wuhrer Manfred , Falck David
Published: May 2020

Fc gamma receptors (FcγR) translate antigen-recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. In this study, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation from primary human material. In contrast to recently published alternatives, we assessed all glycosylation sites in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of FcγRIII glycosylation differences between individuals, cell types, subcellular locations and pathophysiological conditions.
De novo synthesis and salvage pathway coordinately regulates polyamine homeostasis and determines T cell proliferation and function
Authors: Wu Ruohan , Chen Xuyong , Kang Siwen , Wang Tingting , Gnanaprakasam JN Rashida , Yao Yufeng , Liu Lingling , Zheng Song Guo , Fan Gaofeng , Burns Mark R , Wang Ruoning
Published: May 2020

Robust and effective T cell-mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of de novo synthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in polyamine uptake from the environment, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular PA pool, inhibits T cell proliferation, suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine for managing inflammatory and autoimmune diseases.
Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate
Authors: Ladinsky Mark S. , Gnanapragasam Priyanthi N.P. , Yang Zhi , West Anthony P. , Kay Michael S , Bjorkman Pamela J.
Published: May 2020

Fusion of HIV-1 with the membrane of its target cell, an obligate first step in virus infectivity, is mediated by binding of the viral envelope (Env) spike protein to its receptors, CD4 and CCR5/CXCR4, on the cell surface. The process of viral fusion appears to be fast compared with viral egress and has not been visualized by electron microscopy (EM). To capture fusion events for EM, the process must be slowed or stopped by trapping Env-receptor binding at an intermediate stage. Here we describe using fusion inhibitors to trap HIV-1 virions attached to target cells by Envs in an extended pre-hairpin intermediate state. Electron tomography revealed HIV-1 virions bound to TZM-bl cells by 2-4 narrow spokes, with slightly more spokes present when evaluated with mutant virions that lacked the Env cytoplasmic tail. These results represent the first direct visualization of the hypothesized pre-hairpin intermediate and improve our understanding of Env-mediated HIV-1 fusion and infection of host cells.
Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses
Authors: Zonneveld Marijke I. , van Herwijnen Martijn J.C. , Fernandez-Gutierrez Marcela M. , Giovanazzi Alberta , de Groot Anne Marit , Kleinjan Marije , van Capel Toni M.M. , Sijts Alice J.A.M. , Taams Leonie S. , Garssen Johan , de Jong Esther C. , Kleerebezem Michiel , Nolte-’t Hoen Esther N.M. , Redegeld Frank A. , Wauben Marca H.M.
Published: May 2020

Maternal milk is nature’s first functional food. It plays a crucial role in the development of the infant’s gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract.
HumanizedDrosophilamodel of the Meier-Gorlin syndrome reveals conserved and divergent features of the Orc6 protein
Authors: Balasov Maxim , Akhmetova Katarina , Chesnokov Igor
Published: May 2020

Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears and skeletal abnormalities. Patients with MGS often carry mutations in the genes encoding the subunits of the Origin Recognition Complex (ORC), components of the pre-replicative complex (pre-RC) and replication machinery. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. A mutation in conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. Recently, new mutation in Orc6 was also identified however, it is localized in the N-terminal domain of the protein. In order to study the functions of Orc6 we used human gene to rescue theorc6deletion inDrosophila. Using the “humanized” Orc6-basedDrosophilamodel of the Meier-Gorlin syndrome we discovered that unlike previous Y225S MGS mutation in Orc6, the K23E substitution in the N-terminal TFIIB-like domain of Orc6 disrupts the protein ability to bind DNA. Our studies revealed the importance of evolutionary conserved and variable domains of Orc6 protein and allowed the studies of human protein functions and the analysis of the critical amino acids in live animal heterologous system as well as provided novel insights into the mechanisms underlying MGS pathology.
Atypical response inhibition and error processing in 22q11.2 Deletion Syndrome and Schizophrenia: Towards neuromarkers of disease progression and risk
Authors: Francisco Ana A. , Horsthuis Douwe J. , Popiel Maryann , Foxe John J. , Molholm Sophie
Published: May 2020

22q11.2 deletion syndrome (also known as DiGeorge syndrome or velo-cardio-facial syndrome) is characterized by increased vulnerability to neuropsychiatric symptoms, with approximately 30% of individuals with the deletion going on to develop schizophrenia. Clinically, deficits in executive function have been noted in this population, but the underlying neural processes are not well understood. Using a Go/No-Go response inhibition task in conjunction with high-density electrophysiological recordings (EEG), we sought to investigate the behavioral and neural dynamics of inhibition of a prepotent response (a critical component of executive function) in individuals with 22q11.2DS with and without psychotic symptoms, when compared to individuals with idiopathic schizophrenia and age-matched neurotypical controls. Twenty-eight participants diagnosed with 22q11.2DS (14-35 years old; 14 with at least one psychotic symptom), 15 individuals diagnosed with schizophrenia (18-63 years old) and two neurotypical control groups (one age-matched to the 22q11.2DS sample, the other age-matched to the schizophrenia sample) participated in this study. Analyses focused on the N2 and P3 no-go responses and error-related negativity (Ne) and positivity (Pe). Atypical inhibitory processing was shown behaviorally and by significantly reduced P3, Ne, and Pe responses in 22q11.2DS and schizophrenia. Interestingly, whereas P3 was only reduced in the presence of psychotic symptoms, Ne and Pe were equally reduced in schizophrenia and 22q11.2DS, regardless of the presence of symptoms. We argue that while P3 may be a marker of disease severity, Ne and Pe might be candidate markers of risk.
Invasive freshwater snails form novel microbial relationships
Authors: Bankers L. , Dahan D. , Neiman M. , Adrian-Tucci C. , Frost C. , Hurst G.D.D. , King K.C.
Published: May 2020

Resident microbes (microbiota) can shape host organismal function and adaptation in the face of environmental change. Invasion of new habitats exposes hosts to novel selection pressures, but little is known about the impact of invasion on microbiota and the host-microbiome relationship after this transition (e.g., how rapidly symbioses are formed, whether microbes influence invasion success). We used high-throughput 16S rRNA sequencing of New Zealand (native) and European (invasive) populations of the freshwater snailPotamopyrgus antipodarumand found that while invaders do carry over some core microbial taxa from New Zealand, most of their microbial community is distinct. This finding highlights that invasions can result in the formation of novel symbioses. We further show that the native microbiome is composed of fewer core microbes than the microbiome of invasive snails, suggesting that the microbiota is streamlined to essential members. Together, our findings demonstrate that microbiota comparisons across native and invasive populations can reveal the impact of a long coevolutionary history and specialization of microbes in the native host range, as well as new associations occurring after invasion. We lay essential groundwork for understanding how microbial relationships affect invasion success and how microbes may be utilized in the control of invasive hosts.
Eliciting OTUD3/RIPK-Dependent Necroptosis to Prevent Epithelial Ovarian Cancer
Authors: Johnson Joshua , Bales Elise C. , Bitler Benjamin G. , Watson Zachary L.
Published: May 2020

Background

There is an urgent need for early prevention strategies against high grade serous ovarian carcinoma (HGSOC), the deadliest gynecologic malignancy. Transformed p53-null fallopian tube epithelium (FTE) cells are precursors of HGSOC that may be eliminated by inducing necroptosis, a programmed form of inflammatory cell death. Induction of necroptosis is dependent upon activation of receptor-interacting serine/threonine-protein kinases 1 and 3 (RIPK1/3). TNFα and progesterone (P4) effectively promote necroptosis. In this study, we explore the activation of necroptosis as an approach to inhibit HGSOC progression.

Methods

Using gene ontology sets as a reference, we analyzed publicly available datasets of HGSOC to correlate the expression of necroptosis effectors to clinical outcomes. Usingin vitromodels of HGSOC we evaluated the effect of TNFα, P4, and α-eleostearic acid on necroptosis. In parallel, the necroptosis inhibitor Necrostatin-1 was used to confirm necroptosis-specific cell death.

Results

Expression of the P4 receptor (PGR) was sharply reduced in a HGSOC cohort compared to normal, nonmalignant FTE. However, several genes involved in necroptosis signaling were elevated in HGSOC, includingTNFandRIPK1. Increased expression ofPGR, the necroptosis effectorsTNFandRIPK1/3, as well as ovarian tumor domain-containing deubiquitinase 3 (OTUD3) were associated with higher overall survival in 484 HGSOC cases. HGSOC cells activated necroptosis in response to P4, TNFα, and α-eleostearic acid treatment, while P4 or TNFα treatment of HGSOC cells increasedTNF,RIPK1, andOTUD3expression. OTUD3 is a putative tumor suppressor that stabilizes PTEN and is hypothesized to be functionally similar to the necroptosis inducer, OTUD7B. shRNA knockdown ofOTUD3resulted in decreased PTEN protein and RIPK1 protein.

Conclusions

We conclude that necroptosis activation may be a viable prevention strategy that leads to the elimination of transformed FTE “founder” cells and prevents HGSOC tumorigenesis. Our data indicate that HGSOC cells activate necroptosis in response to P4, TNFα, and α-eleostearic acid, suggesting that established HGSOC cells may also be eliminated by activating necroptosis.
CNCDatabase: a database of non-coding cancer drivers
Authors: Liu Eric Minwei , Martinez-Fundichely Alexander , Bollapragada Rajesh , Spiewack Maurice , Khurana Ekta
Published: May 2020

Most mutations in cancer genomes occur in the non-coding regions with unknown impact to tumor development. Although the increase in number of cancer whole-genome sequences has revealed numerous putative non-coding cancer drivers, their information is dispersed across multiple studies and thus it is difficult to bridge the understanding of non-coding alterations, the genes they impact and the supporting evidence for their role in tumorigenesis across multiple cancer types. To address this gap, we have developed CNCDatabase, Cornell Non-Coding Cancer driver Database (https://cncdatabase.med.cornell.edu/) that contains detailed information about predicted non-coding drivers at gene promoters, 5’ and 3’ UTRs (untranslated regions), enhancers, CTCF insulators and non-coding RNAs. CNCDatabase documents 1,111 protein-coding genes and 90 non-coding RNAs with reported drivers in their non-coding regions from 32 cancer types by computational predictions of positive selection in whole-genome sequences; differential gene expression in samples with and without mutations; or another set of experimental validations including luciferase reporter assays and genome editing. The database can be easily modified and scaled as lists of non-coding drivers are revised in the community with larger whole-genome sequencing studies, CRISPR screens and further experimental validations. Overall, CNCDatabase provides a helpful resource for researchers to explore the pathological role of non-coding alterations and their associations with gene expression in human cancers.
Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2
Authors: Korber B , Fischer WM , Gnanakaran S , Yoon H , Theiler J , Abfalterer W , Foley B , Giorgi EE , Bhattacharya T , Parker MD , Partridge DG , Evans CM , Freeman TM , de Silva TI ,   on behalf of the Sheffield COVID-19 Genomics Group, LaBranche CC , Montefiori DC
Published: May 2020

We have developed an analysis pipeline to facilitate real-time mutation tracking in SARS-CoV-2, focusing initially on the Spike (S) protein because it mediates infection of human cells and is the target of most vaccine strategies and antibody-based therapeutics. To date we have identified thirteen mutations in Spike that are accumulating. Mutations are considered in a broader phylogenetic context, geographically, and over time, to provide an early warning system to reveal mutations that may confer selective advantages in transmission or resistance to interventions. Each one is evaluated for evidence of positive selection, and the implications of the mutation are explored through structural modeling. The mutation Spike D614G is of urgent concern; it began spreading in Europe in early February, and when introduced to new regions it rapidly becomes the dominant form. Also, we present evidence of recombination between locally circulating strains, indicative of multiple strain infections. These finding have important implications for SARS-CoV-2 transmission, pathogenesis and immune interventions.
Removing the Gβγ-SNAP25 brake on exocytosis enhances insulin action, promotes adipocyte browning, and protects against diet-induced obesity
Authors: Ceddia Ryan P. , Zurawski Zack , Gray Analisa Thompson , Adegboye Feyisayo , McDonald-Boyer Ainsley , Shi Fubiao , Liu Dianxin , Maldonado Jose , Feng Jiesi , Li Yulong , Alford Simon , Ayala Julio E. , McGuinness Owen P. , Collins Sheila , Hamm Heidi E.
Published: May 2020

Negative regulation of exocytosis from secretory cells throughout the body is accomplished through inhibitory signals from Gi/oG protein-coupled receptors by Gβγ subunit inhibition of two common mechanisms: (i) decreased calcium entry and (ii) direct interaction of Gβγ with the SolubleN-ethylmaleimide-sensitive factor Attachment Protein (SNAP) Receptor (SNARE) plasma membrane fusion machinery. We have previously shown that disabling the second mechanism with a truncation of SNAP25 (SNAP25Δ3/Δ3) decreases the affinity of Gβγ for the SNARE complex, leaving exocytotic fusion as well as modulation of calcium entry intact but disabling GPCR inhibition of exocytosis. Here we report significant beneficial metabolic remodeling in mice carrying this mutation. Chow-fed SNAP25Δ3/Δ3mice exhibit enhanced insulin sensitivity and increased beiging of white fat. In response to a high fat diet, the metabolic protection was amplified in SNAP25Δ3/Δ3mice. Glucose homeostasis, whole body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. This metabolic protection in SNAP25Δ3/Δ3mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting basal autonomic activity is required. Direct electrode stimulation of sympathetic neurons exocytosis from SNAP25Δ3/Δ3inguinal adipose depot resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as a new avenue for combatting metabolic disease.

GRAPHICAL ABSTRACT
Optimal prediction with resource constraints using the information bottleneck
Authors: Sachdeva Vedant , Mora Thierry , Walczak Aleksandra M. , Palmer Stephanie
Published: May 2020

Responding to stimuli requires that organisms encode information about the external world. Not all parts of the signal are important for behavior, and resource limitations demand that signals be compressed. Prediction of the future input is widely beneficial in many biological systems. We compute the trade-offs between representing the past faithfully and predicting the future for input dynamics with different levels of complexity. For motion prediction, we show that, depending on the parameters in the input dynamics, velocity or position coordinates prove more predictive. We identify the properties of global, transferrable strategies for time-varying stimuli. For non-Markovian dynamics we explore the role of long-term memory of the internal representation. Lastly, we show that prediction in evolutionary population dynamics is linked to clustering allele frequencies into non-overlapping memories, revealing a very different prediction strategy from motion prediction.
Critical Residues of Gβγ for the interaction with the SNARE Complex
Authors: Mueller Benjamin K. , Kaya Ali I , Zurawski Zack , Yim Yun Young , Meiler Jens , Hamm Heidi E.
Published: May 2020

The mechanisms and regulation of neurotransmitter release is a complex process involving many co-factors and proteins. One critical interaction is the regulation of exocytosis when G-protein βγ (Gβγ) dimers bind to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complex. The complex is comprised of N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Herein we probe across the entire family of human Gβ and Gγ proteins for residues critical for the interaction with SNARE, by systematically screening peptide sequences for their ability to bind to tSNARE. The coiled-coil region of Gβγ showed high affinity to tSNARE, along with the propeller region of Gβ on the opposite side from the coiled-coil region. Peptides based on Gβ1γ2, shown to have high affinity to SNARE, tSNARE were screened further by alanine scanning to probe for residues critical for binding to tSNARE. Full length Gβ1γ2and SNARE were docked computationally using Rosetta, to examine the experimentally determined binding sites. Docking converged on two possible sites of interaction using two distinct regions of both Gβ1γ2and SNARE.
Clustering NMR: Machine learning assistive rapid (pseudo) two-dimensional relaxometry mapping
Authors: Peng Weng Kung
Published: May 2020

Low-field nuclear magnetic resonance (NMR) relaxometry is an attractive approach for point-of-care testing medical diagnosis, industrial food science, andin situoil-gas exploration. One of the problem however is, the inherently long relaxation time of the (liquid) sample, (and hence low signal-to-noise ratio) causes unnecessarily long repetition time. In this work, we present a new class of methodology for rapid and accurate object classification using NMR relaxometry with the aid of machine learning. We demonstrate that the sensitivity and specificity of the classification is substantially improved with higher order of (pseudo)-dimensionality (e.g., 2D or multidimensional). This new methodology (termed as Clustering NMR) is extremely useful for rapid and accurate object classification (in less than a minute) using the low-field NMR.
Nature and effective range of non-cell autonomous activator and inhibitor peptides specifying plant stomatal patterning
Authors: Zeng Scott , Lo Emily K. W. , Hazelton Bryna J. , Morales Miguel F. , Torii Keiko U.
Published: May 2020

Stomata are epidermal valves that facilitate gas exchange between plants and their environment. Stomatal patterning is regulated by EPIDERMAL PATTERING FACTOR (EPF)-family of secreted peptides: EPF1 enforcing stomatal spacing, whereas EPF-LIKE9, also known as Stomagen, promoting stomatal development. It remains unknown, however, how far these signaling peptides act. Utilizing Cre-Lox recombination-based mosaic sectors that overexpress either EPF1 or Stomagen in Arabidopsis cotyledons, we reveal a range within the epidermis and across the cell layers in which these peptides influence patterns. To quantitatively determine their effective ranges, we developed a computational pipeline, SPACE (Stomata Patterning AutoCorrelation on Epidermis), that describes probabilistic two-dimensional stomatal distributions based upon spatial autocorrelation statistics used in Astrophysics. The SPACE analysis shows that, whereas both peptides act locally, the inhibitor, EPF1, exerts longer-range effects than the activator, Stomagen. Furthermore, local perturbation of stomatal development has little influence on global two-dimensional stomatal patterning. Our findings conclusively demonstrate the nature and extent of EPF peptides as non-cell autonomous local signals and provides a means to quantitatively characterize complex spatial patterns in development.

Summary Statement

Non-cell autonomous effects of activator and inhibitor peptides on 2-D spatial patterning of stomata were quantitatively characterized using chimeric sectors and a SPACE computational pipeline.
Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome
Authors: Shaiber Alon , Willis Amy D. , Delmont Tom O. , Roux Simon , Chen Lin-Xing , Schmid Abigail C. , Yousef Mahmoud , Watson Andrea R. , Lolans Karen , Esen Özcan C. , Lee Sonny T. M. , Downey Nora , Morrison Hilary G. , Dewhirst Floyd E. , Mark Welch Jessica L. , Eren A. Murat
Published: May 2020

Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and their intriguing patterns of site specificity amidst the lack of dispersal limitation. Yet, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among the taxa that belong to recently discovered branches of microbial life. Here we assembled metagenomes from daily tongue and dental plaque samples from multiple individuals and reconstructed 790 non-redundant genomes, 43 of which resolved to TM7 that formed six monophyletic clades distinctly associated either with plaque or with tongue. Both pangenomic and phylogenomic analyses grouped tongue-specific TM7 clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 grouped together with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and the mobilome of the cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of human associated microbes. Additionally, we report that prophages are widespread amongst oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment.
A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues
Authors: Nims Robert J. , Pferdehirt Lara , Ho Noelani B. , Savadipour Alireza , Lorentz Jeremiah , Sohi Sima , Kassab Jordan , Ross Alison K. , O’Conor Christopher J. , Liedtke Wolfgang B. , Zhang Bo , McNulty Amy L. , Guilak Farshid
Published: May 2020

Mechanobiologic signals regulate cellular responses under physiologic and pathologic conditions. Using synthetic biology and tissue engineering, we developed a mechanically-responsive bioartificial tissue that responds to mechanical loading to produce a pre-programmed therapeutic biologic drug. By deconstructing the signaling networks induced by activation of the mechanically-sensitive ion channel transient receptor potential vanilloid 4 (TRPV4), we created synthetic TRPV4-responsive genetic circuits in chondrocytes. We engineered these cells into living tissues that respond to mechanical loading by producing the anti-inflammatory biologic drug, interleukin-1 receptor antagonist. Chondrocyte TRPV4 is activated by osmotic loading and not direct cellular deformation, suggesting tissue loading is transduced into an osmotic signal that activates TRPV4. Either osmotic or mechanical loading of tissues transduced with TRPV4-responsive circuits protected constructs from inflammatory degradation by interleukin-1α. This synthetic mechanobiology approach was used to develop a mechanogenetic system to enable long-term, autonomously regulated drug delivery driven by physiologically-relevant loading.
Hypothalamic circuitry underlying stress-induced insomnia and peripheral immunosuppression
Authors: Li Shi-Bin , Borniger Jeremy C , Yamaguchi Hiroshi , Hédou Julien , Gaudillière Brice , de Lecea Luis
Published: May 2020

The neural substrates of insomnia/hyperarousal induced by stress remain unknown. Here, we show that restraint stress leads to hyperarousal associated with strong activation of corticotropin-releasing hormone neurons in the paraventricular nucleus of hypothalamus (CRHPVN) and hypocretin neurons in the lateral hypothalamus (HcrtLH). CRHPVNneurons are quiescent during natural sleep-wake transitions but are strongly active under restraint stress. CRISPR-Cas9-mediated knockdown of thecrhgene in CRHPVNneurons abolishes hyperarousal elicited by stimulating LH-projecting CRHPVNneurons. Genetic ablation of Hcrt neurons orcrhgene knockdown significantly reduces insomnia/hyperarousal induced by restraint stress. Given the association between stress and immune function, we used single-cell mass cytometry by time of flight (CyTOF) to analyze peripheral blood and found extensive changes to immune cell distribution and functional responses during wakefulness upon optogenetic stimulation of CRHPVNneurons. Our findings suggest both central and peripheral systems are synergistically engaged in the response to stress via CRHPVNcircuitry.
Conformational Selection in Ligand Recognition by the First Tudor Domain of PHF20L1
Authors: Lv Mengqi , Gao Jia , Li Mingwei , Ma Rongsheng , Li Fudong , Liu Yaqian , Liu Mingqing , Zhang Jiahai , Yao Xuebiao , Wu Jihui , Shi Yunyu , Tang Yajun , Pan Yueyin , Zhang Zhiyong , Ruan Ke
Published: May 2020

The first Tudor domain of PHF20L1 (PHF20L1 Tudor1) recognizes both histone methylation and non-histone methylation to play versatile roles, e.g., PHF20L1 Tudor1 binds to the oncogenic target DNA (cytosine-5) methyltransferase 1 (DNMT1) to prevent it from degradation. However, the crystal structure of the PHF20 Tudor domain, a homolog of PHF20L1, reveals a closed aromatic cage of the Tudor domain. It is thus highly desirable to interrogate the ligand-recognition mechanism of PHF20L1 Tudor1, which will in turn validate the potential druggability of this target. Here, we solved the crystal structure of the free form PHF20L1 Tudor1, which adopts the closed conformation similar to PHF20. NMR relaxation dispersion and molecular dynamics (MD) simulations suggest a pre-existing low-population conformation with a remarkable rearrangement of aromatic cage residues. Such structural rearrangement is further revealed by the crystal structures of PHF20L1 Tudor1 in complex with the lysine 142 methylated (K142me1) DNMT1, and a small molecule cosolvent 2-(N-morpholino)ethanesulfonic acid (MES), respectively. This result thus ignites interest in the discovery of small molecule inhibitors against PHF20L1 Tudor1. The hit identified from NMR fragment-based screening protrudes into the same open form aromatic cage of PHF20L1 Tudor1, and blocks the interaction between PHF20L1 Tudor1 and methylated DNMT1. Further free form crystal structures of key mutants reveal one open form and one closed form aromatic cage, which is energetically trapped observed in the NMR relaxation dispersion and MD simulations. The binding of DNMT1 with PHF20L1 Tudor1 mutants was also recapitulated in cancer cells. The mutagenesis thus alters the structure, dynamics and eventually the function of PHF20L1 Tudor1. Our results demonstrate that PHF20L1 Tudor1 utilizes the same conformational selection mechanism to recognize ligands, regardless of whether it is a natural substrate or a small molecule identified from fragment-based screening. Albeit at a low population, the pre-existing ligand-binding conformation shall shift the paradigm in the druggability assessment of a dynamic protein, even though it may lack a small molecule binding pocket in its free form structure. The inhibition of PHF20L1 paves an alternative way to target DNMT1 degradation.
Leveraging biological complexity to predict patch occupancy in a recent host range expansion
Authors: Forister M. L. , Philbin C. S. , Marion Z. H. , Buerkle C. A. , Dodson C. D. , Fordyce J. A. , Forister G. W. , Lebeis S. L. , Lucas L. K. , Nice C. C. , Gompert Z.
Published: May 2020

Specialized plant-insect interactions are a defining feature of life on earth, yet we are only beginning to understand the factors that set limits on host ranges in herbivorous insects. To understand the colonization of alfalfa by the Melissa blue butterfly, we quantified arthropod assemblages and plant metabolites across a wide geographic region, while controlling for climate and dispersal inferred from population genomic variation. The presence of the butterfly is successfully predicted by direct and indirect effects of plant traits and interactions with other species. Results are consistent with the predictions of a theoretical model of parasite host range in which specialization is an epiphenomenon of the many barriers to be overcome rather than a consequence of trade-offs in developmental physiology.

One sentence summary

The formation of a novel plant-insect interaction can be predicted with a combination of biotic and abiotic factors, with comparable importance revealed for metabolomic variation in plants and interactions with mutualists, competitors and enemies.
Experimental Stroke Induces Chronic Gut Dysbiosis and Neuroinflammation in Male Mice
Authors: Brichacek Allison L. , Nwafor Divine C. , Benkovic Stanley A. , Chakraborty Sreeparna , Kenney Sophia M. , Mace Maria E. , Jun Sujung , Gambill Catheryne A. , Wang Wei , Hu Heng , Ren Xuefang , Povroznik Jessica M. , Engler-Chiurazzi Elizabeth B. , Primerano Donald A. , Denvir James , Percifield Ryan , Infante Aniello , Franko Jennifer , Schafer Rosana , Gemoets Darren E. , Brown Candice M.
Published: May 2020

Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke. We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology up to 36 days post-stroke compared to sham controls. Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham surgery. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Bioinformatics analysis demonstrate significant differences in abundance among Firmicutes and Bacteroidetes taxa at the phylum, family, and species levels in tMCAO compared to sham mice that persisted up to one month post-stroke. The most persistent changes in post-stroke microbial abundance were a decrease in bacteria family S24-7 and significant increases inRuminococcaceae. Overall, these changes resulted in a persistently increased Firmicutes:Bacteroidetes ratio in stroke animals. Intestinal histopathology showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting. Increased astrocyte and microglial activity were also detected one-month post-stroke. These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis, intestinal inflammation, and chronic neuroinflammation.

Clinical Perspectives

The microbiota-gut-brain axis, recently implicated in several neurological disorders, remains largely unexplored at chronic time points post-tMCAO.

Our results demonstrate chronic gut dysbiosis, prolonged behavioral deficits, and persistent cerebral and intestinal inflammation post-tMCAO in male C57BL/6J mice.

These results suggest that manipulation of microbiota may help reduce poor outcomes after stroke and lead to improved post-stroke functional recovery.
ACE2 and TMPRSS2 expression by clinical, HLA, immune, and microbial correlates across 34 human cancers and matched normal tissues: implications for SARS-COV-2 COVID-19
Authors: Bao Riyue , Hernandez Kyle , Huang Lei , Luke Jason J.
Published: May 2020

Background

Pandemic COVID-19 by SARS-COV-2 infection is facilitated by the ACE2 receptor and protease TMPRSS2. Modestly sized case series have described clinical factors associated with COVID-19, while ACE2 and TMPRSS2 expression analyses have been described in some cell types. Cancer patients may have worse outcomes to COVID-19.

Methods

We performed an integrated study of ACE2 and TMPRSS2 gene expression across and within organ systems, by normal versus tumor, across several existing databases (The Cancer Genome Atlas, Census of Immune Single Cell Expression Atlas, The Human Cell Landscape, and more). We correlated gene expression with clinical factors (including but not limited to age, gender, race, BMI and smoking history), HLA genotype, immune gene expression patterns, cell subsets, and single-cell sequencing as well as commensal microbiome.

Results

Matched normal tissues generally display higher ACE2 and TMPRSS2 expression compared with cancer, with normal and tumor from digestive organs expressing the highest levels. No clinical factors were consistently identified to be significantly associated with gene expression levels though outlier organ systems were observed for some factors. Similarly, no HLA genotypes were consistently associated with gene expression levels. Strong correlations were observed between ACE2 expression levels and multiple immune gene signatures including interferon-stimulated genes and the T cell-inflamed phenotype as well as inverse associations with angiogenesis and transforming growth factor-β signatures. ACE2 positively correlated with macrophage subsets across tumor types. TMPRSS2 was less associated with immune gene expression but was strongly associated with epithelial cell abundance. Single-cell sequencing analysis across nine independent studies demonstrated little to no ACE2 or TMPRSS2 expression in lymphocytes or macrophages. ACE2 and TMPRSS2 gene expression associated with commensal microbiota in matched normal tissues particularly from colorectal cancers, with distinct bacterial populations showing strong associations.

Conclusions

We performed a large-scale integration of ACE2 and TMPRSS2 gene expression across clinical, genetic, and microbiome domains. We identify novel associations with the microbiota and confirm host immunity associations with gene expression. We suggest caution in interpretation regarding genetic associations with ACE2 expression suggested from smaller case series.
A Workflow for Protein Structure Determination from Thin Crystal Lamella by Micro-Electron Diffraction
Authors: Beale Emma V. , Waterman David G. , Hecksel Corey , van Rooyen Jason , Gilchrist James B. , Parkhurst James M. , de Haas Felix , Buijsse Bart , Evans Gwyndaf , Zhang Peijun
Published: May 2020

Micro-Electron Diffraction (MicroED) has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as needles or plates measuring only a few hundred nanometres in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimised for handling compound effects that are unique to electron diffraction data. Here, we present an integrated workflow for microED, from sample preparation by cryo-focused ion beam milling, through data collection with a standard Ceta-D detector, to data processing using the DIALS software suite, thus enabling routine atomic structure determination of protein crystals of any size and shape using microED. We demonstrate the effectiveness of the workflow by determining the structure of proteinase K to 2.0 Å resolution and show the advantage of using protein crystal lamellae over nanocrystals.
Connectomes across development reveal principles of brain maturation
Authors: Witvliet Daniel , Mulcahy Ben , Mitchell James K. , Meirovitch Yaron , Berger Daniel R. , Wu Yuelong , Liu Yufang , Koh Wan Xian , Parvathala Rajeev , Holmyard Douglas , Schalek Richard L. , Shavit Nir , Chisholm Andrew D. , Lichtman Jeff W. , Samuel Aravinthan D.T. , Zhen Mei
Published: May 2020

From birth to adulthood, an animal’s nervous system changes as its body grows and its behaviours mature. The form and extent of circuit remodelling across the connectome is unknown. We used serial-section electron microscopy to reconstruct the full brain of eight isogenicC. elegansindividuals across postnatal stages to learn how it changes with age. The overall geometry of the brain is preserved from birth to adulthood. Upon this constant scaffold, substantial changes in chemical synaptic connectivity emerge. Comparing connectomes among individuals reveals substantial connectivity differences that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. Over development, the central decision-making circuitry is maintained whereas sensory and motor pathways substantially remodel. With age, the brain progressively becomes more feedforward and discernibly modular. Developmental connectomics reveals principles that underlie brain maturation.
OMAmer: tree-driven and alignment-free protein assignment to subfamilies outperforms closest sequence approaches
Authors: Rossier Victor , Vesztrocy Alex Warwick , Robinson-Rechavi Marc , Dessimoz Christophe
Published: May 2020

Assigning new sequences to known protein families and subfamilies is a prerequisite for many functional, comparative and evolutionary genomics analyses. Such assignment is commonly achieved by looking for the closest sequence in a reference database, using a method such as BLAST. However, ignoring the gene phylogeny can be misleading because a query sequence does not necessarily belong to the same subfamily as its closest sequence. For example, a hemoglobin which branched out prior to the hemoglobin alpha/beta duplication could be closest to a hemoglobin alpha or beta sequence, whereas it is neither. To overcome this problem, phylogeny-driven tools have emerged but rely on gene trees, whose inference is computationally expensive.

Here, we first show that in multiple animal and plant datasets, 18 to 62% of assignments by closest sequence are misassigned, typically to an over-specific subfamily. Then, we introduce OMAmer, a novel alignment-free protein subfamily assignment method, which limits over-specific subfamily assignments and is suited to phylogenomic databases with thousands of genomes. OMAmer is based on an innovative method using evolutionarily-informedk-mers for alignment-free mapping to ancestral protein subfamilies. Whilst able to reject non-homologous family-level assignments, we show that OMAmer provides better and quicker subfamily-level assignments than approaches relying on the closest sequence, whether inferred exactly by Smith-Waterman or by the fast heuristic DIAMOND.

OMAmer is available from the Python Package Index (as omamer), with the source code and a precomputed database available athttps://github.com/DessimozLab/omamer.
Piezo1 activation attenuates thrombin-induced blebbing in breast cancer cells
Authors: O’Callaghan Paul , Engberg Adam , Fatsis-Kavalopoulos Nikos , Sanchez Gonzalo , Idevall-Hagren Olof , Kreuger Johan
Published: May 2020

Cancer cells exploit a variety of migration modes to leave primary tumors and establish metastases, including amoeboid cell migration facilitated by bleb formation. Here we demonstrate that thrombin induces dynamic blebbing in the MDA-MB-231 breast cancer cell line, and confirm that PAR2 activation is sufficient to induce this effect. Cell confinement has been implicated as a driving force in bleb-based migration. Unexpectedly, we find that gentle contact compression, exerted using a “Cell Press” to mechanically stimulate cells, attenuated thrombin-induced blebbing with an associated increase in cytosolic calcium. Thrombin-induced blebbing was similarly attenuated using Yoda1, an agonist of the mechanosensitive calcium channel Piezo1, and its capacity to attenuate blebbing was impaired in Piezo1 depleted cells. Additionally, Piezo1 activation suppressed and reversed the thrombin-induced phosphorylation of ERM proteins, which are implicated in the blebbing process, and this activity was in part mediated through activation of the PP1A/PP2A family of serine/threonine phosphatases. Our results provide mechanistic insights into Piezo1 activation as a suppressor of dynamic blebbing, specifically that which is induced by thrombin.
Structural basis of Rgg protein binding to their regulatory pheromones and target DNA promoters
Authors: Capodagli Glenn C. , Tylor Kaitlyn M. , Kaelber Jason T. , Petrou Vasileios I. , Federle Michael J. , Neiditch Matthew B.
Published: May 2020

Rgg family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors inStreptococcusspecies, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryo-EM structure ofStreptococcus thermophilusRgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures to that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of bothS. dysgalactiaeRgg2 andS. thermophilusRgg3 in complex with their target DNA promoters. The physiological importance of the observed Rgg-DNA interactions was dissected usingin vivogenetic experiments andin vitrobiochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of the SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.

Significance Statement

Secreted peptide pheromones regulate critical biological processes in Gram-positive bacteria. In streptococci such as the human pathogenS. pyogenes, oligopeptide pheromones, like the short hydrophobic peptides (SHPs), regulate virulence, antimicrobial resistance, and biofilm formation. SHPs directly regulate the activity of transcription factors called Rgg2 and Rgg3. We present the cryo-EM structure of Rgg3 in complex with SHP3, as well as X-ray crystal structures of Rgg2 bound to target promoter DNA, Rgg3 bound to target promoter DNA, and Rgg3 alone. Based on the cryo-EM, X-ray crystallographic, biochemical, and genetic studies presented here, we provide not only detailed mechanistic insight into the molecular basis of Rgg3-SHP3, Rgg2-DNA, and Rgg3-DNA binding specificity, but also a new model of transcription factor regulatory interplay.