bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-cell evidence for plasmid addiction mediated by toxin-antitoxin systems
Authors: Fraikin Nathan , Van Melderen Laurence 
Published: August 2023

AbstractToxin-antitoxin (TA) systems are small selfish genetic modules that increase vertical stability of their replicons. They have long been thought to stabilize plasmids by killing cells that fail to inherit a plasmid copy through a phenomenon called post-segregational killing (PSK) or addiction. While this model has been widely accepted, no direct observation of PSK was reported in the literature. Here, we devised a system that enables visualization of plasmid loss and PSK at the single-cell level using meganuclease-driven plasmid curing. Using theccdsystem, we show that cells deprived of accd-encoding plasmid show hallmarks of DNA damage,i.e. filamentation and induction of the SOS response. Activation ofccd-triggered cell death in most plasmid-free segregants, although some intoxicated cells were able to resume growth, showing that PSK-induced damage can be repaired in a SOS-dependent manner. Damage induced byccdactivates resident lambdoid prophages, which potentiate the killing effect ofccd. The loss of a model plasmid containing TA systems encoding toxins presenting various molecular mechanisms induced different morphological changes, growth arrest and loss of viability. Our experimental setup enables further studies of TA-induced phenotypes and suggests that PSK is a general mechanism for plasmid stabilization by TA systems.
Mapping protein–exopolysaccharide binding interaction inStaphylococcus epidermidisbiofilms by live cell proximity labeling
Authors: Vo Luan H. , Hong Steven , Stepler Kaitlyn E. , Liyanaarachchi Sureshee M. , Yang Jack , Nemes Peter , Poulin Myles B. 
Published: August 2023

AbstractBacterial biofilms consist of cells encased in an extracellular polymeric substance (EPS) composed of exopolysaccharides, extracellular DNA, and proteins that are critical for cell–cell adhesion and protect the cells from environmental stress, antibiotic treatments, and the host immune response. Degrading EPS components or blocking their production have emerged as promising strategies for prevention or dispersal of bacterial biofilms, but we still have little information about the specific biomolecular interactions that occur between cells and EPS components and how those interactions contribute to biofilm production.Staphylococcus epidermidisis a leading cause of nosocomial infections as a result of producing biofilms that use the exopolysaccharide poly- (1→6)-β-N-acetylglucosamine (PNAG) as a major structural component. In this study, we have developed a live cell proximity labeling approach combined with quantitative mass spectrometry-based proteomics to map the PNAG interactome of liveS. epidermidisbiofilms. Through these measurements we discovered elastin-binding protein (EbpS) as a major PNAG-interacting protein. Using live cell binding measurements, we found that the lysin motif (LysM) domain of EbpS specifically binds to PNAG present inS. epidermidisbiofilms. Our work provides a novel method for the rapid identification of exopolysaccharide-binding proteins in live biofilms that will help to extend our understanding of the biomolecular interactions that are required for bacterial biofilm formation.
HIV-1 binds dynein directly to hijack microtubule transport machinery
Authors: Badieyan Somayesadat , Lichon Drew , Andreas Michael P. , Gillies John P. , Peng Wang , Shi Jiong , DeSantis Morgan E. , Aiken Christopher R. , Böcking Till , Giessen Tobias W. , Campbell Edward M. , Cianfrocco Michael A. 
Published: August 2023

SummaryViruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet the molecular mechanism that describes how viruses connect to the motor machinery is unknown. Here, we demonstrate the first example of viral microtubule trafficking from purified components: HIV-1 hijacking microtubule transport machinery. We discover that HIV-1 directly binds to the retrograde microtubule-associated motor, dynein, and not via a cargo adaptor, as previously suggested. Moreover, we show that HIV-1 motility is supported by multiple, diverse dynein cargo adaptors as HIV-1 binds to dynein light and intermediate chains on dynein’s tail. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, like HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results introduce a new model of viral trafficking wherein a pathogen opportunistically ‘hijacks’ the microtubule transport machinery for motility, enabling multiple transport pathways through the host cytoplasm.
Toxoplasma gondiiactin filaments are tuned for rapid disassembly and turnover
Authors: Hvorecny Kelli L. , Sladewski Thomas E. , De La Cruz Enrique M. , Kollman Justin M. , Heaslip Aoife T. 
Published: August 2023

AbstractThe cytoskeletal protein actin plays a critical role in the pathogenicity ofToxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there is conflicting data regarding the biochemical and biophysical properties ofToxoplasmaactin. Here, we imaged thein vitroassembly of individualToxoplasmaactin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin,Toxoplasmafilaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of stabilized and unstabilized filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune dynamic properties of actin filaments without disrupting their conserved structure.
Oxidative regulation of TDP-43 self-association by a β-to-α conformational switch
Authors: Gu Jinge , Zhou Xiaoming , Sutherland Lillian , Kato Masato , Jaczynska Klaudia , Rizo Josep , McKnight Steven L. 
Published: August 2023

SummaryAn evolutionarily conserved region of the TDP-43 low complexity domain twenty residues in length can adopt either an α-helical or β-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-β structure. Self-association can be monitored via the formation of phase separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the low complexity domain. Oxidation disassembles the cross-β structure, thus eliminating both self-association and phase separation. Here we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate β-strand mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent, and that exposure to lipids enhances the β-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of low complexity domains.Significance StatementThe TDP-43 protein is a constituent of RNA granules involved in regulated translation. TDP-43 contains a C-terminal domain of 150 amino acids of low sequence complexity conspicuously decorated with ten methionine residues. An evolutionarily conserved region (ECR) of 20 residues within this domain can adopt either of two forms of labile secondary structure. Under normal conditions wherein methionine residues are reduced, the ECR forms a labile cross-β structure that enables RNA granule condensation. Upon methionine oxidation, the ECR undergoes a conformational switch to become an α-helix incompatible with self-association and granule integrity. Oxidation of the TDP-43 low complexity domain is hypothesized to occur proximal to mitochondria, thus facilitating dissolution of RNA granules and activation of localized translation.
Heparan sulfate modifications of betaglycan promote TIMP3-dependent ectodomain shedding to fine-tune TGF-β signaling
Authors: Choi Alex S , Jenkins-Lane Laura M , Barton Wade , Kumari Asha , Lancaster Carly , Raulerson Calen , Ji Hao , Altomare Diego , Starr Mark D , Whitaker Regina , Phaeton Rebecca , Arend Rebecca , Shtutman Michael , Nixon Andrew B , Hempel Nadine , Lee Nam Y , Mythreye Karthikeyan 
Published: August 2023

AbstractIn pathologies such as cancer, aberrant Transforming Growth Factor-β (TGF-β) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pivotal pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-β responses. Betaglycan/type III TGF-β receptor (TβRIII), is an established co-receptor for the TGF-β superfamily known to bind directly to TGF-βs 1-3 and inhibin A/B. While betaglycan can be membrane-bound, it can also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. The extracellular domain of betaglycan undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. Here we report the unexpected discovery that the heparan sulfate modifications are critical for the ectodomain shedding of betaglycan. In the absence of such modifications, betaglycan is not shed. Such shedding is indispensable for the ability of betaglycan to suppress TGF-β signaling and the cells’ responses to exogenous TGF-β ligands. Using unbiased transcriptomics, we identified TIMP3 as a key regulator of betaglycan shedding and thereby TGF-β signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-β signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan modifications of betaglycan for shedding and influence on TGF-β signaling responses. Dysregulated shedding of TGF-β receptors plays a vital role in determining the response and availability of TGF-βs’, which is crucial for prognostic predictions and understanding of TGF-β signaling dynamics.
Rab7a activation promotes degradation of select tight junction proteins at the blood-brain barrier after ischemic stroke
Authors: Cottarelli Azzurra , Shahriar Sanjid , Arac Ahmet , Glendinning Michael , Tuohy Mary Claire , Prochilo Grace , Neal Jason B. , Edinger Aimee L. , Agalliu Dritan 
Published: August 2023

SUMMARYThe stability of tight junctions (TJs) between endothelial cells (ECs) is essential to maintain blood-brain barrier (BBB) function in the healthy brain. Following ischemic stroke, TJ strand dismantlement due to protein degradation leads to BBB dysfunction, yet the mechanisms driving this process are poorly understood. Here, we show that endothelial-specific ablation of Rab7a, a small GTPase that regulates endolysosomal protein degradation, reduces stroke-induced TJ strand disassembly resulting in decreased paracellular BBB permeability and improved neuronal outcomes. Two pro-inflammatory cytokines, TNFα and IL1β, but not glucose and oxygen deprivation, induce Rab7a activation via Ccz1 in brain ECsin vitro, leading to increased TJ protein degradation and impaired paracellular barrier function. Silencing Rab7a in brain ECsin vitroreduces cytokine-driven endothelial barrier dysfunction by suppressing degradation of a key BBB TJ protein, Claudin-5. Thus, Rab7a activation by inflammatory cytokines promotes degradation of select TJ proteins leading to BBB dysfunction after ischemic stroke.
The mechanism ofGαqregulation ofPLCβ3-catalyzedPIP2hydrolysis
Authors: Falzone Maria E. , MacKinnon Roderick 
Published: August 2023

AbstractPLCβenzymes cleavePIP2producing IP3 and DAG.PIP2modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca2+levels and protein phosphorylation by protein kinase C, respectively.PLCβenzymes are under the control of GPCR signaling through direct interactions with G proteinsGβγandGαqand have been shown to be coincidence detectors for dual stimulation ofGαqand Gαicoupled receptors.PLCβsare aqueous-soluble cytoplasmic enzymes, but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed thatGβγactivatesPLCβ3by recruiting it to the membrane. Using these same methods, here we show thatGαqincreases the catalytic rate constant,kcat, ofPLCβ3. Since stimulation ofPLCβ3byGαqdepends on an autoinhibitory element (the X-Y linker), we propose thatGαqproduces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of thePLCβ3-Gαq,andPLCβ3-Gβγ(2)-Gαqcomplexes, which show that these G proteins can bind simultaneously and independently of each other to regulatePLCβ3activity. The structures rationalize a finding in the enzyme assay, that co-stimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity ofPLCβ3is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.Significance StatementFor certain cellular signaling processes, the background activity of signaling enzymes must be minimal and stimulus-dependent activation robust. Nowhere is this truer than in signaling byPLCβ3, whose activity regulates intracellular Ca2+, phosphorylation by Protein Kinase C, and the activity of numerous ion channels and membrane receptors. In this study we show howPLCβ3enzymes are regulated by two kinds of G proteins,GβγandGαq. Enzyme activity studies and structures on membranes show how these G proteins act by separate, independent mechanisms, leading to a product rule of co-stimulation when they act together. The findings explain how cells achieve robust stimulation ofPLCβ3in the setting of very low background activity, properties essential to cell health and survival.
Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor
Authors: Aspacio Derek , Zhang Yulai , Cui Youtian , King Edward , Black William B. , Perea Sean , Luu Emma , Siegel Justin B. , Li Han 
Published: August 2023

AbstractNatural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+and NADP+, to orchestrate life-essential redox reaction directions. However, in whole cells the reliance on these canonical cofactors limits flexible control of redox reaction direction as these reactions are permanently tied to catabolism or anabolism. In cell-free systems, NADP+is too expensive in large scale. We have previously reported the use of nicotinamide mononucleotide, (NMN+) as a low-cost, noncanonical redox cofactor capable of specific electron delivery to diverse chemistries.Here, we present Nox Ortho, an NMNH-specific water-forming oxidase, that completes the toolkit to modulate NMNH/NMN+ratio. This work uncovers an enzyme design principle that succeeds in parallel engineering of six butanediol dehydrogenases as NMN(H)-orthogonal biocatalysts consistently with a 103– 106-fold cofactor specificity switch from NAD(P)+to NMN+. We combine these to produce chiral-pure 2,3-butanediol (Bdo) isomers without interference from NAD(H) or NADP(H) in vitro and inE. colicells. We establish that NMN(H) can be held at a distinct redox ratio on demand, decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo.
Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases
Authors: Koh Duk-Su , Stratiievska Anastasiia , Jana Subhashis , Otto Shauna C. , Swanson Teresa M. , Nhim Anthony , Carlson Sara , Raza Marium , Naves Lígia Araujo , Senning Eric N. , Mehl Ryan A. , Gordon Sharona E. 
Published: August 2023

ABSTRACTLigands such as insulin, epidermal growth factor, platelet derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell- impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.
Targeting the glycan epitope type IN-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells
Authors: Rossdam Charlotte , Brand Smilla , Beimdiek Julia , Oberbeck Astrid , Albers Marco Darius , Naujok Ortwin , Buettner Falk F. R. 
Published: August 2023

AbstractCell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galβ1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markersOCT4andNANOGwas highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.
The assessment of biophilic features in residential buildings: a case from Dubai
Authors: Shakhshir Kulthoum , Sheta Wael 
Published: August 2023

PurposeThis study explores the difference in occupants' satisfaction with biophilic features in high-rises, townhouses, and villas in Dubai to identify new strategies that promote more human-nature connections. The study also serves as a benchmark for applying Biophilic Design in the Gulf region and bridging between biophilic theory and practice.Design/methodology/approachA mixed-mode approach was used, combining surveys from occupants and interviews with experts (3 policymakers, a developer, and a biophilic design educator). The survey collected occupants' satisfaction and acceptance of proposed strategies, and the interviews validated the potentially accepted solutions.FindingsThe results indicated high overall satisfaction with biophilic features despite neutral/low satisfaction with specific features. In high-rise buildings, respondents showed high acceptance for different proposed strategies; 76.4% for adaptive control of balconies by season, 82.7% for utilizing outdoor roof gardens, 78.9% for indoor gardens, and 71.8% for vertical farming. Additionally, respondents accepted social strategies; 63.6% agreed to join clubs promoting environmental events, and 75.4% accepted participating in outdoor recreational activities accompanied by local environmental education. The interviews showed an inclination toward incentive programs instead of obligations.Originality/valueUnlike former studies that assessed indoor environmental qualities without referring to biophilic theory or limited reference to biophilic design, this study provides a novel approach for bridging theory and practice by integrating biophilic design into residential buildings for Dubai's future developments. Additionally, the authors suggest validated proposals to enhance the occupants' biophilic living experience, serving as a benchmark for biophilic application in the Gulf region.
How might responsible management education (RME) be used to develop responsible leadership skills among students in business schools? Evidence from non-Western business schools
Authors: Mousa Mohamed , del Aguila Levy , Abdelgaffar Hala 
Published: August 2023

PurposeThis paper aims to find an answer to the questions: To what extent is the implementation of responsible management education (RME) perceived to be adequate for developing responsible leadership skills among business school students? How should it be used effectively to guarantee such an outcome?Design/methodology/approachThe authors conducted semi-structured interviews with 24 management educators working at three public business schools. Thematic analysis was used to analyse the collected data.FindingsThe findings show that the implementation of RME alone is not adequate to ensure the development of responsible leadership skills among students in business schools. However, management educators do perceive it as a considerable step towards that outcome if accompanied with internship and training opportunities to exercise and observe how social roles and activities are practiced in business, not-for-profit and civil society organisations.Originality/valueThis study is a pioneering attempt to address the relationship between RME and developing responsible leadership skills among students in non-Western business schools.