bluuebunny/crossref_metadata_2025_split huggingface dataset:
Derivation of Steady-State First-order Rate Constant Equations for Enzyme-Substrate Complex Dissociation, as well as Zero-order Rate Constant Equations in Relation to Background Assumptions
Authors: Udema Ikechukwu I. 
Published: December 2022

ABSTRACTThe maximum velocity (Vmax) of catalysis and the substrate concentration ([ST]) at half theVmax, theKM, are regarded as steady-state (SS) parameters even though they are the outcomes of zero-order kinetics (ZOK). The research was aimed at disputing such a claim with the following objectives: To: 1) carry out an overview of issues pertaining to the validity of assumptions; 2) derive the needed steady-state (SS) equations distinct from Michaelian equations that can be fitted to both experimental variables and kinetic parameters; 3) calculate the SS first-order rate constant for the dissociation of enzyme-substrate complex (ES) to free substrate, S and enzyme, E; 4) derive the equation of rate constant as a function of the reciprocal of the duration of each catalytic event in the reaction pathway. The experimental values of the data were generated by Bernfeld and Lineweaver-Burk methods. The calculated SS 1storder-order rate constant was « the zero-order Michaelian value, and the difference is ≈ 97.59 % of the zero-order value; the SS catalytic rate differed from the zero-order catalytic rate by ≈ 76.41 % of the latter value; and it was ≈ 93.87 % with respect to the 2ndorder rate constant for the formation of enzyme-substrate complex. The equations of time-dependent rate constants,KM, and dissociation constants were derived. The concentration [ST] of the S must be > the concentration ([E0]) of the E for the quasi-steady-state assumption (or approximation) to hold. The SS kinetic parameters are not equivalent to zero-order parameters.
Risk of Infection Due to Airborne Virus in Classroom Environments Lacking Mechanical Ventilation
Authors: Goldblatt Alexandra , Loccisano Michael J. , Mahe Mazharul I. , Dennehy John J. , Spagnolo Fabrizio 
Published: December 2022

ABSTRACTThe COVID-19 pandemic highlighted the role of indoor environments on disease transmission. Enclosed spaces where pathogen-laden aerosols accumulate was strongly linked to increased transmission events. Here we employ a surrogate non-pathogenic virus, the bacteriophage phi6, to interrogate aerosol transmission in classroom environments that do not have any natural or mechanical ventilation in order to determine how effectively aerosols facilitate new infections. We find that virus-laden aerosols establish new infections over all distances tested within minutes and that the time of exposure did not change transmission rate. We further find that humidity, but not temperature nor a UV-based disinfection device, significantly impacted transmission rates. Our data suggest that, even without mechanical ventilation, relative humidity remains a highly effective mitigation strategy while UV air treatment did not.Practical ImplicationsTransmission of pathogens through airborne particles is a major source of disease transmission. People now spend much of their time indoors, thus understanding indoor airborne transmission is vital to managing outbreaks. Most classrooms in the U.S. do not have any mechanical ventilation systems and so here, we test airborne transmission of a virus in such classrooms. Infection transmission rates are not greatly impacted by distance, time or even some UV treatment, but are curbed by the amount of moisture in the air.
Light-activated assembly of connexon nanopores in synthetic cells
Authors: Sihorwala Ahmed , Lin Alexander , Stachowiak Jeanne C. , Belardi Brian 
Published: December 2022

AbstractDuring developmental processes and wound healing, activation of living cells occurs with spatiotemporal precision and leads to rapid release of soluble molecular signals, allowing communication and coordination between neighbors. Non-living systems capable of similar responsive release hold great promise for information transfer in materials and site-specific drug delivery. One non-living system that offers a tunable platform for programming release is synthetic cells. Encased in a lipid bilayer structure, synthetic cells can be outfitted with molecular conduits that span the bilayer and lead to material exchange. While previous work expressing membrane pore proteins in synthetic cells demonstrated content exchange, user-defined control over release has remained elusive. In mammalian cells, connexon nanopore structures drive content release and have garnered significant interest since they can direct material exchange through intercellular contacts. Here, we focus on connexon nanopores and present activated release of material from synthetic cells in a light-sensitive fashion. To do this, we re-engineer connexon nanopores to assemble after post-translational processing by a protease. By encapsulating proteases in light-sensitive liposomes, we show that assembly of nanopores can be triggered by illumination, resulting in rapid release of molecules encapsulated within synthetic cells. Controlling connexin nanopore activity provides an opportunity for initiating communication with extracellular signals and for transferring molecular agents to the cytoplasm of living cells in a rapid, light-guided manner.
Characterization of Novel Recombinant Mycobacteriophages derived from Homologous Recombination between two Temperate Phages
Authors: Mohammed Hamidu T , Mageeney Catherine , Korenberg Jamie , Graham Lee , Ware Vassie C 
Published: December 2022

ABSTRACTComparative analyses of mycobacteriophage genomes reveals extensive genetic diversity in genome organization and gene content, contributing to widespread mosaicism. We previously reported that the prophage of mycobacteriophage Butters (cluster N) provides defense against infection by Island3 (subcluster I1). To explore the anti-Island3 defense mechanism, we attempted to isolate Island3 defense escape mutants on a Butters lysogen, but only uncovered phages with recombinant genomes comprised of regions of Butters and Island3 arranged from left arm to right arm as Butters-Island3-Butters (BIBs). Recombination occurs within two distinct homologous regions that encompasslysin A, lysin B, andholingenes in one segment, andRecEandRecTgenes in the other. Structural genes of mosaic BIB genomes are contributed by Butters while the immunity cassette is derived from Island3. Consequently, BIBs are morphologically identical to Butters (as shown by transmission electron microscopy) but are homoimmune with Island3. A reverse experiment where an Island3 lysogen was infected with Butters yielded Butters phages and no recombinants, demonstrating directionality to the recombination phenomenon. Recombinant phages overcome antiphage defense and silencing of the lytic cycle. We leverage this observation to propose a stratagem to generate novel phages for therapeutic use.GRAPHICAL ABSTRACT
Catching thoughts: self-caught experience sampling preferentially captures characteristic features of off-task experiences across the life span
Authors: Martinon Léa M. , Smallwood Jonathan , Riby Leigh M. 
Published: December 2022

AbstractUnderstanding transient states, like off-task mind-wandering, is assumed to be improved by capitalizing on our ability to recognize changes in our stream of thought, a process known as meta-awareness. We test this assumption by comparing mind-wandering content when noticed by the participant (self-caught) against those thoughts reported after externally initiated probes (probe-caught). Thirty-eight older and 36 younger individuals completed a cognitive task. At the same time, multiple feature descriptions of thoughts (task-relevance, temporal focus, and self-referential) were captured using self and probe-caught methods. Using a pattern-learning approach, we established that self-caught experiences produce similar but generally “noisier” estimates compared to those reported at probes. However, self-caught experiences contained more off-task characteristics relative to reports at probes. Importantly, despite reductions in off-task thought, older adults retain the ability to self-catch experiences with these features. Our study establishes self-catching ability as an essential means of revealing the detailed content of off-task states, an ability relatively well maintained into old age.
The compact Casπ (Cas12l) ‘bracelet’ provides a unique structural platform for DNA manipulation
Authors: Sun Ao , Li Cheng-Ping , Chen Zhihang , Zhang Shouyue , Li Danyuan , Yang Yun , Li Long-Qi , Zhao Yuqian , Wang Kaichen , Li Zhaofu , Liu Jinxia , Liu Sitong , Wang Jia , Liu Jun-Jie Gogo 
Published: December 2022

AbstractCRISPR-Cas modules serve as the adaptive nucleic-acid immune systems for prokaryotes, and provide versatile tools for nucleic-acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (∼860 aa), from the environmental metagenome. Complexed with a large guide-RNA (∼170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5’ C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a ‘bracelet’ architecture of Casπ effector encircling the DNA target at 3.4-Å resolution, substantially different from the canonical ‘two-lobe’ architectures of Cas12 and Cas9 nucleases. And the large guide-RNA serves as a ‘two-arm’ scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulations.
Real-time dynamics of individual chemoreceptor mRNA molecules reveals translation hotspots at the inner membrane ofEscherichia coli
Authors: Bergmiller Tobias , Krasnopeeva Ekaterina , Sarikas Srdjan , Nikolic Nela , Guet Calin C. 
Published: December 2022

AbstractSince bacteria lack a nucleus, the location of mRNA molecules is determined by the different characteristics of the encoded proteins, and the transcriptome is spatially arranged into cytosolic and membrane-associated mRNA. While translation of membrane protein-encoding mRNA has been studied in great mechanistic detail using biochemical methods, the spatiotemporal dynamics of this process remains poorly understood at the subcellular level. Here, we investigate the dynamics of individual fluorescently labelled mRNA molecules encoding the transmembrane serine chemoreceptor Tsr, to probe the mechanism of membrane protein translation. Analysis oftsrmRNA diffusion in the proximity of the plasma membrane revealed distinct diffusive modes that reflect the state of the mRNA molecule and its involvement in the process of active translation into the Sec secretion system. We find that the composition, and hence the fluidity of the membrane affects diffusion of membrane targeted mRNAs. Moreover, Tsr translation occurs in localized membrane regions, similar to eukaryotic hotspots. The hotspot localization coincides with the physical location of the transcribed gene, which itself is displaced towards the inner membrane. These findings suggest that inner membrane protein translation is a spatially defined process that occurs in hotspots, indicative of long-lived transertion sites. Our results show an additional layer of spatio-temporal structuring within the bacterial cell, thus revealing a qualitatively different understanding of the basic process of transcription and translation in bacteria.Significance StatementA large fraction of the bacterial proteome is directly synthesized into the inner membrane, and this process shapes the overall distribution of mRNA transcripts within the cell. Although highly dynamic in their nature, bacterial transcriptomes have mostly been studied in fixed cells. Here, we track individual mRNA molecules encoding the serine chemoreceptor in living bacterial cells and find that translation occurs in membrane hotspots that were previously exclusive to eukaryotes. Our results indicate an additional layer of spatio-temporal structuring within the bacterial cell that impacts our understanding of transcription and translation in bacteria.
Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K+-Responsive Fluorescent Protein Sensor
Authors: Eisenhauer Klara , Weber Wadim , Kemp Philipp , Gebhardt Carolin , Kaufmann Marwan , Tewes Noel , Zhadanowa Hanna , Tietze Alesia , Rauh Oliver , Stein Viktor 
Published: December 2022

AbstractThe functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanoporesin vitrostill constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analyzed using the functional nanopore (FuN) screen exploiting genetically-encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+in- and efflux patterns across the inner membrane ofEscherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analyzed with the FuN screen and to what extent alternative fluorescent protein sensors can be adapted. Focussing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally-designed nanopores. Further, the FuN screen is extended to K+-specific fluorescent protein sensors to provide a complementary read-out to Ca2+. Finally, a comparison with high-resolution biophysical and electrophysiological studies in planar lipid bilayers provides an experimental benchmark for future studies.
Morphogenesis of bullet-shaped rabies virus particles requires a functional interplay between the viral matrix protein and ESCRT-I component TSG101
Authors: Itakura Yukari , Tabata Koshiro , Saito Takeshi , Intaruck Kittiya , Kawaguchi Nijiho , Kishimoto Mai , Torii Shiho , Kobayashi Shintaro , Ito Naoto , Harada Michiko , Inoue Satoshi , Maeda Ken , Takada Ayato , Hall William W. , Orba Yasuko , Sawa Hirofumi , Sasaki Michihito 
Published: December 2022

AbstractViral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host ESCRT (endosomal sorting complex required for transport) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection, and specifically the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a siRNA screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late-domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology.ImportanceEnveloped-viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT (endosomal sorting complex required for transport) complexes. Some enveloped-viruses utilize their late (L)-domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped-virions, but the underlying molecular mechanisms involved remain elusive. Herein, we showed that TSG101, one of ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.
Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs
Authors: Yared Marcel-Joseph , Yoluç Yasemin , Catala Marjorie , Tisné Carine , Kaiser Stefanie , Barraud Pierre 
Published: December 2022

ABSTRACTAs essential components of the cellular protein synthesis machineries, tRNAs undergo a tightly controlled biogenesis process, which include the incorporation of a large number of posttranscriptional chemical modifications. Maturation defaults resulting in lack of modifications in the tRNA core may lead to the degradation of hypomodified tRNAs by the rapid tRNA decay (RTD) and nuclear surveillance pathways. Although modifications are typically introduced in tRNAs independently of each other, several modification circuits have been identified in which one or more modifications stimulate or repress the incorporation of others. We previously identified m1A58 as a late modification introduced after more initial modifications, such as Ѱ55 and T54 in yeast elongator tRNAPhe. However, previous reports suggested that m1A58 is introduced early along the tRNA modification process, with m1A58 being introduced on initial transcripts of initiator tRNAiMet, and hence preventing its degradation by the nuclear surveillance and RTD pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m1A58 incorporation, we examined the m1A58 modification pathways in yeast elongator and initiator tRNAs. For that, we first implemented a generic approach enabling the preparation of tRNAs containing specific modifications. We then used these specifically modified tRNAs to demonstrate that the incorporation of T54 in tRNAPheis directly stimulated by Ѱ55, and that the incorporation of m1A58 is directly and individually stimulated by Ѱ55 and T54, thereby reporting on the molecular aspects controlling the Ѱ55 → T54 → m1A58 modification circuit in yeast elongator tRNAs. We also show that m1A58 is efficiently introduced on unmodified tRNAiMet, and does not depend on prior modifications. Finally, we show that the m1A58 single modification has tremendous effects on the structural properties of yeast tRNAiMet, with the tRNA elbow structure being properly assembled only when this modification is present. This rationalizes on structural grounds the degradation of hypomodified tRNAiMetlacking m1A58 by the nuclear surveillance and RTD pathways.
An optimized and validated workflow for developing stable producer cell lines with >99.99% assurance of clonality and high clone recovery
Authors: Scherzinger Julia , Türk Daniel , Aprile-Garcia Fernando 
Published: December 2022

AbstractThere is a constant pressure to reduce timelines in mammalian cell line development (CLD) for biotherapeutic protein production. Demonstration of clonal derivation of the generated cell lines is key for health authorities’ approval. To meet these regulatory and process-oriented demands, single-cell dispensers have become vital instruments for single-cell cloning. We conducted validation experiments with the UP.SIGHT (CYTENA GmbH) to determine this instrument’s single-cell dispensing efficiency (SCDE) and probability of clonal derivation (p(clonal)). Process optimization to maximize clone recovery with several cell lines was also performed, focusing on cloning media and plate type. With a SCDE >97%, p(clonal) >99.99% and clone recovery values of up to 80%, the data reported here support the notion that the UP.SIGHT covers all steps in the single-cell dispensing process with assurance of clonality and colony tracking, leading to faster and more efficient CLD workflows. This work also serves as a guideline for instrument validation and guidance towards process optimization.
ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis and meiosis
Authors: Guo Chenxi , Xiao Yuan , Gu Jingkai , Hu Zhe , Zheng Jiahuan , Hua Renwu , Hai Zhuo , Su Jiaping , Zhang Jian V. , Yeung William S.B. , Wang Tianren 
Published: December 2022

AbstractCaseinolytic protease proteolytic subunit (ClpP) and caseinolytic protease X (ClpX) are mitochondrial matrix peptidases that activate mitochondrial unfolded protein response to maintain protein homeostasis in the mitochondria. However, the role of ClpP and ClpX in spermatogenesis remains largely unknown. In this study, we demonstrated the importance of ClpP/ClpX for meiosis and spermatogenesis with two conditional knockout (cKO) mouse models. We found that ClpP/ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply during meiosis and attenuated zygotene-pachytene transformation of the male germ cells. The dysregulated spermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within the seminiferous tubules. We found mTORC1 pathway was over-activated after deletion of ClpP/ClpX in spermatocytes. Long-term inhibition of the mTORC1 signaling via rapamycin treatmentin vivopartially rescue spermatogenesis. The data reveal the novel roles of ClpP and ClpX in regulating meiosis and spermatogenesis.One-Sentence SummaryClpP/ClpX is required for maintaining mitochondrial functions in spermatocytes during meiosis and spermatogenesis.
Distinct molecular profile of the chick Organizer as a stem zone during axial elongation
Authors: Wood Timothy R. , Kucinski Iwo , Voiculescu Octavian 
Published: December 2022

AbstractThe vertebrate Organizer plays a crucial role in defining the main axes of embryo: it neuralizes the surrounding ectoderm, and is the site of emigration for cells making axial and paraxial mesendoderm during elongation. The chick Organizer becomes a stem zone at the onset of elongation: it stops recruiting cells from the neighbouring ectoderm, and generates all its derivatives from the small number of resident cells it contains at the end of gastrulation stages. Nothing is known about the molecular identity of this stem zone. Here, we specifically labelled long-term resident cells of the Organizer, and compared their RNA-seq profile to that of the neighbouring cell populations. Screening by RT-PCR and in situ hybridisation identified four genes (WIF1, PTGDS, ThPOandUCKL1), which are upregulated only in the Organizer region when it becomes a stem zone, and remain expressed there during axial elongation. In experiments specifically labelling the resident cells of the mature Organizer, we show that only these cells express these genes. These findings molecularly define the Organizer as a stem zone, and offer a key to understanding how this zone is set up, the molecular control of its cells’ behaviour, and the evolution of axial growth zones.