bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers using chemistry
Authors: Kumar Pratik , Gutu Alina , Waring Amelia , Brown Timothy A. , Lavis Luke D. , Tebo Alison G. 
Published: January 2024

AbstractChemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.
DNA-PKcs Suppresses Illegitimate Chromosome Rearrangements
Authors: Wang Jinglong , Sadeghi Cheyenne A , Frock Richard L 
Published: January 2024

AbstractTwo DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ involved in human and A-EJ in mice predominantly. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in humans is not known. We find partial DNA-PKcs inhibition in human cells leads to increased translocations and the continued involvement of a dampened NHEJ. In contrast, complete DNA-PKcs inhibition substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse progenitor B cell lines, significantly impairs V(D)J recombination and generated higher rates of translocations as a consequence of dysregulated coding and signal end joining. Genetic DNA-PKcs inhibition suppresses NHEJ involvement entirely, with repair phenotypically resembling Ku70-deficient A-EJ. In contrast, we find DNA-PKcs necessary in generating the near-excusive MMEJ associated with Lig4 deficiency. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement while also contributing to MMEJ in both species.
Structure of a lasso peptide bound ETB receptor provides insights into the mechanism of GPCR inverse agonism
Authors: Shihoya Wataru , Akasaka Hiroaki , Jordan Peter A. , Lechner Anna , Okada Bethany K. , Cruz Gabriella Costa Machado da , Sano Fumiya K. , Tanaka Tatsuki , Kawahara Ryo , Chaudhari Rajan , Masamune Hiroko , Burk Mark J. , Nureki Osamu 
Published: January 2024

AbstractLasso peptides exhibit a unique lariat-like knotted structure imparting exceptional stability and thus show promise as therapeutic agents that target cell-surface receptors. One such receptor is the human endothelin ETBreceptor, which is implicated in challenging cancers with poor immunotherapy responsiveness. TheStreptomyces-derived lasso peptide, RES-701-3, is a selective inhibitor for ETBand a compelling candidate for therapeutic development. However, meager production from a genetically recalcitrant host has limited further structure-activity relationship studies of this potent inhibitor. Here, we report cryo-electron microscopy structures of ETBreceptor in both its apo form and complex with RES-701-3, facilitated by a calcineurin-fusion strategy. Hydrophobic interactions between RES-701-3 and the transmembrane region of the receptor, especially involving two tryptophan residues, play a crucial role in RES-701-3 binding. Furthermore, RES-701-3 prevents conformational changes associated with G-protein coupling, explaining its inverse agonist activity. A comparative analysis with other lasso peptides and their target proteins highlights the potential of lasso peptides as precise drug candidates for G-protein-coupled receptors. This structural insight into RES-701-3 binding to ETBreceptor offers valuable information for the development of novel therapeutics targeting this receptor and provides a broader understanding of lasso peptide interactions with human cell-surface receptors.
Discovery of Waddington’s developmental canals elucidates the embryogenesis stability inCaenorhabditis elegans
Authors: Wang Jianguo , Xiao Long , Yao Zeqi , Deng Shanjun , Yang Jianrong , Wu Chung-I , Du Zhuo , He Xionglei 
Published: January 2024

AbstractDuring embryogenesis, the cells in an embryo need to make numerous spatiotemporal decisions. However, there is inherent noise in each decision due to genetic or environmental fluctuations. How to suppress the noise accumulation to achieve stable embryonic end-products, a process known as Waddington’s developmental canalization, has been a major puzzle in biology since the 1940s. Previous studies have focused on the molecular noise within a cell instead of the cell noise within an embryo, thus providing indirect understandings (e,g., the well-known genetic capacitor Hsp90). In this study, we applied time-lapse microscopic imaging to capturing the spatiotemporal features of single cells, including cell position and cell cycle length, during the embryogenesis of approximately 2,100Caenorhabditis elegansembryos exposed to various genetic or environmental perturbations. By treating the deviation of a cell’s spatiotemporal feature from the expected value as noise, we modeled the transmission of noise from each mother cell to their daughters. We discovered pervasive mother-daughter negative feedbacks, which collectively constitute continuous and comprehensive ‘canals’ for suppressing noise accumulation along the developmental cell lineages, with the steepness (measuring noise suppression efficacy) and depth (measuring noise tolerance level) of the canals quantitatively defined. The learned quantitative rules enabled us to develop a cell-noise-based model that accurately predicts the nematode hatching phenotype, revealing how embryonic stochasticity could cause phenotypic disparity. With a high-dimensional mathematical tool we then proved the system stability of embryogenesis against the cell spatiotemporal noise. We also revealed several dozen canal-maintaining genes and proposed a novel association study framework that links embryonic cells rather than genetic variants with organismal traits. In sum, this study discovered and quantitatively characterized the developmental canals that directly stabilize embryogenesis in a metazoan, illuminating an 80-year-old puzzle and paving a way for studying the phenotypic plasticity and robustness of multicellular organisms from the perspective of embryogenesis.
Precision Genome Editing Unveils a Breakthrough in Reversing Antibiotic Resistance: CRISPR/Cas9 Targeting of Multi-Drug Resistance Genes in Methicillin-ResistantStaphylococcus aureus
Authors: Ates Aysegul , Tastan Cihan , Ermertcan Safak 
Published: January 2024

AbstractAntibiotic resistance poses a global health crisis, limiting the efficacy of available therapeutic agents and hindering the development of new antibiotics. The pharmaceutical industry’s waning interest in antibiotic production further exacerbates this challenge. Addressing antibiotic resistance demands innovative solutions. Here, we explore the application of CRISPR-Cas-based antimicrobials as a pioneering approach to combat multidrug resistance. Our study focuses on the methicillin-resistant clinical Staphylococcus aureus (MRSA), a significant clinical threat. Utilizing CRISPR/Cas9 technology, we aimed to concurrently target methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes, thereby altering the resistance profile and enhancing sensitivity to antibiotics. We engineered CRISPR plasmids containing sgRNAs specific to the target regions, which were subsequently electroporated into MRSA strains. Real-time Polymerase Chain Reaction (RT-PCR) assessed changes in resistance gene expression, while disk diffusion and broth microdilution methods determined alterations in resistance status. Western blotting analyzed changes in PBP2a expression, and Sanger sequence analysis confirmed mutations in target regions. Results revealed a statistically significant 1.5-fold decrease in mecA gene expression, 5.5-fold decrease in grlA gene, 6-fold decrease in grlB gene, and 4-fold decrease in aacA gene compared to the wild type strain, as determined by RT-PCR. Antibiotic susceptibility tests demonstrated the suppression of mecA, grlA, grlB, and aacA genes, resulting in the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis showed 70% decrease in PBP2a expression, indicating the breakdown of beta-lactam resistance. Sanger sequence analysis confirmed point mutations in grlB and aacA genes, along with three-base mutations in grlA and mecA genes. Our findings underscore the potential of CRISPR/Cas9 technology to mitigate antibiotic resistance, offering a transformative strategy to restore the efficacy of existing antibiotics in the face of multidrug-resistant pathogens.
Flagella are required to coordinately activate competition and host colonization factors in response to a mechanical signal
Authors: Speare Lauren , Zhao Liang , Pavelsky Morgan N. , Jackson Aundre , Smith Stephanie , Tyagi Bhavyaa , Sharpe Garrett C. , Woo Madison , Satkowiak Lizzie , Bolton Trinity , Gifford Scott M. , Septer Alecia N. 
Published: January 2024

AbstractBacteria employ antagonistic strategies to eliminate competitors of an ecological niche. Contact-dependent mechanisms, such as the type VI secretion system (T6SS), are prevalent in host-associated bacteria, yet we know relatively little about how T6SS+ strains make contact with competitors in highly viscous environments, such as host mucus. To better understand how cells respond to and contact one another in such environments, we performed a genome-wide transposon mutant screen of the T6SS-wielding beneficial bacterial symbiont,Vibrio fischeri, and identified two sets of genes that are conditionally required for killing. LPS/capsule and flagellar-associated genes do not affect T6SS directly and are therefore not required for interbacterial killing when cell contact is forced yet are necessary for killing in high-viscosity liquid (hydrogel) where cell-cell contact must be biologically mediated. Quantitative transcriptomics revealed thatV. fischerisignificantly increases expression of both T6SS genes and cell surface modification factors upon transition from low-to high-viscosity media. Consistent with coincubation and fluorescence microscopy data, flagella are not required for T6SS expression in hydrogel. However, flagella play a key role in responding to the physical environment by promoting expression of the surface modification genes identified in our screen, as well as additional functional pathways important for host colonization including uptake of host-relevant iron and carbon sources, and nitric oxide detoxification enzymes. Our findings suggest that flagella may act as a mechanosensor forV. fischerito coordinately activate competitive strategies and host colonization factors, underscoring the significance of the physical environment in directing complex bacterial behaviors.SignificanceThe physical environment can have dramatic effects on bacterial behavior, but little is known about how mechanical signals impact antagonistic interactions. Symbiotic bacteria use molecular weapons to eliminate competitors for limited space within highly viscous host tissue and mucus.To better understand how the physical environment affects competition and adhesion within eukaryotic hosts, we used quantitative transcriptomics to reveal the flagella-dependent transcriptional response to bacterial transition from lower to a higher viscosity environment. This work revealed the T6SS interbacterial weapon is coordinately activated with host colonization factors, emphasizing the importance of integrating activation of interbacterial weapons into host colonization pathways to enhance a symbiont’s ability to successfully colonize the host while efficiently eliminating potential competitors from the host niche.
Hunting for peptide binders of specific targets with data-centric generative language models
Authors: Nie Zhiwei , Li Daixi , Chen Jie , Xu Fan , Liu Yutian , Fu Jie , Liu Xudong , Wang Zhennan , Ma Yiming , Wang Kai , Zhang Jingyi , Hu Zhiheng , Song Guoli , Ye Yuxin , Yin Feng , Zhou Bin , Liu Zhihong , Li Zigang , Gao Wen , Tian Yonghong 
Published: January 2024

AbstractThe increasing frequency of emerging viral infections calls for more efficient and low-cost drug design methods. Peptide binders have emerged as a strong contender to curb the pandemic due to their efficacy, safety, and specificity. Here, we propose a customizable low-cost pipeline incorporating model auditing strategy and data-centric methodology for controllable peptide generation.A generative protein language model, pretrained on approximately 140 million protein sequences, is directionally fine-tuned to generate peptides with desired properties and binding specificity. The subsequent multi-level structure screening reduces the synthetic distribution space of peptide candidates regularly to identify authentic high-quality samples, i.e. potential peptide binders, atin silicostage. Paired with molecular dynamics simulations, the number of candidates that need to be verified in wet-lab experiments is quickly reduced from more than 2.2 million to 16. These potential binders are characterized by enhanced yeast display to determine expression levels and binding affinity to the target. The results show that only a dozen candidates need to be characterized to obtain the peptide binder with ideal binding strength and binding specificity. Overall, this work achieves efficient and low-cost peptide design based on a generative language model, increasing the speed ofde novoprotein design to an unprecedented level. The proposed pipeline is customizable, that is, suitable for rapid design of multiple protein families with only minor modifications.
Multiparameter-based photosynthetic state transitions of single phytoplankton cells
Authors: Harris Paul David , Eliezer Nadav Ben , Keren Nir , Lerner Eitan 
Published: January 2024

AbstractPhytoplankton are a major source of primary production. Their photosynthetic fluorescence uniquely reports on their type, physiological state and response to environmental conditions. Changes in phytoplankton photophysiology are commonly monitored by bulk fluorescence spectroscopy, where gradual changes are reported in response to different perturbations such as light intensity changes. What is the meaning of such trends in bulk parameters if their values report ensemble averages of multiple unsynchronized cells? To answer this, we developed an experimental scheme that enables acquiring multiple fluorescence parameters, from multiple excitation sources and spectral bands. This enables tracking fluorescence intensities, brightnesses and their ratios, as well as mean photon nanotimes equivalent to mean fluorescence lifetimes, one cell at a time. We monitored three different phytoplankton species during diurnal cycles and in response to an abrupt increase in light intensity. Our results show that we can define specific subpopulations of fluorescence parameters for each of the phytoplankton species and in response to varying light conditions. Importantly, we identify the cells undergo well-defined transitions between these subpopulations that characterize the different light behaviors. The approach shown in this work will be useful in the exact characterization of phytoplankton cell states and parameter signatures in response to different changes these cells experience in marine environments, which will be useful in monitoring marine-related effects of global warming.Significance StatementUsing three representatives of red-linage phytoplankton we demonstrate distinct photophysiological behaviors at the single cell level. The results indicate cell wide coordination into discrete cell states. We test cell state transitions as a function of light acclimation during diurnal cycle and in response to large intensity increases, which stimulate distinct photoprotective response mechanisms. The analysis was made possible through the development of flow-based confocal detection at multiple excitation and emission wavelengths monitoring both pigment composition and photosynthetic performance. Our findings show that with enough simultaneously recorded parameters per each cell, the detection of multiple phytoplankton species at their distinct cell states is possible. This approach will be useful in examining the response of complex natural marine populations to environmental perturbations.
Straightforward semi-quantitative MALDI-TOF MS based screening approach for selection of recombinant protein producing E. Coli
Authors: Kravtsov Ivan N. , Solovyev Andrey I. , Potemkina Eugenia A. , Kartashova Anastasya V. , Dmitrieva Maria A. , Danilova Kseniya V. , Tutykhina Irina L. , Polyakov Nikita B. , Egorova Daria A. 
Published: January 2024

AbstractMALDI-TOF MS represents a rapid and cost-effective method for identifying proteins and microorganisms. When obtaining recombinant protein producers, differences in expression levels among transformants necessitate the conduction of a small-scale screening of expression levels. This study proposes a fast and easy method for screening clones producing recombinant proteins using MALDI-TOF MS. Various recombinant proteins were utilized to test the proposed method
Genomic Transfer via Membrane Vesicle: A Strategy of Giant Phage phiKZ for Early Infection
Authors: Antonova Daria , Nichiporenko Anna , Sobinina Mariia , Vishnyakov Innokentii E. , Moiseenko Andrey , Kurdyumova Inna , Khodorkovskii Mikhail , Sokolova Olga S. , Yakunina Maria V. 
Published: January 2024

AbstractDuring infection, the giant phiKZ phage forms a specialized structure at the center of the host cell called the phage nucleus. This structure is crucial for safeguarding viral DNA against bacterial nucleases and for segregating the transcriptional activities of late genes. Here, we describe a morphological entity, the early phage infection vesicle (EPI vesicle), which appears to be responsible for earlier gene segregation at the beginning of the infection process. Using cryo-electron microscopy, electron tomography, and fluorescence microscopy with membrane-specific dyes, we found that the EPI vesicle is enclosed in a lipid bilayer originating, apparently, from the inner membrane of the bacterial cell. Our investigations further disclose that the phiKZ EPI vesicle contains both viral DNA and viral RNA polymerase (vRNAP). We have observed that the EPI vesicle migrates from the cell pole to the center, displaying co-localization with ChmA, the primary protein of the phage nucleus. While phage DNA is transported into the phage nucleus after phage maturation, the EPI vesicle remains outside. We hypothesized that the EPI vesicle acts as a membrane transport agent, efficiently delivering phage DNA to the phage nucleus while protecting it from the nucleases of the bacterium.
Saturated fatty acid-Coenzyme A supplementation restores neuronal energy levels, synaptic function, and protein homeostasis in hereditary spastic paraplegia
Authors: Saber Saber H. , Yak Nyakuoy , Yong Xuan Ling Hilary , Lu Siyuan , Binder Tobias , Purushothaman Reshinthine , Gaudin Arnaud , Harmer Jeffrey , Talbo Gert Hoy , van Waardenberg Ashley J , Anggono Victor , Balistreri Giuseppe , Joensuu Merja 
Published: January 2024

AbstractHereditary Spastic Paraplegia (HSP) type 54 is a complex childhood autosomal recessive neurodegenerative disorder characterized by impairments in both neuromuscular and cognitive functions. This condition arises from mutations in theDDHD2gene, which encodes for the phospholipase A1 enzyme DDHD2. Previous research has indicated that loss ofDDHD2results in lipid droplet accumulation in the brain, progressive apoptosis of motor neurons in the spinal cord, a reduction in cardiolipin content, and an increase in reactive oxygen species. However, the precise underlying mechanisms of HSP54 remains unclear. Our recent study demonstrated a robust increase in saturated free fatty acids (sFFAs), particularly myristic acid, during neuronal stimulation and memory acquisitionin vivoin the brains of mice andin vitroin primary neurons. This activity-dependent increase of sFFAs was blocked inDDHD2knockout mice (DDHD2-/-), suggesting that disturbed production of sFFAs underlies the neuronal pathology of HSP54. Here, using electron microscopy (EM) and live-cell confocal imaging, mass spectrometry and proteomics, electric field stimulation, as well as fluorometric and mitochondrial function assays in cultured primary neurons, we discovered that loss of DDHD2 leads to reduced levels of acetyl-coenzyme A (CoA) and ATP. Additionally, DDHD2 deficiency results in impaired respiratory function, altered mitochondrial morphology and distribution, a significant defect in synaptic vesicle recycling with an accumulation of large bulk endosomes in the presynapses, as well as an imbalance in global protein homeostasis. Our study further reveals that the combined administration of myristic acid and CoA (Myr-CoA) fully rescues mitochondrial function and ATP production within 48 hours. This intervention also leads to a marked restoration of neuronal protein homeostasis, providing the first demonstration of a potential combinatory therapeutic intervention for HSP54. Our findings demonstrate that the sFFAs released by the activity of DDHD2 play a central role in maintaining neuronal energy levels, synaptic function, and protein balance. The requirement for DDHD2 lipase activity in these processes can, therefore, be bypassed by supplementation of a preconjugated Myr-CoA.
The distribution of Cyclone Idai’s water impacts in Beira, Mozambique
Authors: McCordic Cameron , Raimundo Ines , Judyn Matthew , Willis Duncan 
Published: January 2024

PurposeClimate hazards in the form of cyclones are projected to become more intense under the pressures of future climate change. These changes represent a growing hazard to low lying coastal cities like Beira, Mozambique. In 2019, Beira experienced the devastating impact of Cyclone Idai. One of the many impacts resulting from this Cyclone was disrupted drinking water access. This investigation explores the distribution of Cyclone Idai’s impact on drinking water access via an environmental justice lens, exploring how preexisting water access characteristics may have predisposed households to the impacts of Cyclone Idai in Beria.Design/methodology/approachRelying on household survey data collected in Beira, the investigation applied a decision tree algorithm to investigate how drinking water disruption was distributed across the household survey sample using these preexisting vulnerabilities.FindingsThe investigation found that households that mainly relied upon piped water sources and experienced inconsistent access to water in the year prior to Cyclone Idai were more likely to experience disrupted drinking water access immediately after Cyclone Idai. The results indicate that residents in formal areas of Beira, largely reliant upon piped water supply, experienced higher rates of disrupted drinking water access following Cyclone Idai.Originality/valueThese findings question a commonly held assumption that informal areas are more vulnerable to climate hazards, like cyclones, than formal areas of a city. The findings support the inclusion of informal settlements in the design of climate change adaptation strategies.