bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Molecular-based Methods for the Detection and Quantification ofCryptosporidiumspp. in Wastewater
Authors: Hachimi Oumaima , Falender Rebecca , Davis Gabriel , Vranka Wafula Rispa , Sutton Melissa , Bancroft June , Cieslak Paul , Lininger Noah , Kelly Christine , Kaya Devrim , Radniecki Tyler S. 
Published: March 2024

AbstractCryptosporidium, a eukaryotic protozoan parasite, poses a significant public health risk as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is estimated to be largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Unlike clinical surveillance, wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized methods forCryptosporidiumspp. detection in wastewater challenges the comparability of studies. Additionally, the use of standard methods for the detection and quantification ofCryptosporidiumspp. in surface waters may not be appropriate for wastewater samples due to the more complex composition of the wastewater matrix. Thus, in this study, we evaluated methods for concentratingCryptosporidiumoocysts from wastewater, extracting DNA, and detecting its genetic markers, using wastewater seeded withC. parvumoocysts. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome particles. All methods except the Nanotrap Microbiome particles were conducted with or without additional purification via immunomagnetic separation. For DNA extractions, we tested the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit, while assessing the impact of bead beating and freeze-thaw cycles on DNA yield. Genetic detection was performed using qPCR targeting theCryptosporidium18S rRNA gene. Our results indicated that centrifugation yielded the highest oocyst recoveries (39-77%), followed by the Nanotrap Microbiome particles (24%), electronegative filtration with a phosphate buffered saline with 20% Tween 80 elution (22%), and Envirocheck HV capsule filtration (13%). IMS was found to be unsuitable due to interference from the wastewater matrix, significantly reducing recovery rates to 0.03 to 4%. DNA yields were highest with bead-beating pretreatment with either the DNeasy Powersoil Pro kit (314 gc/μL DNA) or the QIAamp DNA Mini kit (238 gc/μL DNA). In contrast, freeze-thaw pretreatment reduced DNA yields to under 92 gc/μL DNA, likely through DNA degradation. This study is amongst the first to compare different concentration methods, including Nanotrap Microbiome particles, and DNA extraction methods that can be utilized forCryptosporidiumspp. wastewater surveillance, highlighting the importance of method selection for accurate detection and quantification.
Structural Requirements for Activity of Mind bomb1 in Notch Signaling
Authors: Cao Ruili , Gozlan Oren , Tveriakhina Lena , Zhou Haixia , Jiang Hanjie , Cole Philip A. , Aster Jon C. , Sprinzak David , Blacklow Stephen C. 
Published: March 2024

AbstractMind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2∼Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region of MIB1 (ccRING3) drives MIB1 dimerization and that ubiquitin transfer activity of MIB1 relies solely on RING3. We report x-ray crystal structures of a UbcH5B-ccRING3 complex as a fusion protein and of the ANK region. Directly tethering the N-terminal region to ccRING3 forms a minimal MIB1 protein, which is sufficient to induce a Notch response in receiver cells. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.
O-GlcNAcylation of FOXK1 orchestrates the E2F pathway and promotes oncogenesis
Authors: Masclef Louis , Ahmed Oumaima , Iannantuono Nicholas , Gagnon Jessica , Gushul-Leclaire Mila , Boulay Karine , Estavoyer Benjamin , Echbicheb Mohamed , Poy Marty , Ali Boubacar Kalidou , Boubekeur Amina , Menggad Saad , Schcolnik-Cabrera Alejandro , Balsalobre Aurelio , Bonneil Eric , Thibault Pierre , Hulea Laura , Tanaka Yoshiaki , Antoine-Mallette Frédérick , Drouin Jacques , Affar El Bachir 
Published: March 2024

AbstractGene transcription is a highly regulated process, and deregulation of transcription factors activity underlies numerous pathologies including cancer. Albeit near four decades of studies have established that the E2F pathway is a core transcriptional network that govern cell division in multi-cellular organisms1,2, the molecular mechanisms that underlie the functions of E2F transcription factors remain incompletely understood. FOXK1 and FOXK2 transcription factors have recently emerged as important regulators of cell metabolism, autophagy and cell differentiation3–6. While both FOXK1 and FOXK2 interact with the histone H2AK119ub deubiquitinase BAP1 and possess many overlapping functions in normal biology, their specific functions as well as deregulation of their transcriptional activity in cancer is less clear and sometimes contradictory7–13. Here, we show that elevated expression of FOXK1, but not FOXK2, in primary normal cells promotes transcription of E2F target genes associated with increased proliferation and delayed entry into cellular senescence. FOXK1 expressing cells are highly prone to cellular transformation revealing important oncogenic properties of FOXK1 in tumor initiation. High expression of FOXK1 in patient tumors is also highly correlated with E2F gene expression. Mechanistically, we demonstrate that FOXK1, but not FOXK2, is specifically modified by O-GlcNAcylation. FOXK1 O-GlcNAcylation is modulated during the cell cycle with the highest levels occurring during the time of E2F pathway activation at G1/S. Moreover, loss of FOXK1 O-GlcNAcylation impairs FOXK1 ability to promote cell proliferation, cellular transformation and tumor growth. Mechanistically, expression of FOXK1 O-GlcNAcylation-defective mutants results in reduced recruitment of BAP1 to gene regulatory regions. This event is associated with a concomitant increase in the levels of histone H2AK119ub and a decrease in the levels of H3K4me1, resulting in a transcriptional repressive chromatin environment. Our results define an essential role of O-GlcNAcylation in modulating the functions of FOXK1 in controlling the cell cycle of normal and cancer cells through orchestration of the E2F pathway.
Structural architecture of amyloid-β oligomers, curvilinear protofibrils and annular assemblies, imaged by cryo-EM and cryo-ET
Authors: Liang Ruina , Torres-Flores Andrea P. , Qi Shang , Khursheed Anum , Tian Yao , Szwedziak Piotr , Baker Mark D. , Volkov Vladimir A. , Darbari Vidya C. , Viles John H. 
Published: March 2024

ABSTRACTSmall prefibrillar structures of the amyloid-β peptide (Aβ) are believed to be central to cytotoxicity in Alzheimer’s disease, the most common form of dementia. A snapshot of these prefibrillar assemblies has therefore been characterized using a combination of cryo-ET and cryo-EM single particle analysis. This has facilitated an understanding of the relationship between, oligomers, curvilinear protofibrils and annular assemblies. A highly consistent diameter for all curvilinear protofibrils and oligomers of 28 Å, indicates that these assemblies are simply structural extensions from the smaller oligomers. Furthermore, their basic crosssection suggests mature amyloid fibrils might be initiated by the lateral binding of two curvilinear protofibrils.Ab-initio3D reconstruction also reveals ring-shaped annular assemblies. These possess a central internal channel,ca. 14 Å in diameter and 54 Å long, which is capable of traversing lipid membranes. Large conductance recorded using patch-clamp electrophysiology, match the internal diameter of the Aβ annular architecture.
Structure and RNA-binding of the helically extended Roquin CCCH-type zinc finger
Authors: Tants Jan-Niklas , Oberstrass Lasse , Weigand Julia E. , Schlundt Andreas 
Published: March 2024

ABSTRACTZinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3’-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich elements (ARE). We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence specificity through the ROQ-ZnF tandem.
EXCRETE enables deep proteomics of the microbial extracellular environment
Authors: Russo David A. , Oliinyk Denys , Pohnert Georg , Meier Florian , Zedler Julie A. Z. 
Published: March 2024

AbstractExtracellular proteins play a significant role in shaping microbial communities which, in turn, can impact ecosystem function, human health, and biotechnological processes. Yet, for many ubiquitous microbes, there is limited knowledge regarding the identity and function of secreted proteins. Here, we introduce EXCRETE (enhanced exoproteome characterization by mass spectrometry), a workflow that enables comprehensive description of microbial exoproteomes from minimal starting material. Using cyanobacteria as a case study, we benchmark EXCRETE and show a significant increase over current methods in the identification of extracellular proteins. Subsequently, we show that EXCRETE can be miniaturized and adapted to a 96-well high-throughput format. Application of EXCRETE to cyanobacteria from different habitats (Synechocystissp. PCC 6803,Synechococcussp. PCC 11901, andNostoc punctiformePCC 73102), and in different cultivation conditions, identified up to 85% of all predicted secreted proteins. Finally, functional analysis reveals that cell envelope maintenance and nutrient acquisition are central functions of the cyanobacterial secretome. Collectively, these findings challenge the general belief that cyanobacteria lack secretory proteins and point to a functional conservation of the secretome across freshwater, marine, and terrestrial species.
Evaluation ofPeregrinus maidis transformer-2as a target for CRISPR-based control
Authors: Wang Yu-Hui , Rivera Dina Espinoza , Klobasa William , Lorenzen Marcé D. 
Published: March 2024

ABSTRACTThe corn planthopper,Peregrinus maidis, is an economically important pest of corn and sorghum. Here we report the initial steps towards developing a CRISPR-based control method, precision guided sterile insect technique (pgSIT), for this hemipteran pest. Specifically, we evaluated the potential oftransformer-2(tra-2) as a target for sterilizing insects. First, we identifiedtra-2transcripts within ourP. maidistranscriptome database and performed RNA interference (RNAi) to confirm functional conservation. RNAi-mediated knockdown ofPmtra-2in nymphs transformed females into pseudomales with deformed ovipositors resembling male claspers. While males showed no overt difference in appearance, they were indeed sterile. Importantly, the results were similar to those observed in another planthopper,Nilaparvata lugens. We also used CRISPR/Cas9 genome editing to assess the impact oftra-2knockout in injectees. CRISPR-mediated knockout ofPmtra-2had lethal effects on embryos, and hence not many injectees reached adulthood. However, mosaic knockout ofPmtra-2did impact female and male fertility, which supports the use oftra-2as a target for pgSIT in this hemipteran species.
Optically active, paper-based scaffolds for 3D cardiac tissue engineering
Authors: Guo F. , Jooken S. , Ahmad A. , Yu W. , Deschaume O. , Thielemans W. , Bartic C. 
Published: March 2024

AbstractIn this work, we report the design and fabrication of a light-addressable, paper-based, nanocomposite scaffold for optical modulation and read-out of in vitro grown cardiac tissue. The scaffold consists of paper cellulose microfibers functionalized with gold nanorods (GNRs) and semiconductor quantum dots (QDs), embedded into a cell-permissive collagen matrix. The GNRs enable cardiomyocyte activity modulation through local temperature gradients induced by near-infrared (NIR) laser illumination, with the local temperature changes reported by the temperature-dependent QD photoluminescence (PL). The micrometer size paper fibers promote the tubular organization of HL-1 cardiac muscle cells, while the NIR plasmonic stimulation modulates reversibly their activity. Given its high spatial resolution, NIR modulation offers an excellent alternative to electrode-based methods for cell activity modulation and is more compatible with 3D tissue constructs. As such, optical platforms based on nanocomposite scaffolds will have a significant impact on the progress of drug screening, toxicity studies, and heart disease modeling.
The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin resistantStaphylococcus aureus
Authors: Nielsen Tobias Krogh , Petersen Ida Birkjær , Xu Lijuan , Barbuti Maria Disen , Mebus Viktor , Justh Anni , Alqarzaee Abdulelah Ahmed , Jacques Nicolas , Oury Cécile , Thomas Vinai , Kjos Morten , Henriksen Camilla , Frees Dorte 
Published: March 2024

AbstractInfections caused by methicillin resistantStaphylococcus aureus(MRSA) are a leading cause of mortality worldwide. MRSA have acquired resistance to next generation β-lactam antibiotics through the horizontal acquisition of themecAresistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators which through poorly described mechanisms enhance resistance. TheyjbHgene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation ofyjbHincreased β-lactam MICs up to 16-folds and transformed MRSA cells with low level of resistance to being homogenously highly resistant to β-lactams. TheyjbHgene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPRi to knock downspxtranscription, we unambiguously linked hyper-resistance to accumulation of Spx. Spx was previously proposed to be essential, however, our data indicate that Spx is dispensable for growth at 37°C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in β-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum β-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.
Comparison of CcrM-dependent methylation inCaulobacter crescentusandBrucella abortusby nanopore sequencing
Authors: Campbell Maxwell , Barton Ian Scott , Roop R. Martin , Chien Peter 
Published: March 2024

AbstractBacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order,Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such asBrucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation inCaulobacterand show excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation inBrucella abortusfor the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on theBrucellamethylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.ImportanceDNA methylation plays an important role in bacteria to maintain genome integrity and regulate of gene expression. We used nanopore sequencing to directly measure methylated bases inCaulobacter crescentusandBrucella abortus. InCaulobacter, we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in Brucella for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns inBrucella.
Dark-based Optical Sectioning assists Background Removal in Fluorescence Microscopy
Authors: Cao Ruijie , Li Yaning , Wang Wenyi , Zhang Guoxun , Wang Gang , Sun Yu , Ren Wei , Sun Jing , Hou Yiwei , Xu Xinzhu , Hu Jiakui , Lu Yanye , Li Changhui , Wu Jiamin , Li Meiqi , Qu Junle , Xi Peng 
Published: March 2024

AbstractA fundamental challenge in fluorescence microscopy is the defocused background caused by scattering light, optical aberration, or limited axial resolution. Severe defocus backgrounds will submerge the in-focus information and cause artifacts in the following processing. Here, we leverage a priori knowledge about dark channels of biological structures and dual frequency separation to develop a single-frame defocus removal algorithm. It stably improves the signal-to-background ratio and structural similarity index measure of images by approximately 10-fold, and recovers in-focus signal with 85% accuracy, even when the defocus background is 50 times larger than in-focus information. Our Dark-based optical sectioning approach (Dark sectioning) is fully compatible with various microscopy techniques, such as wide-filed microscopy, polarized microscopy, laser-scanning / spinning-disk confocal microscopy, stimulated emission depletion microscopy, lightsheet microscopy, and light-field microscopy. It also complements reconstruction or processing algorithms such as deconvolution, structure illumination microscopy, and super-resolution optical fluctuation imaging.
Synthesis of versatile neuromodulatory molecules by a gut microbial glutamate decarboxylase
Authors: Dadi Pavani , Pauling Clint W. , Shrivastava Abhishek , Shah Dhara D. 
Published: March 2024

ABSTRACTDysbiosis of the microbiome correlates with many neurological disorders, yet very little is known about the chemistry that controls the production of neuromodulatory molecules by gut microbes. Here, we found that an enzyme glutamate decarboxylase (BfGAD) of a gut microbeBacteroides fragilisforms multiple neuromodulatory molecules such as γ-aminobutyric acid (GABA), hypotaurine, taurine, homotaurine, and β-alanine. We evolvedBfGAD and doubled its taurine productivity. Additionally, we increased its specificity towards the substrate L-glutamate. Here, we provide a chemical strategy via which theBfGAD activity could be fine-tuned. In future, this strategy could be used to modulate the production of neuromodulatory molecules by gut microbes.
Structural insights into sphingosine 1-phosphate receptor 4 activation
Authors: Gao Wei , Gan Shiyi , Zhang Mengting , Inoue Asuka , Xie Mengting , He Huan , Zhu Huan , Guo Shanshan , Qiu Chen , Chang Di , Yu Jinling , Deng Zhuo , Ye Fang , Li Shiliang , Zhang Jian , Zhao Zhenjiang , Xue Mengzhu , Ofosuhene Bernard , Xu Yufang , Lin Honghuang , Qian Xuhong , Zhu Lili , Du Yang , Li Honglin 
Published: March 2024

AbstractS1PR4 is one of five subtypes of sphingosine 1-phosphate receptors (S1PRs) that regulate immune cell functioning, with functional distinctions to other subtypes. S1PR1-targeted modulators caused serious cardiac and vascular adverse effects because S1PR1 was expressed throughout the whole body. Since S1PR4 was only expressed in lung and lymphoid cells, S1PR4-targeted modulators might not trigger these side effects. However, the development of S1PR4-specific agonists is greatly hindered because of the lack of activated S1PR4 structure. Here, we resolved cryo-EM structures of activated S1PR4 and revealed the structural mechanism of ligand recognition, receptor activation, and Gαicoupling. Our results offered structural templates for the development of selective S1PR4 agonists with improved safety profiles.
Structure-guided aminoacylation and assembly of chimeric RNAs
Authors: Radakovic Aleksandar , Lewicka Anna , Todisco Marco , Aitken Harry R. M. , Weiss Zoe , Kim Shannon , Bannan Abdullah , Piccirilli Joseph A. , Szostak Jack W. 
Published: March 2024

AbstractCoded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture found in modern tRNA. We demonstrate that the T-loop assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of such chimeric ribozymes would have allowed the greater functionality of amino acids to contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid specific aminoacyl-RNA synthetase enzymes prior to their role in protein synthesis.