bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanistic insight into the ATP hydrolysis cycle of tick-borne encephalitis virus helicase
Authors: Anindita Paulina Duhita , Halbeisen Marco , Řeha David , Tůma Roman , Franta Zdeněk 
Published: March 2022

AbstractHelicase domain of nonstructural protein 3 (NS3H) unwinds double-stranded RNA replication intermediate during flavivirus life cycle in ATP-dependent manner. While the helicases mechanism of Dengue and Zika viruses has been extensively studied, little is known about helicase activity of the tick-borne encephalitis virus NS3. In the current study, we demonstrated that ATP hydrolysis cycle of NS3H is strongly stimulated by ssRNA but not ssDNA, suggesting that NS3H is an RNA-specific helicase. However, ssDNA binding inhibits ATPase activity in a non-competitive manner. We also captured several structural snapshots of key ATP hydrolysis stages. An intermediate, in which the inorganic phosphate (Pi) and ADP resulted from ATP hydrolysis and remain trapped inside the ATPase site, suggests that Pi release is the rate-limiting step and is accelerated by RNA binding and/or translocation. Based on these structures, we modeled NS3H-ssRNA and -ssDNA binding and performed MD simulations. Our model suggests that NS3H-ssRNA binding triggers conformational changes, revealing the coupling between helicase and ATPase activities. The structural models revealed that ssDNA inhibition may occur via non-specific ssDNA binding to several positively charged surface patches which in turn causes repositioning of ATP molecule within the ATPase site.
Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain
Authors: Wörle Elisabeth , Newman Anthony , Burgio Gaetan , Grohmann Dina 
Published: March 2022

AbstractNucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the BH using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the BH is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the BH upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.
Strain stiffening of Ndc80 complexes attached to microtubule plus ends
Authors: Schwietert Felix , Volkov Vladimir A. , Huis in ’t Veld Pim J. , Dogterom Marileen , Musacchio Andrea , Kierfeld Jan 
Published: March 2022

ABSTRACTIn the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time-trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild type Ndc80 and three variants (CH-domains mutated or Hec1-tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening, i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models, which show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.SIGNIFICANCEBy time-tracing the stiffness of microtubule end-tracking Ndc80 complexes in optical trap experiments, we detect strain stiffening, and, thereby, provide new insights into the elastic properties of the Ndc80 complex. The strain stiffening is robust against mutations in the Ndc80 complex. We relate strain stiffening to the structure of the Ndc80 complex by means of a simple polymer model, to thermal fluctuations of the microtubule, and to the flexibility of force-generating flaring protofilaments at the tip of the microtubule. Since Ndc80 complexes play a major role for transmitting force from microtubule ends to the kinetochore, their elastic properties are of great interest for a deeper understanding of chromosome dynamics in the mitotic spindle.
Neonate personality affects early-life resource acquisition in a large social mammal
Authors: Amin Bawan , Jennings Dómhnall J. , Norman Alison , Ryan Andrew , Ioannidis Vasiliki , Magee Alice , Haughey Hayley-Anne , Haigh Amy , Ciuti Simone 
Published: March 2022

AbstractCurrent debate in the field of animal personality revolves around whether personality is reflecting individual differences in resource allocation or acquisition. Despite the large body of literature, the proximate relationships between personality, resource allocation, and acquisition are still unclear, especially during early stages of development. Here we studied how among-individual differences in behaviour develop over the first 6 months of life, and their potential association with resource acquisition in a free-ranging population of fallow deer (Dama dama). We related proxies of neonate personality – i.e. neonate physiological (heart rate) and behavioural (latency to leave at release) anti-predator responses to human handling – to the proportion of time fawns allocated to scanning during their first summer and autumn of life. We then investigated whether there was a trade-off between scanning time and foraging time in these juveniles, and how it developed over their first 6 months of life. We found that neonates with longer latencies at capture (i.e. risk-takers) allocated less time scanning their environment, but that this relationship was only present when fawns were 3-6 months old during autumn, but not when fawns were only 1-2 months old during summer. We also found that time spent scanning was negatively related to time spent foraging – a relationship rarely tested in juveniles of large mammals - and that this relationship becomes stronger over time, as fawns gradually switch from a nutrition rich (milk) to a nutrition poor (grass) diet. Our results highlight a potential mechanistic pathway in which neonate personality may drive differences in early-life resource acquisition, through allocation, of a large social mammal.
OnRamp: rapid nanopore plasmid validation
Authors: Mumm Camille , Drexel Melissa L. , McDonald Torrin L. , Diehl Adam G. , Switzenberg Jessica A. , Boyle Alan P. 
Published: March 2022

AbstractRecombinant plasmid vectors are versatile tools which have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation, however this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids due to read-length limits. While next-generation sequencing (NGS) does provide full-plasmid sequencing at scale, it is impractical and costly when utilized outside of library-scale validation. Here we present OnRamp (Oxford nanopore-based Rapid Analysis of Multiplexed Plasmids), an alternative method for routine plasmid validation which combines the advantages of NGS’s full plasmid coverage and scalability with Sanger’s affordability and accessibility by leveraging nanopore’s novel long-read sequencing technology. We include customized wet-lab protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is built into the OnRamp webapp (http://OnRamp.BoyleLab.org), which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views in a user-friendly manner, precluding the need for programming experience in analyzing nanopore results. Here we describe the OnRamp protocols and pipeline, and demonstrate our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure, at less than half the cost of equivalent Sanger sequencing.
Reproducibility problem with a proposed standard method to measure disinfection efficacy
Authors: Hilgren John , Srikanth Mrudula , Brutofsky Milady , Mainquist Keith , Prew Paul , King William , Jones Rhonda , Hellickson Lisa , Luedtke Josh , Restrepo Kristie , Brill Florian , Quinn Patrick 
Published: March 2022

AbstractA collaborative study was carried out in four laboratories to determine the reproducibility of a proposed ASTM International standard method for quantitatively evaluating the efficacy of disinfectants on hard, non-porous surfaces against bacteria. The method, known as the Quantitative Method, has also been suggested as a future regulatory standard for the United States and internationally. The multi-lab study was carried out using Pseudomonas aeruginosa and an alkyl dimethyl benzyl ammonium chloride antimicrobial product diluted in hard water. Results of the study showed acceptable repeatability in log10 reductions within each laboratory, but unacceptable reproducibility across laboratories despite careful analyst training and standardization of test conditions. A follow-up study ruled out analyst-to-analyst differences as the cause of the poor reproducibility. As it currently exists, the Quantitative Method is not sufficiently reproducible. Ruggedness testing to assess the sensitivity of the method to small changes in operational factors is recommended.
Production of novel biosurfactant by a new yeast species isolated from Prunus mume Sieb. et Zucc
Authors: Kim Jeong-Seon , Lee Miran , Ki Dae-Won , Kwon Soon-Wo , Ko Young-Joon , Kim Jong-Shik , Yun Bong-Sik , Kim Soo-Jin 
Published: March 2022

AbstractBiosurfactants reduce surface and interfacial tension due to their amphiphilic properties, and are an eco-friendly alternative for chemical surfactants. In this study, a novel yeast strain JAF-11 that produces biosurfactant was selected using drop collapse method, and the properties of the material were investigated. The nucleotide sequences of the strain were compared with closely related strains and identified based on the D1/D2 domain of the large-subunit rDNA (LSU) and internal transcribed spacer (ITS) regions. Neodothiora populina CPC 39399T, the closest species with strain JAF-11 in the phylogenetic tree, showed a sequence similarity of 97.75% for LSU and 94.27% for ITS, respectively. The result suggests that the strain JAF-11 represent a distinct species which cannot be assigned to any existing genus or species in the family Dothideaceae. Strain JAF-11 was able to produce biosurfactant reducing the surface tension of medium to 34.5 mN/m on the 6th day of culture and the result of measuring the critical micelle concentration (CMC) by extracting the crude biosurfactant was found to be 24 mg l-1. The molecular weight 502 of the purified biosurfactant was confirmed by measuring the fast atom bombardment mass spectrum (FAB-MS). The chemical structure was analyzed by measuring 1H nuclear magnetic resonance (NMR), 13C NMR, two-dimensional NMRs of the compound. The molecular formula was C26H46O9, and it was composed of one octanoyl group and two hexanoyl group to myo-inositol moiety. The new biosurfactant is the first report of a compound produced by a novel yeast strain JAF-11. This new biosurfactant is proposed as potential candidate for use in a variety field.
Growth inhibition of Akkermansia muciniphila by a secreted pathobiont sialidase
Authors: van Muijlwijk Guus H. , Bröring Emma , van Mierlo Guido , Jansen Pascal W.T.C. , Vermeulen Michiel , Aerts Piet C. , van Putten Jos P.M. , Palm Noah W. , de Zoete Marcel R. 
Published: March 2022

AbstractAkkermansia muciniphila is considered a key constituent of a healthy gut microbiota. In inflammatory bowel disease (IBD), A. muciniphila has a reduced abundance while other, putative pathogenic, mucus colonizers bloom. We hypothesized that interbacterial competition may contribute to this observation. By screening the supernatants of a panel of enteric bacteria, we discovered that a previously uncharacterized Allobaculum species potently inhibits the growth of A. muciniphila. Mass spectrometry analysis identified a secreted Allobaculum sialidase as inhibitor of A. muciniphila growth. The sialidase targets sialic acids on casein O-glycans, thereby altering the accessibility of nutrients critical for A. muciniphila. The altered glycometabolic niche results in distorted A. muciniphila cell division and efficiently arrests its growth. The identification of a novel mechanism of A. muciniphila growth inhibition by a competing bacterial pathobiont may provide a rationale for interventions aimed at restoring and maintaining a healthy microbiota symbiosis in patients with intestinal disease.
Decorating chromatin for enhanced genome editing using CRISPR-Cas9
Authors: Chen Evelyn , Lin-Shiao Enrique , Trinidad Marena , Doost Mohammad Saffari , Colognori David , Doudna Jennifer A. 
Published: March 2022

AbstractCRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by three-fold and HDR:indel ratio by five-fold compared to that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a new strategy to increase HDR efficiency.Significance StatementCRISPR-Cas-mediated homology-directed repair (HDR) enables precision genome editing for diverse research and clinical applications, but HDR efficiency is often low due to competing end-joining pathways. Here, we describe a simple strategy to influence DNA repair pathway choice and improve HDR efficiency by engineering CRISPR-Cas9-methyltransferase fusion proteins. This strategy highlights the impact of histone modifications on DNA repair following CRISPR-Cas-induced double-stranded breaks and adds to the CRISPR genome editing toolbox.
Dual role of an essential HtrA2/Omi protease in the human malaria parasite: maintenance of mitochondrial homeostasis and induction of apoptosis like cell death under cellular stress
Authors: Singh Shweta , Datta Gaurav , Jain Shaifali , Thakur Vandana , Muneer Azhar , Asad Mohd , Ali Shakir , Mohmmed Asif 
Published: March 2022

AbstractHtrA family of serine protease is known to play role in mitochondrial homeostasis as well as in the programmed cell death. Mitochondrial homeostasis and metabolism are crucial for survival and propagation of malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin like protease activity that can be inhibited by its specific inhibitor, ucf-101. Transgenic parasite line was generated, using HA-glmS C-terminal tagging approach, for localization as well as for inducible knockdown of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, ucf-101 treatment had no effect on the parasite growth, suggesting non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase like protease as well as parasite cell death, suggesting involvement of protease activity of PfHtrA2 in apoptosis like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show specific role of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating the stress induced cell death.
Artificial Hsp100-mediated systems for re-localizing protein aggregates
Authors: Fischbach Arthur , Johns Angela , Hao Xinxin , Schneider Kara L. , Nyström Thomas 
Published: March 2022

AbstractSpatial Protein Quality Control (sPQC) sequesters misfolded proteins into specific, organelle-associated inclusions within the cell to harness their toxicity. To approach the role of sPQC in cellular fitness, neurodegenerative diseases and aging, we report on the construction of Hsp100-based systems in yeast cells, which can artificially target protein aggregates to non-canonical locations. We demonstrated that aggregates of mutant Huntingtin (mHtt), the disease-causing agent of Huntington’s disease can be artificially targeted to daughter cells as well as to eisosomes and endosomes with this approach. Removing aggregates from mother cells did not significantly affect their lifespan and targeting mHtt to multiple smaller aggregates rather than one large inclusion did not alter its toxicity. We demonstrated that this approach is able to manipulate mHtt inclusion formation also in human cells and has the potential to be a useful complementation to present therapeutic approaches aimed at alleviating age-related neurodegenerative diseases.
Investigation of wind effect on different quadrotors
Authors: Ercan Hamdi , Ulucan Hamdi , Can Muharrem Selim 
Published: March 2022

Purpose
Although windy weather conditions have a significant effect on the flight safety and stability of any aircraft, the fact that quadrotors are lighter than other aircraft makes them more sensitive to the wind. This study aims to examine the extent to which quadrotors and their sensors, which are used in many fields and whose use is expected to increase significantly in the future, are affected by wind.

Design/methodology/approach
Flight experiments were carried out on different routes assigned by using Pixhawk Holybro 4 and Radiolink flight controllers. In these flight experiments, quadrotors were exposed to winds at different speeds and directions.

Findings
In the flight experiments, the deviation amounts in the quadrotor’s route at which wind speed was determined, and it was seen that these deviations were very serious and affected the safe flight at high wind speeds. According to the sensor information obtained from both different quadrotors’ flight experiments at different wind speeds, it was determined that the wind decreased the sensors’ accuracy.

Practical implications
It is foreseen that the data obtained in this study will be a source to be used in the design of quadrotors to be used in public areas in the future and to take the wind into account for safe flight.

Originality/value
In this study, numerous flight tests were carried out experimentally at various speeds from low speeds to high speeds on different routes using different flight controllers. The deviation data on the obtained routes and the effect of the wind on the sensors are experienced in real atmospheric conditions.
Horizontal take-off of an insect-like FMAV based on stroke plane modulation
Authors: Chen Zihao , Zhang Weiping , Mou Jiawang , Zheng Kexin 
Published: March 2022

Purpose
Vertical take-off is commonly adopted in most insect-mimicking flapping-wing micro air vehicles (FMAV) while insects also adopt horizontal take-off from the ground. The purpose of this paper is to study how insects adjust their attitude in such a short time during horizontal take-off by means of designing and testing an FMAV based on stroke plane modulation.

Design/methodology/approach
An FMAV prototype based on stroke plane rotating modulation is built to test the flight performance during horizontal take-off. Dynamic gain and decoupling mixer is added to compensate for the nonlinearity during the rotation angle of the stroke plane getting too large at the beginning of take-off. Force/torque test based on a six-axis sensor validates the change of aerodynamic force and torque at different rotation angles. High-speed camera and motion capture system test the flight performance of horizontal take-off.

Findings
Stroke plane modulation can provide a great initial pitch toque for FMAV to realize horizontal take-off. But the large range of rotation angles causes nonlinearity and coupling of roll and yaw. A dynamic gain and a mixer are added in the controller, and the FMAV successfully achieves horizontally taking off in less than 1 s.

Originality/value
The research in this paper shows stroke plane modulation is suitable for insect’s horizontal take-off