bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering stringent genetic biocontainment of yeast with a protein stability switch
Authors: Hoffmann Stefan A. , Cai Yizhi 
Published: November 2022

AbstractSynthetic biology holds immense promise to tackle key problems we are facing, for instance in resource use, environmental health, and human health care. However, comprehensive safety measures are needed to deploy genetically engineered microorganisms in open-environment applications. Here, we describe a genetic biocontainment system based on conditional stability of essential proteins. We used a yeast-adapted destabilizing domain degron, which can be stabilized by estradiol addition (ERdd). Leveraging the yeast GFP collection and lab automation platforms, we ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes,SPC110, DIS3andRRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence.SPC110-ERddoffered the most stringent containment, with an escape frequency of 7.0×10-8, and full growth restoration at 100 nM estradiol. By systematically analyzing the containment escapees, we identified the non-essential C-terminal region ofSPC110as target for escape mutations. Its removal decreased the escape frequency with a single ERdd tag further to 4.3×10-9. CombiningSPC110-ERddwith a second ERdd tag on eitherDIS3orRRP46resulted in escape frequencies below the detection limit of the used assay (<2×10-10). Being based on conditional protein stability, this approach is mechanistically orthogonal to previously reported intrinsic biocontainment systems. It thus can be readily combined with other systems, for instance ones based on transcriptional or translational control of essential gene expression, to achieve multiplexed, extremely stringent control over the survival of engineered organisms.SignificanceSynthetic biology holds enormous potential to tackle key issues humanity is facing and can for instance revolutionize agriculture, bioremediation or health care. In each case, the unchecked spread of engineered organisms in natural environments must be prevented. This is particularly problematic with use cases of engineered microbes in open environments. Intrinsic, genetically encoded biocontainment systems, which control cell survival based on environmental cues, can solve this issue. We have developed such a genetic biocontainment system acting on the stability of essential proteins, leveraging a switchable degron. Through a large-scale screening for suitable essential target genes, we were able to create yeast strains that are strictly dependent on estradiol. Supplied with this small molecule, the engineered cells maintain high fitness and grow as robustly as the unmodified strains.
Optimal clustering for quantum refinement of biomolecular structures: Q|R#4
Authors: Wang Yaru , Kruse Holger , Moriarty Nigel W. , Waller Mark P. , Afonine Pavel V. , Biczysko Malgorzata 
Published: November 2022

AbstractQuantum refinement (Q|R) of crystallographic or cryo-EM derived structures of biomolecules within the Q|R project aims at usingab initiocomputations instead of library-based chemical restraints. An atomic model refinement requires the calculation of the gradient of the objective function. While it is not a computational bottleneck in classic refinement it is a roadblock if the objective function requiresab initiocalculations. A solution to this problem adopted in Q|R is to divide the molecular system into manageable parts and do computations for these parts rather than using the whole macromolecule. This work focuses on the validation and optimization of the automaticdivide-and-conquerprocedure developed within the Q|R project. Also, we propose an atomic gradient error score that can be easily examined with common molecular visualization programs. While the tool is designed to work within the Q|R setting the error score can be adapted to similar fragmentation methods. The gradient testing tool presented here allowsa prioridetermination of the computationally efficient strategy given available resources for the potentially time-expensive refinement process. The procedure is illustrated using a peptide and small protein models considering different quantum mechanical (QM) methodologies from Hartree-Fock, including basis set and dispersion corrections, to the modern semi-empirical method from the GFN-xTB family. The results obtained provide some general recommendations for the reliable and effective quantum refinement of larger peptides and proteins.
iLID-antiGFP-nanobody is a flexible targeting strategy for recruitment to GFP-tagged proteins
Authors: Mahlandt Eike K. , Toereppel Maarten , Haydary Tayeba , Goedhart Joachim 
Published: November 2022

AbstractOptogenetics is a fast-growing field, that applies light-sensitive proteins to manipulate cellular processes. A popular optogenetics tool is the improved light-induced dimer (iLID). It comprises two components, iLID and SspB, which heterodimerize upon illumination with blue light. This system is often used to recruit proteins to a specific subcellular location, e.g. by targeting the iLID to the plasma membrane. The targeting requires modification of the iLID with a targeting sequence. To skip the modification of the iLID and use existing GFP fusion as targets, we fuse an antiGFP nanobody to the iLID. We show that the antiGFP nanobody is able to locate iLID to GFP-tagged proteins. Plus, the light-dependent recruitment of SspB to iLID, localized by the antiGFP nanobody to a GFP-tagged protein, is still functioning efficiently. This approach increases flexibility, enabling the recruitment of any GFP-tagged protein, without the necessity of protein engineering.
Recent evolution of a maternally-acting sex-determining supergene in a fly with single-sex broods
Authors: Baird Robert B. , Urban John M. , Mongue Andrew J. , Jaron Kamil S. , Hodson Christina N. , Grewoldt Malte , Martin Simon H. , Ross Laura 
Published: November 2022

AbstractSex determination is a key developmental process, yet it is remarkably variable across the tree of life. The dipteran family Sciaridae exhibits one of the most unusual sex determination systems in which mothers control offspring sex through selective elimination of paternal X chromosomes. Whereas in some members of the family females produce mixed-sex broods, others such as the dark-winged fungus gnatBradysia coprophilaare monogenic, with females producing single-sex broods. Female-producing females were previously found to be heterozygous for a large X-linked paracentric inversion (X’), which is maternally inherited and absent from male-producing females. Here we assembled and characterized the X’ sequence. As close sequence homology between the X and X’ made identification of the inversion challenging, we developed a k-mer-based approach to bin genomic reads before assembly. We confirmed that the inversion spans most of the X’ chromosome (approximately 55Mb) and encodes around 3500 genes. Analysis of the divergence between the inversion and the homologous region of the X revealed that it originated very recently (<0.5 mya). Surprisingly, we found that the X’ is more complex than previously thought and is likely to have undergone multiple rearrangements that have produced regions of varying ages, resembling a supergene composed of evolutionary strata. We found functional degradation of around 7.3% of genes within the region of recombination suppression, but no evidence of accumulation of repetitive elements. Our findings provide an indication that sex-linked inversions are driving turnover of the strange sex determination system in this family of flies.
Ultra Low Power, Event-Driven Data Compression of Multi-Unit Activity
Authors: Savolainen Oscar W. , Zhang Zheng , Constandinou Timothy G. 
Published: November 2022

AbstractRecent years have demonstrated the feasibility of using intracortical Brain-Machine Interfaces (iBMIs), by decoding thoughts, for communication and cursor control tasks. iBMIs are increasingly becoming wireless due to the risk of infection and mechanical failure, typically associated with percutaneous connections. The wireless communication itself, however, increases the power consumption further; with the total dissipation being strictly limited due to safety heating limits of cortical tissue. Since wireless power is typically proportional to the communication bandwidth, the output Bit Rate (BR) must be minimised. Whilst most iBMIs utilise Multi-Unit activity (MUA), i.e. spike events, and this in itself significantly reduces the output BR (compared to raw data), it still limits the scalability (number of channels) that can be achieved. As such, additional compression for MUA signals are essential for fully-implantable, high-information-bandwidth systems. To meet this need, this work proposes various hardware-efficient, ultra-low power MUA compression schemes. We investigate them in terms of their BRs and hardware requirements as a function of various on-implant conditions such as MUA Binning Period (BP) and number of channels. It was found that for BPs ≤ 10 ms, the delta-asynchronous method had the lowest total power and reduced the BR by almost an order of magnitude relative to classical methods (e.g. to approx. 151 bps/channel for a BP of 1 ms and 1000 channels on-implant.). However, at larger BPs the synchronous method performed best (e.g. approx. 29 bps/channel for a BP of 50 ms, independent of channel count). As such, this work can guide the choice of MUA data compression scheme for BMI applications, where the BR can be significantly reduced in hardware efficient ways. This enables the next generation of wireless iBMIs, with small implant sizes, high channel counts, low-power, and small hardware footprint. All code and results have been made publicly available.
retro-Tango enables versatile retrograde circuit tracing inDrosophila
Authors: Sorkaç Altar , A Moșneanu Rareș , Crown Anthony M , Savaş Doruk , Okoro Angel M , Talay Mustafa , Barnea Gilad 
Published: November 2022

AbstractTranssynaptic tracing methods are crucial tools in studying neural circuits. Although a couple of anterograde tracing methods and a targeted retrograde tool have been developed inDrosophila melanogaster, there is still need for an unbiased, user-friendly, and flexible retrograde tracing system. Here we describeretro-Tango, a method for transsynaptic, retrograde circuit tracing and manipulation inDrosophila. In this genetically encoded system, a ligand-receptor interaction at the synapse triggers an intracellular signaling cascade that results in reporter gene expression in presynaptic neurons. Importantly, panneuronal expression of the elements of the cascade renders this method versatile, enabling its use not only to test hypotheses but also to generate them. We validateretro-Tango in various circuits and benchmark it by comparing our findings with the electron microscopy reconstruction of theDrosophilahemibrain. Our experiments establishretro-Tango as a key method for circuit tracing in neuroscience research.
Lineage plasticity enables low-ER luminal tumors to evolve and gain basal-like traits
Authors: Mohamed Gadisti Aisha , Mahmood Sundis , Ognjenovic Nevena B. , Lee Min Kyung , Wilkins Owen M. , Christensen Brock C. , Muller Kristen E. , Pattabiraman Diwakar R. 
Published: November 2022

Abstract/summaryStratifying breast cancer into specific molecular or histological subtypes aids in therapeutic decision-making and predicting outcomes, however, these subtypes may not be as distinct as previously thought. Patients with luminal-like, Estrogen Receptor (ER)- expressing tumors have better prognosis than patients with more aggressive, triple- negative or basal-like tumors. There is, however, a subset of luminal-like tumors that express lower levels of ER, which exhibit more basal-like features. We have found that breast tumors expressing lower levels of ER, traditionally considered to be luminal-like, represent a distinct subset of breast cancer characterized by the emergence of basal-like features. Lineage tracing of low-ER tumors in the MMTV-PyMT mouse mammary tumor model revealed that basal marker expressing cells arose from normal luminal epithelial cells, suggesting that luminal-to-basal plasticity is responsible for the evolution and emergence of basal-like characteristics. This plasticity allows tumor cells to gain a new lumino-basal phenotype, thus leading to intratumoral lumino-basal heterogeneity. Single- cell RNA sequencing revealed SOX10 as a potential driver for this plasticity, which is known among breast tumors to be almost exclusively expressed in Triple Negative Breast Cancer (TNBC) and was also found to be highly expressed in low-ER tumors. These findings suggest that basal-like tumors may result from the evolutionary progression of luminal tumors with low ER expression.
Identification of heterokaryon incompatibility genes inAspergillus fumigatushighlights a narrow footprint of ancient balancing selection
Authors: Auxier Ben , Zhang Jianhua , Marquez Francisca Reyes , Senden Kira , van den Heuvel Joost , Aanen Duur K. , Snelders Eveline , Debets Alfons J.M. 
Published: November 2022

AbstractIn fungi, a phenomenon termed heterokaryon incompatibility restricts hyphal fusion to occur within an individual since fusion between individuals leads to cell death. Generally, the genes involved are found to be under balancing selection from negative frequency dependent fitness. Here, we assess this inAspergillus fumigatus, a human pathogenic fungus with an extremely high crossover rate. Using auxotrophic markers we screened sexual progeny for compatibility to identify genes involved in this process, the so-calledhetgenes. In total, 5/148 (3.4%) offspring were compatible with a parent and 166/2142 (7.7%) sibling pairs were compatible, consistent with several segregating incompatibility loci. Genetic mapping resulted in five loci, four of which could be fine mapped to individual genes, of which we tested three through heterologous expression, confirming their causal relationship. Surprisingly, a population-level analysis of two available independent datasets did not show an increase in Tajima’s D near these loci, normally a hallmark of balancing selection. However, analysis of closely related species did show trans-species polymorphisms across >10 million years, and equal allele frequencies withinA. fumigatus. Using availablede novoassemblies, we show that these balanced polymorphisms are restricted to within several hundred base pairs flanking the coding sequence, potentially due to this species’ high crossover rate. In addition to identifying the firsthetgenes in anAspergillusspecies, this work highlights the interaction of long-term balancing selection with a high recombination rate. Future mechanistic work on thesehetgenes may provide novel routes for clinical therapies, as well as opportunities for strain improvement in biotechnology.
RNAlysis: analyze your RNA sequencing data without writing a single line of code
Authors: Teichman Guy , Cohen Dror , Ganon Or , Dunsky Netta , Shani Shachar , Gingold Hila , Rechavi Oded 
Published: November 2022

AbstractBackgroundAmongst the major challenges in next-generation sequencing experiments are exploratory data analysis, interpreting trends, identifying potential targets/candidates, and visualizing the results clearly and intuitively. These hurdles are further heightened for researchers who are not experienced in writing computer code, since the majority of available analysis tools require programming skills. Even for proficient computational biologists, an efficient and replicable system is warranted to generate standardized results.ResultsWe have developedRNAlysis, a modular Python-based analysis software for RNA sequencing data.RNAlysisallows users to build customized analysis pipelines suiting their specific research questions, going all the way from raw FASTQ files, through exploratory data analysis and data visualization, clustering analysis, and gene-set enrichment analysis.RNAlysisprovides a friendly graphical user interface, allowing researchers to analyze data without writing code. We demonstrate the use ofRNAlysisby analyzing RNA data from different studies usingC. elegansnematodes. We note that the software is equally applicable to data obtained from any organism.ConclusionsRNAlysisis suitable for investigating a variety of biological questions, and allows researchers to more accurately and reproducibly run comprehensive bioinformatic analyses. It functions as a gateway into RNA sequencing analysis for less computer-savvy researchers, but can also help experienced bioinformaticians make their analyses more robust and efficient, as it offers diverse tools, scalability, automation, and standardization between analyses.
Kinetic control in amyloid polymorphism: Different agitation and solution conditions promote distinct amyloid polymorphs of alpha-synuclein
Authors: Devi Santosh , Garg Dushyant Kumar , Bhat Rajiv 
Published: November 2022

AbstractAggregation of neuronal protein α-synuclein is implicated in synucleinopathies, including Parkinson’s disease. Despite abundant in vitro studies, the mechanism of α-synuclein assembly process remains ambiguous. In this work, α-synuclein aggregation was induced by its constant mixing in two separate modes, either by agitation in a 96-well microplate reader (MP) or in microcentrifuge tubes using a shaker incubator (SI). Aggregation in both modes occurred through a sigmoidal growth pattern with a well-defined lag, growth, and saturation phase. The end-stage MP- and SI-derived aggregates displayed distinct differences in morphological, biochemical, and spectral signatures as discerned through AFM, proteinase-K digestion, FTIR, Raman, and CD spectroscopy. The MP-derived aggregates showed irregular morphology with a significant random coil conformation, contrary to SI-derived aggregates, which showed typical β-sheet fibrillar structures. The end-stage MP aggregates convert to β-rich SI-like aggregates upon 1) seeding with SI-derived aggregates and 2) agitating in SI. We conclude that end-stage MP aggregates were in a kinetically trapped conformation, whose kinetic barrier was bypassed upon either seeding by SI-derived fibrils or shaking in SI. We further show that MP-derived aggregates that form in the presence of sorbitol, an osmolyte, displayed a β-rich signature, indicating that the preferential exclusion effect of osmolytes helped overcome the kinetic barrier. Our findings help in unravelling the kinetic origin of different α-synuclein aggregated polymorphs (strains) that encode diverse variants of synucleinopathies. We demonstrate that kinetic control shapes the polymorphic landscape of α-synuclein aggregates, both through de novo generation of polymorphs, and by their interconversion.
Cryo-EM Structure of the 50S-HflX Complex Reveals a Novel Mechanism of Antibiotic Resistance inE. coli
Authors: Wu Damu , Dai Yuhao , Gao Ning 
Published: November 2022

AbstractBacterial HflX is a conserved ribosome-binding GTPase involved in splitting ribosomal complexes accumulated under stress condition. However, the atomic details of its ribosomal interaction remain to be elucidated. In this work, we present a high-resolution structure of theE. coli50S subunit bound with HflX. The structure reveals highly specific contacts between HflX and the ribosomal RNA, and in particular, an insertion loop of the N-terminal domain of HflX is situated in the peptidyl transferase center (PTC) and makes direct interactions with PTC residues. Interestingly, this loop displays steric clash with a few PTC-targeting antibiotics on the 50S subunit, such as chloramphenicol. Deletion ofhflXresults in hypersensitivity to chloramphenicol treatment, and a loop residue G154 of HflX is important for the observed chloramphenicol resistance. Overall, our results suggest that HflX could be a general stress response factor that functions in both stalled ribosome splitting and PTC antibiotic displacing.
Systematic review of mobile travel apps and their smart features and challenges
Authors: Sia Phoebe Yueng-Hee , Saidin Siti Salina , Iskandar Yulita Hanum P. 
Published: November 2022

PurposeMobile travel apps (MTA) smart features were identified based on recent travel application (app) trends and a literature review of MTA smart features. Subsequently, the MTA features that could be prioritised to increase user interest in MTA were determined. The MTA smart feature development challenges that should be mitigated were also identified.Design/methodology/approachThe app identification and selection were based on the one-stop solution characteristics containing the common function of travel apps and eight MTA smart features. A total of 193 Apple apps and 250 Google apps were identified, where 36 apps that met the inclusion and exclusion criteria based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses flowchart were selected for evaluation.FindingsThe high user ratings for apps from both app stores revealed the acceptance of smart technology in the tourism industry. Geolocation tracking services, travel itinerary generators, and real-time personalisation and recommendation were the three major features available in the included MTA. The challenges of MTA with smart features were highlighted from the tourism organisation, app developer and user perspectives.Practical implicationsThe findings can guide tourism organisations and app developers on the smart features that MTA should offer for user engagement. Technological organisations could optimise their technology stack by considering the identified smart features. The findings are valuable for scholars in terms of MTA aesthetics and usability to gain acceptability. The development challenges included significant investment in technology, location accuracy and privacy concerns when implementing MTA smart features.Originality/valueThe previous literature mainly focused on evaluating app quality, assessing app functionality, and user ratings using the Mobile Application Rating Scale, and scoping reviews of MTA articles. Contrastingly, this study is among the first in which MTA smart features were examined from a developer-centric perspective. Moreover, it is suggested that MTA includes integrated smart features for better tourism services and market penetration in the tourism industry.