bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development and optimisation of cationic lipid nanoparticles for mRNA delivery
Authors: Yan Dongnan , Lu Haonan , Kaur Apanpreet , Fu Ruisi , Wang Ning , Teh Jin Hui , Lou Hantao , Aboagye Eric O , Chen Rongjun 
Published: February 2023

AbstractMessenger RNA (mRNA) has been proposed as a therapeutic agent for various diseases, including cancer. To ensure effective transfection of cancer cells, mRNA needs to be transported with a delivery system that protects its integrity and functionality. In this regard, cationic lipid nanoparticles composed of dioleoylphosphatidylethanolamine (DOPE) and 3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) have emerged as common vectors to deliver mRNA. In this project, we aim to use luciferase mRNA as a reporter to synthesise mRNA-loaded cationic lipid nanoparticles, and optimise their mRNA encapsulation and transfection efficiency in ovarian cancer cells. The optimisation process included: 1) adjusting the lipid formulation; 2) adjusting the input mRNA concentration before lipid nanoparticle extrusion; and 3) adjusting the extrusion methods. After optimisation, the encapsulation efficiency was optimised to 62%, thus achieving a relatively high transfection luminescence signal (9.4 times compared to baseline). The lipid nanoparticles also demonstrated stable physical characteristics and high biocompatibility (above 75% cell viability after treatment) within 24 hours. Overall, this project evaluated the synthesis of DOPE/DC-Chol cationic lipid nanoparticles, and optimised their mRNA encapsulation and transfection efficiency in ovarian cancer cell lines. The optimised lipid nanoparticles can be utilised as an ideal system for mRNA delivery, which could be further developed as a potential platform for the immunotherapy in ovarian cancer.
Full-length isoform sequencing for resolving the molecular basis of Charcot-Marie-Tooth 2A
Authors: Stergachis Andrew B. , Blue Elizabeth E. , Gillentine Madelyn A , Wang Lee-kai , Schwarze Ulrike , Cortés Adriana Sedeño , Ranchalis Jane , Allworth Aimee , Bland Austin E. , Chanprasert Sirisak , Chen Jingheng , Doherty Daniel , Folta Andrew B. , Glass Ian , Horike-Pyne Martha , Huang Alden Y. , Khan Alyna T. , Leppig Kathleen A. , Miller Danny E. , Mirzaa Ghayda , Parhin Azma , Raskind Wendy , Rosenthal Elisabeth A. , Sheppeard Sam , Strohbehn Samuel , Sybert Virginia P. , Tran Thao T. , Wener Mark ,   University of Washington Center for Mendelian Genomics (UW-CMG), Undiagnosed Diseases Network (UDN), Byers Peter H. , Nelson Stanley F. , Bamshad Michael J. , Dipple Katrina M. , Jarvik Gail P. , Hoppins Suzanne , Hisama Fuki M. 
Published: February 2023

AbstractObjectivesTranscript sequencing of patient derived samples has been shown to improve the diagnostic yield for solving cases of likely Mendelian disorders, yet the added benefit of full-length long-read transcript sequencing is largely unexplored.MethodsWe applied short-read and full-length isoform cDNA sequencing and mitochondrial functional studies to a patient-derived fibroblast cell line from an individual with neuropathy that previously lacked a molecular diagnosis.ResultsWe identified an intronic homozygousMFN2c.600-31T>G variant that disrupts a branch point critical for intron 6 spicing. Full-length long-read isoform cDNA sequencing after treatment with a nonsense-mediated mRNA decay (NMD) inhibitor revealed that this variant creates five distinct altered splicing transcripts. All five altered splicing transcripts have disrupted open reading frames and are subject to NMD. Furthermore, a patient-derived fibroblast line demonstrated abnormal lipid droplet formation, consistent with MFN2 dysfunction. Although correctly spliced full-lengthMFN2transcripts are still produced, this branch point variant results in deficient MFN2 protein levels and autosomal recessive Charcot-Marie-Tooth disease, axonal, type 2A (CMT2A).DiscussionThis case highlights the utility of full-length isoform sequencing for characterizing the molecular mechanism of undiagnosed rare diseases and expands our understanding of the genetic basis for CMT2A.
An exploratory data analysis from ovine and bovine RNA-seq identifies pathways and key genes related to cervical dilatation
Authors: Gonçalves Joedson Dantas , Sterman Ferraz José Bento , Meirelles Flávio Vieira , Nociti Ricardo Perecin , Franco Oliveira Maria Emilia 
Published: February 2023

AbstractThe present study developed a review and exploration of data in public and already validated repositories. The main objective is to identify the pathways involved in ruminant’s cervical dilatation, which are conserved between cattle and sheep in the follicular and luteal phases of the reproductive cycle. In cattle, 1961 genes were found to be more expressed in the follicular phase and 1560 in the luteal phase. 24 genes were considered exclusively expressed from these 18 genes were in the follicular phase and 6 genes were in the luteal phase. In sheep, 2126 genes are more expressed in the follicular phase and 2469 genes are more expressed in the luteal phase. Hoxb genes were identified in both species and are correlated with the PI3K/Akt pathway. PI3K/Akt was also found in both cattle and sheep, appearing prominently in the follicular and luteal phases of both species. Our analyzes have pointed out that the PI3K/Akt pathway and the Hoxb genes appear in prominence, in modulating mechanisms that involve estrus alterations in the cervix. PI3K/Akt appears to be an important pathway in the cervical relaxation process.
Engineering bacteriophages through deep mining of metagenomic motifs
Authors: Huss Phil , Kieft Kristopher , Meger Anthony , Nishikawa Kyle , Anantharaman Karthik , Raman Srivatsan 
Published: February 2023

AbstractBacteriophages can adapt to new hosts by altering sequence motifs through recombination or convergent evolution. Where such motifs exist and what fitness advantage they confer remains largely unknown. We report a new method, Metagenomic Sequence Informed Functional Scoring (Meta-SIFT), to discover sequence motifs in metagenomic datasets that can be used to engineer phage activity. Meta-SIFT uses experimental deep mutational scanning data to create sequence profiles to enable deep mining of metagenomes for functional motifs which are otherwise invisible to searches. We experimentally tested over 17,000 Meta-SIFT derived sequence motifs in the receptor-binding protein of the T7 phage. The screen revealed thousands of T7 variants with novel host specificity with functional motifs sourced from distant families. Position, substitution and location preferences dictated specificity across a panel of 20 hosts and conditions. To demonstrate therapeutic utility, we engineered active T7 variants against foodborne pathogenE. coliO121. Meta-SIFT is a powerful tool to unlock the functional potential encoded in phage metagenomes to engineer bacteriophages.
Colocalization of Cancer Associated Biomarkers on Single Extracellular Vesicles for Early Cancer Detection
Authors: Salem Daniel P. , Bortolin Laura T. , Gusenleitner Dan , Grosha Jonian , Zabroski Ibukunoluwapo O. , Biette Kelly M. , Banerjee Sanchari , Sedlak Christopher R. , Byrne Delaney M. , Hamzeh Bilal F. , King MacKenzie S. , Cuoco Lauren T. , Santos-Heiman Timothy , Duff Peter A. , Winn-Deen Emily S. , Guettouche Toumy , Mattoon Dawn R. , Huang Eric K. , Schekman Randy , Couvillon Anthony D. , Sedlak Joseph C. 
Published: February 2023

AbstractDetection of cancer early, when it is most treatable, remains a significant challenge due to the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. Detection of their presence is made more difficult by a lack of abundant tumor-specific indicators (i.e., protein biomarkers, circulating tumor DNA, etc.) that would enable detection using a non-invasive diagnostic assay. In addition, many benign conditions manifest in a similar manner, thus discriminating an early-stage cancerous lesion from a benign tumor can present additional challenges and result in unnecessary medical procedures. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. Extracellular vesicles from all cell types, including early-stage tumors, are known to be abundant in blood, are remarkably stable, and serve as a biopsy of their cell of origin. The detection of a colocalized combination of cancer associated biomarkers that provide tumor specificity on the surface of extracellular vesicles enables the discrimination of early- and late-stage cancer from non-malignant conditions.
Different approaches to estimate benthic metazoan diversity associated with free-living macroalgae (Fucus vesiculosus) on shallow soft sediments
Authors: Preston Roxana , Majaneva Markus , Halonen Viivi , Rodil Iván F 
Published: February 2023

AbstractHabitat complexity can boost biodiversity by providing a wide range of niches allowing species co-existence. Baltic Sea benthic communities are characterised by low species diversity. Thus the occurrence of the habitat forming macroalgaFucus vesiculosusmay influence benthic communities and promote diversity. Here we obtain biodiversity estimates through conventional and eDNA approaches for the benthic assemblages associated with free-livingFucusand the adjacent bare-sediment habitats at six sites from the Northern Baltic Proper and the Gulf of Finland. Free-livingF. vesiculosushabitats are heterogeneous with biodiversity estimates varying considerably among sites. The additional habitat complexity provided byF. vesiculosustends to improve taxa richness as a result of additional epifauna assemblages, although infaunal taxa richness and abundance is often reduced. Consequently the complex habitats provided by free-livingF. vesiculosusoften improves biodiversity, yet alters the composition of assemblages in soft sediment habitats and consequential ecosystem functioning. We emphasise the disparity in biodiversity estimates achieved when employing different biodiversity approaches. Biodiversity estimates were more similar within approaches compared to between habitat types, with each approach detecting exclusive taxa. We suggest that biodiversity estimates benefit from a multi-approach design where both conventional and eDNA approaches are employed in complement.
Tracking components of bilingual language control in speech production: an fMRI study using functional localizers
Authors: Wolna Agata , Szewczyk Jakub , Diaz Michele , Domagalik Aleksandra , Szwed Marcin , Wodniecka Zofia 
Published: February 2023

AbstractWhen bilingual speakers switch back to speaking in their native language (L1) after having used their second language (L2), they often experience difficulty in retrieving words in their L1: this phenomenon is referred to as theL2 after-effect.We used the L2 after-effect as a lens to explore the neural bases of bilingual language control mechanisms. Our goal was twofold: first, to explore whether bilingual language control draws on domain-general or language-specific mechanisms; second, to investigate the precise mechanism(s) that drive the L2 after-effect. We used a precision fMRI approach based onfunctional localizersto measure the extent to which the brain activity that reflects the L2 after-effect overlaps with the language network (Fedorenko et al., 2010) and the domain-general Multiple Demand network (Duncan et al., 2010), as well as three task-specific networks that tap into interference resolution, lexical retrieval, and articulation. Forty-two Polish-English bilinguals participated in the study. Our results show that the L2 after-effect reflects increased engagement of domain-general but not language-specific resources. Furthermore, contrary to previously proposed interpretations, we did not find evidence that the effect reflects increased difficulty related to lexical access, articulation, and the resolution of lexical interference. We propose that difficulty of speech production in the picture naming paradigm – manifested as the L2 after-effect – reflects interference at a non-linguistic level of task schemas or a general increase of cognitive control engagement during speech production in L1 after L2.
Deconvolving feeding niches and strategies of abyssal holothurians from their stable isotope, amino acid, and fatty acid composition
Authors: Stratmann Tanja , van Breugel Peter , van Oevelen Dick 
Published: February 2023

AbstractHolothurians are the dominant megabenthic deposit feeders in the Peru Basin (South-East Pacific) and feed to various degrees of selectively on the heterogenous pool of sedimentary detritus, but diet preferences for most holothurian species are unknown. This study reconstructs the diets of 13 holothurian species of the orders Elasipodida, Holothuriida, and Synallactida, from bulk stable isotope analyses (δ13C, δ15N) of holothurian body walls and guts, gut contents, and feces that were combined with compound-specific stable isotope analyses of amino acids, phospholipid-derived fatty acids, and neutral lipid-derived fatty acids in the body wall. Fatty acid concentrations showed high levels of storage lipids, an likely adaption to limited food supply to abyssal plains. Amino acid δ15N isotope values allowed estimating trophic levels of holothurian species and calculating heterotrophic re-synthesis of amino acids. Fatty acids served as trophic markers for feeding on diatom- and dinoflagellate derived phytodetritus, bacteria, Foraminifera, and detritus containing the PUFA C22:1ω9-cis. Several holothurian species seemed to be secondary consumers of detritus, while bacteria in their guts were primary consumers of this detritus. A Sørensen–Dice coefficient based cluster analysis using data of trophic levels, levels of heterotrophic re-synthesis of amino acids, feeding selectivity, and food sources/ diet suggested three trophic groups, characterized by different trophic levels. We show that this multi-biomarker driven approach allows to deconvolve trophic niches and feeding selectivity in one of the most challenging environments on earth and to identify dependence of deep-sea species to organic matter inputs that vary with season and/or climate.
Inter-kingdom microbial interactions revealed by a comparative machine-learning guided multi-omics analysis of industrial-scale biogas plants
Authors: Wirth Roland , Bagi Zoltán , Shetty Prateek , Szuhaj Márk , Cheung Teur Teur Sally , Kovács Kornél L. , Maróti Gergely 
Published: February 2023

AbstractMulti-omics analysis is a powerful tool for the detection and study of inter-kingdom interactions, such as those between bacterial and archaeal members of complex biogas-producing microbial communities. In the present study, the microbiomes of three industrial-scale biogas digesters, each fed with different substrates, were analysed using a machine-learning guided genome-centric metagenomics framework complemented with metatranscriptome data. This data permitted us to elucidate the relationship between abundant core methanogenic communities and their syntrophic bacterial partners. In total, we detected 297 high-quality, non-redundant metagenome-assembled genomes (nrMAGs). Moreover, the assembled 16S rRNA gene profiles of these nrMAGs showed that the phylum Firmicutes possessed the highest copy number, while the representatives of the Archaeal domain had the lowest. Further investigation of the three anaerobic microbial communities showed characteristic alterations over time but remained specific to each industrial-scale biogas plant. The relative abundance of various microbes as revealed by metagenome data were independent from corresponding metatranscriptome activity data. Interestingly, Archaea showed considerably higher activity than was expected from their abundance. We detected 53 nrMAGs that were present in all three biogas plant microbiomes with different abundances. The core microbiome correlated with the main chemical fermentation parameters and no individual parameter emerged as a predominant shaper of community composition. Various interspecies H2/electron transfer mechanisms were assigned to hydrogenotrophic methanogens in the biogas plants that ran on agricultural biomass and wastewater. Analysis of metatranscriptome data revealed that methanogenesis pathways were the most active of all main metabolic pathways. These findings highlight the importance of a combinatorial omics data framework to identify and characterise the activity of specific microbes in complex environments.
IVT generation of guideRNAs for Cas9-enrichment Nanopore Sequencing
Authors: Gilpatrick Timothy , Wang Josh Zhiyong , Weiss David , Norris Alexis L , Eshleman James , Timp Winston 
Published: February 2023

ABSTRACTGenerating high-coverage sequencing coverage at select genomic loci has extensive applications in both research science and genetic medicine. Long-read sequencing technologies (e.g. nanopore sequencing) have expanded our ability to generate sequencing data in regions (e.g. repetitive elements) that are difficult to interrogate with short-read sequencing methods. In work presented here, we expand on our previous work using CRISPR/Cas9 for targeted nanopore sequencing by usingin vitrotranscribed guideRNAs, with 1100 guideRNAs in a single experiment. This approach decreases the cost per guideRNA, increases the number of guideRNAs that can be multiplexed in a single experiment, and provides a way to rapidly screen numerous guideRNAs for cutting efficiency. We apply this strategy in multiple patient-derived pancreatic cancer cell lines, demonstrating its ability to unveil structural variation in “deletion hotspots” around the tumor suppressor genesp16(CDKN2A), andSMAD4.
GeneDART: Extending gene coverage in image-based spatial transcriptomics by deep learning-based domain adaptation with barcode-based RNA-sequencing data
Authors: Ohn Jungyoon , Lee Daeseung , Choi Hongyoon 
Published: February 2023

AbstractSpatial transcriptomics (ST) technologies provide comprehensive biological insights regarding cell-cell interactions and peri-cellular microenvironments. ST technologies are divided into two categories: imaging-based (I-B) and barcode-based (B-B). I-B ST technologies provide high resolution and sensitivity but have limited gene coverage. B-B ST technologies can analyze the whole transcriptome but have lower spatial resolution. To address these limitations, we propose a deep learning-based model that integrates I-B and B-B ST technologies to increase gene coverage while preserving high resolution. A model, trained by a neural network with an adversarial loss based on I-B and B-B datasets from human breast cancer tissue, was able to extend gene coverage to whole transcripts-level and accurately predict gene expression patterns in the I-B dataset with a high resolution. This novel methodology, named GeneDART, could enable researchers to utilize B-B and I-B ST datasets in a complementary way.
Systematic part transfer by extending a modular toolkit to diverse bacteria
Authors: Keating Kevin , Young Eric M. 
Published: February 2023

ABSTRACTIt is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection forE. coli. This enabled testing of a library of DNA constructs across the Pseudomonadota –Escherichia coli, Pseudomonas putida, Cupriavidus necator, andKomagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms – meaning that the same parts can be used to programE. coli, P. putida, C. necator, andK. nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently inK. nataicolathan in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, making it possible to create a small number of parts sets that can span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.
Selection of ade novogene that can promote survival ofE. coliby modulating protein homeostasis pathways
Authors: Frumkin Idan , Laub Michael T. 
Published: February 2023

AbstractCells sometime adapt to challenging environments by turning non-functional loci into functional genes in a process termedde novogene birth. But how proteins with random amino acid sequences integrate into existing cellular pathways to provide a benefit remains poorly understood. Here, we screened ∼108random genes for their ability to rescue growth arrest ofE. colicells producing the ribonuclease toxin MazF. Approximately 2,000 random genes could promote growth by reducing transcription from the promoter drivingmazFexpression. Additionally, one gene, named randomantitoxin ofMazF(ramF), whose protein product was well-tolerated in cells and neutralized MazF by interacting with chaperones, leading to MazF proteolysis. We show that the specificity of RamF for MazF relative to other toxins relies on the degron-like function of MazF’s first 10 amino acids. Finally, we demonstrate that random proteins can improve during evolution by identifying beneficial mutations that turned RamF into a more efficient inhibitor. Our work provides a mechanistic basis for howde novogene birth can produce new, functional proteins that are integrated into complex cellular systems and provide a benefit to cells.