bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thermostablein vitrotranscription-translation for enzyme screening in microdroplets
Authors: Ribeiro Ana L. J. L. , Pérez-Arnaiz Patricia , Sánchez-Costa Mercedes , Pérez Lara , Almendros Marcos , van Vliet Liisa , Gielen Fabrice , Lim Jesmine , Charnock Simon , Hollfelder Florian , González-Pastor J. Eduardo , Berenguer José , Hidalgo Aurelio 
Published: February 2024

AbstractBackgroundIn vitroexpression involves the utilization of the transcription and translation machinery derived from the cell to produce one or more proteins of interest and has found widespread application in the optimization of gene circuits or metabolic pathways in synthetic biology but also in pharmaceutical manufacturing. Mostin vitroexpression systems available are active at moderate temperatures but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Moreover, given the fact that the main barrier toward the general use ofin vitroexpression is its high price compared with host-based recombinant expression, there is a need to develop alternativein vitroexpression systems operating at high temperatures and compatible with technologies that enable ultrahigh-throughput screening in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.ResultsTo this end, we produced high-expression cell-free extracts fromThermus thermophilusforin vitrotranslation and supplemented them with thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable to other thermostablein vitroexpression systems, while the preparation procedure is simpler and can be suited to differentThermus thermophilusstrains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Although the composition of these extracts showed a high background in carboxyl esterase assays, β-glucosidase and cellobiose hydrolase activities could be measured with minimal background.ConclusionsCell-free extracts fromThermus thermophilusrepresent a simpler alternative to heavily optimized or pure component thermostablein vitroexpression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.
Unravelling the roadblocks to 1,2-propanediol biosynthesis in select solventogenicClostridiumspecies
Authors: Agyeman-Duah Eric , Kumar Santosh , Ujor Victor C. 
Published: February 2024

AbstractBackgroundThe compound 1,2-propanediol is an important industrial bulk chemical that has proven particularly recalcitrant to bio-production. Solvent-producingClostridiumspecies represent promising candidates for engineering 1,2-propaediol production. Co-production of 1,2-popanediol and butanol has the potential to improve the economics of the acetone-butanol-ethanol (ABE) fermentation.ResultsIn this study, the methylglyoxal synthase gene (mgsA) fromClostridium beijerinckiiNCIMB 8052 was homologously expressed in this organism. Additionally, a separate strain ofClostridium beijerinckiiNCIMB 8052 was engineered by cloning and expressingmgsAand methylglyoxal/glyoxal reductase (mgR) fromClostridium pasteurianumATCC 6013 as a fused protein linked by polyglycine linker in the former. Both strains ofC. beijerinckiiNCIMB 8052 failed to produce 1,2-propaneol. Instead, traces of acetol—the precursor of 1,2-propanediol—were detected in cultures of both strains. When the recombinant strains were exposed to acetol, both strains exhibited ∼100% acetol-to-1,2-propanediol conversion efficiency. Conversely, methylglyoxal supplementation led to the production of traces of acetol but not lactaldehyde or 1,2-propanediol. When wildtypeC. beijerinckiiNCIMB 8052,C. pasteurianumATCC 6013 andClostridium tyrobutyricumATCC 25755 were challenged with methylglyoxal,C. beijerinckiiproduced ∼0.1 g/L (S)-(+)-1,2-Propanediol, whileC. tyrobutyricumproduced traces of lactate.C. pasteurianumproduced neither 1,2-propanediol nor lactate. The wild types of all three species above exhibited ∼100% acetol-to-1,2-propanediol conversion efficiency. The recombinant strain ofC. beijerinckiiexpressing fused MgsA and MgR fromC. pasteurianumATCC 6013 showed enhanced growth and solvent production, producing as high as 88% more butanol on both glucose and lactose than the control strain and the recombinant strain of the same organism expressing the native MgsA.ConclusionsRecombinant and native strains ofC. beijerinckii,C. pasteurianumandC. tyrobutyricumstudied in this work exhibit extremely poor capacity to catalyze the conversion of the intermediates of the methylglyoxal bypass to 1,2-propanediol. This is indicative of lack of appropriate enzymes to catalyze the reactions from methylglyoxal to acetol or lactaldehyde. Inability to detect methylglyoxal in the recombinant strains harboringmgsA(both homologous and heterologous)— whereas the strain expressing bothmgsAandmgRfromC. pasteurianum, under the same promoter (Padc) produced higher concentrations of butanol—suggests thatC. beijerinckiimight possess a regulatory mechanism that limits the activity of methylglyoxal-producing MgsA. The protein product ofmgRfromC. pasteurianumrepresents a promising metabolic engineering candidate towards increasing butanol production.
Synaptic vesicle characterization of iPSC-derived dopaminergic neurons provides insight into distinct secretory vesicle pools
Authors: Fujise Kenshiro , Rosenfeld Martin Shaun , Rafiq Nisha Mohd 
Published: February 2024

ABSTRACTThe impairment of dopaminergic (DA) neurons plays a central role in the development of Parkinson’s disease. Evidence for distinct populations of synaptic vesicles (SVs) differing in neurotransmitter content (glutamate versus dopamine) has been attributed to differences in trafficking pathways and their exocytosis kinetics. However, the molecular and ultrastructural organization of the two types of vesicles remains poorly understood. Here we examined the development of axonal varicosities in human iPSC-derived DA neurons and glutamatergic neurons (i3Neurons). While i3Neurons are comprised of 40-50 nm small clear SVs, DA neurons are predominantly comprised of large pleiomorphic vesicles including empty and dense core vesicles, in addition to the classical SVs. The large vesicles were positive for VMAT2, the monoamine vesicular transporter responsible for loading dopamine, and are distinctly larger in size and spatially segregated from the VGLUT1/2-positive vesicles when expressed in an ectopic SV-like organelle reconstitution system. Moreover, these VMAT2-positive vesicles were also colocalized to known SV markers such as Rab3, SCAMP5, VAMP2, SV2C and can be clustered by the matrix protein synapsin. Our results show that DA neurons display inherent differences in their populations of neurotransmitter-containing secretory vesicles, and iPSC-derived neurons are powerful models for the study of presynaptic structures.
A multi-symptomatic model of heroin use disorder in rats reveals distinct behavioral profiles and neuronal correlates of heroin vulnerability versus resiliency
Authors: Kuhn Brittany N. , Cannella Nazzareno , Crow Ayteria D. , Lunerti Veronica , Gupta Arkobrato , Walterhouse Stephen J. , Allen Carter , Chalhoub Reda M. , Dereschewitz Eric , Roberts Analyse T. , Cockerham Mackenzie , Beeson Angela , Nall Rusty W. , Palmer Abraham A. , Hardiman Gary , Solberg Woods Leah C. , Chung Dongjun , Ciccocioppo Roberto , Kalivas Peter W. 
Published: February 2024

AbstractObjectiveThe behavioral and diagnostic heterogeneity within human opioid use disorder (OUD) diagnosis is not readily captured in current animal models, limiting translational relevance of the mechanistic research that is conducted in experimental animals. We hypothesize that a non-linear clustering of OUD-like behavioral traits will capture population heterogeneity and yield subpopulations of OUD vulnerable rats with distinct behavioral and neurocircuit profiles.MethodsOver 900 male and female heterogeneous stock rats, a line capturing genetic and behavioral heterogeneity present in humans, were assessed for several measures of heroin use and rewarded and non-rewarded seeking behaviors. Using a non-linear stochastic block model clustering analysis, rats were assigned to OUD vulnerable, intermediate and resilient clusters. Additional behavioral tests and circuit analyses using c-fos protein activation were conducted on the vulnerable and resilient subpopulations.ResultsOUD vulnerable rats exhibited greater heroin taking and seeking behaviors relative to those in the intermediate and resilient clusters. Akin to human OUD diagnosis, further vulnerable rat sub-clustering revealed subpopulations with different combinations of behavioral traits, including sex differences. Lastly, heroin cue-induced neuronal patterns of circuit activation differed between resilient and vulnerable phenotypes. Behavioral sex differences were recapitulated in patterns of circuitry activation, including males preferentially engaging extended amygdala stress circuitry, and females cortico-striatal drug cue-seeking circuitry.ConclusionUsing a non-linear clustering approach in rats, we captured behavioral diagnostic heterogeneity reflective of human OUD diagnosis. OUD vulnerability and resiliency were associated with distinct neuronal activation patterns, posing this approach as a translational tool in assessing neurobiological mechanisms underpinning OUD.
Transcription factor-mediated generation of dopaminergic neurons from human iPSCs – a comparison of methods
Authors: McDonald Kirstin O. , Lyons Nikita M.A. , Schoderboeck Lucia , Hughes Stephanie M. , Basak Indranil 
Published: February 2024

AbstractDopaminergic neurons are the predominant brain cells affected in Parkinson’s disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson’s disease, dopaminergic neurons have been generated from human skin cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, transcription factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compare and contrast three protocols to generate induced pluripotent stem cell-derived dopaminergic neurons using transcription factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate transcription factors into induced pluripotent stem cells, followed by differentiation using different media compositions. We introduced three transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells, and in combination with small molecules, we generated more than 80% neurons in the culture, out of which more than 80% neurons were dopaminergic neurons. Therefore, a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons, a prerequisite for cell replacement therapies.
A novel truncated CHAP modular endolysin, CHAPSAP26-161, that lysesStaphylococcus aureus,Acinetobacter baumannii,andClostridioides difficile
Authors: Choi Yoon-Jung , Kim Shukho , Dahal Ram Hari , Kim Jungmin 
Published: February 2024

AbstractBackgroundDevelopment of novel antimicrobial agents is imperative due to the increasing threat of antibiotic-resistant pathogens. This study aimed to validate the enhanced antibacterial activity andin vivoefficacy of a novel truncated endolysin, CHAPSAP26-161, derived from the CHAP domain of LysSAP26, against multidrug-resistant bacteria.MethodsTwo deletion mutants, CHAPSAP26-139 and CHAPSAP26-161, were constructed by deleting the C-terminal portion of LysSAP26. These were cloned and expressed, and their antibacterial activities, together with protein purification efficiency, were evaluated against 12 bacterial species under various environmental conditions. To test the temperature and pH stability of the three recombinant proteins, the antibacterial effects of the proteins at various temperatures (4°C–60°C) and pH values (3–10) were measured. Time-kill assay measured the optical density (600 nm) and colony-forming units after incubation for 0, 2, 4, 6, 8, and 24 h. We verified this throughin vivoexperiments using mouse models to evaluate the therapeutic potential of CHAPSAP26-161 againstAcinetobacter baumannii.ResultsCHAPSAP26-161 exhibited higher protein purification efficiency and antibacterial activity than LysSAP26. Moreover, CHAPSAP26-161 showed the highest lytic activity againstA. baumanniiwith a minimal bactericidal concentration (MBC) of 5–10 µg/mL, followed byStaphylococcus aureuswith an MBC of 10–25 µg/mL. Interestingly, CHAPSAP26-161 could lyse anaerobic bacteria, such asC. difficile, with an MBC of 25–50 µg/mL. At pH 4–8 and temperatures of 4°C–45°C, CHAPSAP26-161 exhibited optimal hydrolase activity. The lytic activity of CHAPSAP26-161 was dependent on divalent metal ions, especially Zn2+, and increased in the presence of ethylenediamine tetraacetic acid. CHAPSAP26-161 demonstrated superior protein purification efficiency and antibacterial activity than LysSAP26. It showed high lytic activity against gram-positive, gram-negative, and anaerobic bacteria, includingS. aureusandClostridioides difficile.Enhanced stability under varied temperatures and pH conditions.In vivo,tests demonstrated promising therapeutic effects of CHAPSAP26-161 in murine systemicA. baumanniiinfection models.ConclusionsCHAPSAP26-161, a truncated modular endolysin containing only the CHAP domain of LysSAP26, demonstrated higher protein purification efficiency and antibacterial activity than LysSAP26. It also exhibited extended-spectrum antibacterial activity against gram-positive, gram-negative, and anaerobic bacteria, such asS. aureus,A. baumannii, andC. difficile. Its successfulin vivoapplication in murine models highlights its potential as an alternative therapeutic agent in combating antibiotic resistance.
stMMR: accurate and robust spatial domain identification from spatially resolved transcriptomics with multi-modal feature representation
Authors: Zhang Daoliang , Yu Na , Li Wenrui , Sun Xue , Zou Qi , Li Xiangyu , Liu Zhiping , Yuan Zhiyuan , Zhang Wei , Gao Rui 
Published: February 2024

AbstractDeciphering spatial domains using spatially resolved transcriptomics (SRT) is of great value for the characterizing and understanding of tissue architecture. However, the inherent heterogeneity and varying spatial resolutions present challenges in the joint analysis of multi-modal SRT data. We introduce a multi-modal geometric deep learning method, named stMMR, to effectively integrate gene expression, spatial location and histological information for accurate identifying spatial domains from SRT data. stMMR uses graph convolutional networks (GCN) and self-attention module for deep embedding of features within unimodal and incorporates similarity contrastive learning for integrating features across modalities. Comprehensive benchmark analysis on various types of spatial data shows superior performance of stMMR in multiple analyses, including spatial domain identification, pseudo-spatiotemporal analysis, and domain-specific gene discovery. In chicken heart development, stMMR reconstruct the spatiotemporal lineage structures indicating accurate developmental sequence. In breast cancer and lung cancer, stMMR clearly delineated the tumor microenvironment and identified marker genes associated with diagnosis and prognosis. Overall, stMMR is capable of effectively utilizing the multi-modal information of various SRT data to explore and characterize tissue architectures of homeostasis, development and tumor.
Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing
Authors: De Rijk Peter , Watzeels Tijs , Küçükali Fahri , Van Dongen Jasper , Faura Júlia , Willems Patrick , De Deyn Lara , Duchateau Lena , Grones Carolin , Eekhout Thomas , De Pooter Tim , Joris Geert , Rombauts Stephane , De Rybel Bert , Rademakers Rosa , Van Breusegem Frank , Strazisar Mojca , Sleegers Kristel , De Coster Wouter 
Published: February 2024

AbstractWe introducescywalker, an innovative and scalable package developed to comprehensively analyze long-read nanopore sequencing data of full-length single-cell or single-nuclei cDNA. Existing nanopore single-cell data analysis tools showed severe limitations in handling current data sizes. We developed novel scalable methods for cell barcode demultiplexing and single-cell isoform calling and quantification and incorporated these in an easily deployable package. Scywalker streamlines the entire analysis process, from sequenced fragments in FASTQ format to demultiplexed pseudobulk isoform counts, into a single command suitable for execution on either server or cluster. Scywalker includes data quality control, cell type identification, and an interactive report. Assessment of datasets from the human brain, Arabidopsis leaves, and previously benchmarked data from mixed cell lines, demonstrate excellent correlation with short-read analyses at both the cell-barcoding and gene quantification levels. At the isoform level, we show that scywalker facilitates the direct identification of cell-type-specific expression of novel isoforms.
Centriolar satellites are sites of translation of centrosomal and ciliary proteins
Authors: Pachinger Claudia , Dobbelaere Jeroen , Rumpf-Kienzl Cornelia , Raina Shiviya , Garcia-Baucells Júlia , Sarantseva Marina , Brauneis Andrea , Dammermann Alexander 
Published: February 2024

AbstractCentriolar satellites are cytoplasmic particles found in the vicinity of centrosomes and cilia whose functional contribution to the formation of these cellular structures has long been unclear. By characterizing the main scaffolding component of satellites, PCM1 or Combover inDrosophila, we show that satellites are not involved in cellular trafficking as previously thought but rather act as sites for the coordinate translation of centrosomal and ciliary proteins through the interaction with a set of RNA binding proteins and proteins involved in quality control. Strikingly, the concentration of satellites near centrosomes and cilia in vertebrates is not a conserved feature and therefore dispensable for satellite function. Such coordinate synthesis may be a general feature in eukaryotic cells to facilitate protein complex formation and cellular compartmentalization.One-Sentence SummaryCentriolar satellites facilitate the coordinate synthesis of centrosomal and ciliary proteins.
Metabolic reprogramming of hypoxic tumor-associated macrophages through CSF-1R targeting favours treatment efficiency in colorectal cancers
Authors: Gharzeddine Khaldoun , Gonzalez Pietro Cristina , Malier Marie , Hennot Clara , Grespan Renata , Yamaryo-Botté Yoshiki , Botté Cyrille Y. , Thomas Fabienne , Laverriere Marie-Hélène , Girard Edouard , Roth Gael , Millet Arnaud 
Published: February 2024

AbstractTumor-associated macrophages participate in the complex network of support that favors tumor growth. Among the various strategies that have been developed to target these cells, blockade of the CSF-1R receptor is one of the most promising one. Here, we characterize the resulting state of human macrophages exposed to a CSF-1R kinase inhibitor. We find that CSF-1R receptor inhibition in human macrophages is able to impair cholesterol synthesis, fatty acid metabolism and hypoxia-driven expression of dihydropyrimidine dehydrogenase, an enzyme responsible for the 5-fluorouracil macrophage-mediated chemoresistance. We show that this inhibition of the CSF-1R receptor leads to a downregulation of the expression of SREBP2, a transcription factor that controls cholesterol and fatty acid synthesis. We also show that the inhibition of ERK1/2 phosphorylation resulting from targeting the CSF-1R receptor destabilizes the expression of HIF2α in hypoxia resulting in the downregulation of dihydropyrimidine dehydrogenase expression restoring the sensitivity to 5-fluorouracil in colorectal cancer. These results reveal the unexpected metabolic rewiring resulting from the CSF-1R receptor targeting of human macrophages in tumors.
NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis
Authors: Parolia Abhijit , Eyunni Sanjana , Verma Brijesh Kumar , Young Eleanor , Liu Lianchao , George James , Aras Shweta , Das Chandan Kanta , Mannan Rahul , ur Rasool Reyaz , Luo Jie , Carson Sandra E. , Mitchell-Velasquez Erick , Liu Yihan , Xiao Lanbo , Gajjala Prathibha R. , Jaber Mustapha , Wang Xiaoju , He Tongchen , Qiao Yuanyuan , Pang Matthew , Zhang Yuping , Alhusayan Mohammed , Cao Xuhong , Tavana Omid , Hou Caiyun , Wang Zhen , Ding Ke , Chinnaiyan Arul M. , Asangani Irfan A. 
Published: February 2024

AbstractThe androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here, we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 expression is abnormally gained in prostate cancer cells and its functional inhibition impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites, which is greater than 65% of the AR tumor cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and instead contain the canonical palindromic motifs. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in the physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Concordantly, a dual NSD1/2 PROTAC degrader, called LLC0150, was preferentially cytotoxic in AR-dependent prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the ARneo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 as viable paralog co-targets in advanced prostate cancer.