bluuebunny/crossref_metadata_2025_split huggingface dataset:
R-Loop control and mitochondria genome stability require the 5’-3’ exonuclease/flap endonuclease OEX1
Authors: Schatz-Daas Déborah , Le Blevenec Anaïs , Moratti Fabio G. , Chung Kin Pan , Mercier Pierre , Iqbal Rana Khalid , Vallet Elody , Dietrich André , Bock Ralph , Weber-Lotfi Frédérique , Gualberto José M. 
Published: November 2024

AbstractMaintenance of the plant organelle genomes involves factors mostly inherited from their bacterial symbiotic ancestors. In bacteria, a major player in genome maintenance is DNA Polymerase I (Pol I), which provides a 5’-3’-exonuclease/flap-endonuclease activity required for multiple replication and repair functions. In plant organelles, DNA polymerases POL1A and POL1B are evolutionarily derived from DNA Pol I but lack this domain. In Arabidopsis, OEX1 and OEX2 (Organellar Exonucleases 1 and 2) represent this missing domain and are targeted to mitochondria and chloroplasts, respectively. Anoex1mutant allele shows developmental and fertility defects that correlate with the differential segregation of mitochondrial DNA (mtDNA) subgenomes generated by recombination, suggesting that OEX1 processes replication and recombination intermediates whose accumulation results in genome instability. Alternative splicing generates two OEX1 isoforms that can differentially interact with POL1A and POL1B and variably affect mtDNA repair.Recombinant OEX1 has 5’-3’-exonuclease and flap endonuclease activities, the latter being a key function in replication and repair. Furthermore, OEX1 has high affinity for RNA:DNA hybrids, rapidly degrading RNA in Okazaki-like structures and R-loops. Consistent with a role in suppressing R-loops,oex1plants accumulate R-loops in highly transcribed mtDNA regions. Taken together, our results show that OEX1 plays multiple important roles in the processes required to maintain mtDNA stability.
Characterizing the effect of impeller design in plant cell fermentations using CFD modeling
Authors: Manickavasagam Vidya Muthulakshmi , Anupindi Kameswararao , Bhatt Nirav , Srivastava Smita 
Published: November 2024

AbstractCultivation of plant cell cultures in conventional bioreactors designed for microbial cells often results in the decrease of biomass productivity as compared to that in shake flasks, presumably due to the imbalance between the mass transfer requirements and compromise with cell viability. Hit and trial methods are usually adapted to achieve high biomass productivity in the bioreactor. In this study, a rational approach has been developed to choose a suitable impeller forViola odoratacell suspension culture using computational fluid dynamics (CFD). A two-phase CFD model was employed to characterize the non-Newtonian fluid dynamics in a stirred tank reactor using different impeller designs, a setric, Rushton and marine impeller. The simulations were performed adopting Euler-Euler approach for the two-phase flow and dispersedκ− ε turbulence model. The numerical model was validated with good agreement with experimental determination of volumetric mass transfer coefficient. The impact of impeller design was then investigated on critical process parameters like mixing, oxygen mass transfer and shear. The developed CFD model demonstrated that setric impeller is a suitable choice forV. odoratacell cultivation offering low-shear environment at equivalent velocity magnitudes at reactor bottom with higher cell-lift capabilities which is preferable in high cell-density plant cell cultivations.
Essential role for HSP40 in asexual replication and thermotolerance of malaria parasites
Authors: Roper Brianne , Kannan Deepika , Mathews Emily S. , John Audrey R. Odom 
Published: November 2024

AbstractPlasmodium falciparum,the parasite responsible for nearly all cases of severe malaria, must survive challenging environments to persist in its human host. Symptomatic malaria is characterized by periodic fevers corresponding to the 48-hour asexual reproduction ofP. falciparumin red blood cells. As a result,P. falciparumhas evolved a diverse collection of heat shock proteins to mitigate the stresses induced by temperature shifts. Among the assortment of heat shock proteins inP. falciparum, there is only one predicted canonical cytosolic J domain protein, HSP40 (PF3D7_1437900). Here, we generate a HSP40 tunable knockdown strain ofP. falciparumto investigate the biological function of HSP40 during the intraerythrocytic lifecycle. We determine that HSP40 is required for malaria parasite asexual replication and survival of febrile temperatures. Previous reports have connected proteotoxic and thermal stress responses in malaria parasites. However, we find HSP40 has a specific role in heat shock survival and does not mitigate the proteotoxic stresses induced by artemisinin or proteosome inhibition. Following HSP40 knockdown, malaria parasites have a cell cycle progression defect and reduced nuclear replication. Untargeted proteomics reveal HSP40 depletion leads to a multifaceted downregulation of DNA replication and repair pathways. Additionally, we find HSP40 knockdown sensitizes parasites to DNA replication inhibition. Overall, these studies define the specialized role of the J domain protein HSP40 in malaria parasites during the blood stages of infection.Author SummaryMalaria parasites have evolved to survive and cause disease in humans even when they are challenged by the temperature shifts of fever. We are interested in uncovering how malaria parasites persist despite exposure to febrile temperatures. We know the parasite has a diverse collection of heat shock proteins that are important for proper folding of many proteins within cells during stress conditions. In this study, we define the role of one specific heat shock protein, HSP40, by generating a strain of the human malaria parasiteP. falciparumwhere we control the expression of HSP40 during the red blood cell stages of infection. We find that HSP40 is required for malaria parasites to replicate in red blood cells. We demonstrate HSP40 expression to be vital for malaria parasite survival of febrile temperature stress. Additionally, we determine that without HSP40, DNA replication and repair is disrupted. Our work has uncovered essential parasite biology that may be exploited for the development of new antimalarials.
A green lifetime biosensor for calcium that remains bright over its full dynamic range
Authors: van der Linden Franka H. , Thornquist Stephen C. , Beek Rick M. ter , Huijts Jelle Y. , Hink Mark A. , Gadella Theodorus W.J. , Maimon Gaby , Goedhart Joachim 
Published: November 2024

AbstractFluorescent biosensors toggle between two states and for the vast majority of biosensors one state is bright and the other state is dim. As a consequence, there is a substantial difference in the signal-to-noise ratio (SNR) for the two states. The dim state has a low signal-to-noise ratio, which is problematic when precise, quantitative measurements are needed. During the engineering of a red-shifted variant of an mTurquoise-based calcium sensor, we serendipitously generated a green-emitting sensor that shows high brightness in both the calcium-bound and -unbound state, while still showing a calcium dependent lifetime change of >1 nanosecond. This sensor, named G-Ca-FLITS, is comparable in brightness to the bright state of GCaMP3 and jGCaMP7c in mammalian cells. The calcium induced loss in fluorescence intensity is only around 30% and therefore we observe little variation in the SNR when calcium levels change. G-Ca-FLITS shows negligible sensitivity to pH in the physiological range, like its turquoise parent. Using fluorescence lifetime imaging (FLIM), we measured the calcium concentration with G-Ca-FLITS in various organelles and observed in HeLa cells transient and spatially heterogeneous calcium elevations in mitochondria. Finally, we evaluated the use of G-Ca-FLITS and its turquoise predecessor for two-photon FLIM inDrosophilabrains.Abstract Figure
CD3, CD4, and CD8 staining, but not PD-L1, are positively correlated with overall survival in Asian breast cancer
Authors: Pan Jia-Wern , Makmur Haslina , Tai Ju-Yi , Ng Pei-Sze , Leong Herman Xin Yang , Hasan Siti Norhidayu , Yip Cheng-Har , Rajadurai Pathmanathan , Teo Soo-Hwang 
Published: November 2024

AbstractGiven the fundamental importance of T lymphocytes in the effectiveness of cancer immunotherapy, and potentially in the response to chemotherapy and radiotherapy, immune cell markers have been evaluated as predictive and prognostic biomarkers, with conflicting results in the different subtypes of breast cancer and in different studies. In this study, we report the association of T lymphocyte markers with overall survival in a cross-sectional cohort of Asian breast cancer patients. Formalin-fixed, paraffin-embedded tumour samples from 576 breast cancer patients from a Malaysian hospital were stained with CD3, CD4, CD8 and PD-L1 antibodies, and area-based positive staining was quantified from digitized whole slide images. At a median follow up of 68 months, we report that CD3, CD4 and CD8 scores, but not PDL1 scores, were associated with overall survival, with the strongest associations seen in triple-negative breast cancer (TNBC). CD3, CD4, and CD8 scores were also compared to genomic and transcriptomic data from previous studies, and found to have a negative association with copy number aberrations, but no association with tumour mutational burden or neoantigen load. Taken together, T lymphocyte markers may be associated with good prognosis in Asian breast cancer patients.
Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions between Non-Coding RNAs in Cancer Cells
Authors: Schloßhauer Jeffrey L. , Shamloo Sama , Schamrin Katja , Imig Jochen 
Published: November 2024

ABSTRACTLong non-coding RNAs (lncRNAs) represent a vast and functionally diverse class of RNA molecules, with over 100,000 predicted in the human genome. Although lncRNAs are less conserved across species compared to protein-coding genes, they play critical roles in gene regulation, chromatin interactions, and cancer progression. Their involvement in cancer make them promising therapeutic targets. CRISPR interference (CRISPRi), utilizing catalytically inactive Cas9 fused with a transcriptional repressor such as KRAB-MeCP2, offers a precise method for targeting nuclear lncRNAs and assessing their functions. This study introduces a dual CRISPRi system using orthogonal CRISPRi technologies fromStaphylococcus aureusandStreptococcus pyogenesdCas9-KRAB, optimized for combinatorial targeting of lncRNAs in human melanoma cells. The protocol facilitates combinatorial gene knockdown or synthetic lethal screening of lncRNA pairs, providing a novel tool for cancer research. By exploring synthetic lethality between lncRNAs, this approach can help identify lncRNA interactions critical for cancer cell survival, offering new therapeutic strategies. The dual system’s functionality is demonstrated, highlighting its potential in identifying critical cancer-specific lncRNA interactions.SUMMARYThis study presents a dual CRISPRi system targeting long non-coding RNAs in melanoma cells. It enables combinatorial gene knockdown and synthetic lethal screening, identifying cancerspecific lncRNA interactions for potential therapeutic strategies.
Predicting Tumor Mutational Burden from UHF-Dielectrophoresis Crossover Frequency
Authors: Daverat Héloïse , Blasco Nina , Robert Sandrine , Pothier Arnaud , Rovini Amandine , Boutaud Marie , Jemfer Charlotte , Dalmay Claire , Lalloué Fabrice , Durand Karine , Naves Thomas 
Published: November 2024

ABSTRACTTumor Mutational Burden (TMB) has emerged as a crucial biomarker to guide patient eligibility for immunotherapy. However, whole exome sequencing, the gold-standard method for TMB measurement, remains limited in accessibility due to its high costs, operational complexity, and lengthy processing times. To address these limitations, we investigated whether Ultra-High-Frequency (UHF) technology could serve as a novel approach to assess TMB by analyzing the crossover frequencies or electromagnetic signature (EMS) of cancer cells on a lab-on-a-chip biosensor, integrating microfluidics and dielectrophoresis.In a panel of 12 cancer cell lines with varying TMB levels, we observed that EMS showed an upward shift correlating with higher TMB, particularly in solid tumor cell lines. This finding suggests a potential relationship between TMB and EMS. To further explore this hypothesis, we artificially increased mutation levels by treating cells with the highly mutagenic compound N-ethyl-N-nitrosourea (ENU). Results showed that EMS captured significant TMB variations in ENU-treated cells with enhanced proliferative capacity compared to their parental counterparts. These results underscore the importance of matched control samples for reliable EMS measurements. Altogether, our findings highlight the potential of EMS to detect TMB variations associated with proliferative activity, a key hallmark of cancer cells, thereby enabling a more precise stratification of cancer cells.HighlightsWe propose a new biosensor to improve patient stratification for ICI eligibilityHigh frequency fields and dielectric spectroscopy can estimate TMB in cancer cellsSignificant changes in UHF-DEP signatures correlate with varying TMB levelsUHF-DEP provides a cheap, rapid and label-free method to predict ICI responseOffers an innovative and complementary marker to conventional diagnostic approachesGraphical abstract
Chemogenetic manipulation of the ventral pallidum-nucleus accumbens shell pathway modulates sucrose consumption independent of motivation in female rats
Authors: Peroutka Markie , Rivero Covelo Ignacio 
Published: November 2024

AbstractHere we investigated the role of the ventral pallidum (VP) to nucleus accumbens shell (AcbSh) pathway in mediating sucrose consumption and motivation for food in female rats. Using chemogenetic techniques, excitatory and inhibitory designer receptors were expressed in VP neurons projecting to the AcbSh. Behavioral assays assessed both lever pressing under a progressive ratio schedule and free sucrose consumption following activation or inhibition of this pathway. Results showed that inhibition of VP-AcbSh projections increased sucrose intake, while excitation reduced it, without affecting motivation to work for food. These findings indicate a specific influence of the VP-AcbSh pathway on sucrose consumption independent of food-seeking motivation. The study highlights the importance of differentiating VP efferents in understanding distinct roles in feeding behaviors and suggests that the VP-AcbSh pathway modulates consumption based on food type and potentially other variables.
Vcfexpress: flexible, rapid user-expressions to filter and format VCFs
Authors: Pedersen Brent S. , Quinlan Aaron R. 
Published: November 2024

AbstractMotivationVariant Call Format (VCF) files are the standard output format for various software tools that identify genetic variation from DNA sequencing experiments. Downstream analyses require the ability to query, filter, and modify them simply and efficiently. Several tools are available to perform these operations from the command line, including BCFTools, vembrane, slivar, and others.ResultsHere, we introduce vcfexpress, a new, high-performance toolset for the analysis of VCF files, written in the Rust programming language. It is nearly as fast as BCFTools, but adds functionality to execute user expressions in the lua programming language for precise filtering and reporting of variants from a VCF or BCF file. We demonstrate performance and flexibility by comparing vcfexpress to other tools using the vembrane benchmark.Availabilityvcfexpress is available under the MIT license athttps://github.com/brentp/vcfexpressContactbpederse@gmail.com,aquinlan@genetics.utah.eduSupplementary informationSupplementary data are available atBioinformaticsonline.
Hydrophobins fromAspergillusmediate fungal interactions with microplastics
Authors: Klauer Ross R. , Silvestri Rachel , White Hanna , Hayes Richard D. , Riley Robert , Lipzen Anna , Barry Kerrie , Grigoriev Igor V. , Talag Jayson , Bunting Victoria , Stevenson Zachary , Solomon Kevin V. , Blenner Mark 
Published: November 2024

AbstractMicroplastics present myriad ecological and human health risks including serving as a vector for pathogens in human and animal food chains. However, the specific mechanisms by which pathogenic fungi colonize these microplastics have yet to be explored. In this work, we examine the opportunistic fungal pathogen,Aspergillus fumigatus, and other common soil and marineAspergilli, which we found bind microplastics tightly. Up to 3.85+/-1.48 g microplastic plastic/g fungi were bound and flocculated for polypropylene (PP), polyethylene (PE), and polyethylene terephthalate (PET) powders and particles ranging in size from 0.05 – 5 mm. Gene knockouts revealed hydrophobins as a key biomolecule driving microplastic-fungi binding. Moreover, purified hydrophobins were still able to flocculate microplastics independent of the fungus. Our work elucidates a role for hydrophobins in fungal colonization of microplastics and highlights a potential target for mitigating the harm of microplastics through engineered fungal-microplastic interactions.Abstract FigureSignificance StatementMicroplastics pose serious ecological and human health effects by introducing pathogens and toxins into animal and human food chains. Many pathogenic microorganisms preferentially form biofilms on microplastic particles that are then ingested. Here, we demonstrate that hydrophobins, highly hydrophobic, cell surface proteins, enable microplastic binding and colonization by the opportunistic pathogenAspergillus fumigatusand other fungi within theAspergillusgenus. Our work recognizes a novel role for hydrophobin proteins, identifying potential strategies for pathogen control and protein-based microplastics recovery.
Comparative RT-qPCR and qPCR reveals early infection, low-titer infection, and relative cell activity of the HLB bacterium,CandidatusLiberibacter asiaticus
Authors: Patterson Rachel , Zheng Desen , Luo Weiqi , Deng Zhanao , Bock Clive , Li Ruhui , Duan Yongping 
Published: November 2024

AbstractCandidatusLiberibacter asiaticus (Las) is one of the causal agents of citrus huanglongbing (HLB) epidemics worldwide. Due to its fastidious nature, intracellular and systemic infection, detecting Las at early and/or low-titer infection, as well as differentiating between live or dead cells in the host psyllids and citrus plants is critical for effective HLB management. To achieve both sensitive Las detection and differentiation, we employed one-step reverse transcription-quantitative PCR (RT-qPCR) using total nucleic acids as template. This method allows use of both Las 16S rRNA and rDNA as template in the same reaction and increases detection sensitivity by up to 1000-fold in comparison with quantitative PCR (qPCR). The increased sensitivity significantly reduces false negative detection and detects the otherwise undetectable low-titer Las infections. Furthermore, the greater the abundance of 16S rRNA present in the samples, the bigger the Ct gap obtained between RT-qPCR and qPCR results. The numerical Ct gap can be used to deduce relative Las cellular activity and indirectly infer whether cells are alive or dead. In addition, this comparative detection method also can be used to select inoculum and monitor relative cell activity duringin vitroLas culture and evaluate the effectiveness of antimicrobial treatments against Las bacteria.
Molecular impact of nicotine and smoking exposure on the developing and adult mouse brain
Authors: Gonzalez-Padilla Daianna , Eagles Nicholas J. , Cano Marisol , Pertea Geo , Jaffe Andrew E. , Maynard Kristen R. , Hancock Dana B. , Handa James T. , Martinowich Keri , Collado-Torres Leonardo 
Published: November 2024

ABSTRACTMaternal smoking during pregnancy (MSDP) is associated with significant cognitive and behavioral effects on offspring. While neurodevelopmental outcomes have been studied for prenatal exposure to nicotine, the main psychoactive component of cigarette smoke, its contribution to MSDP effects has never been explored. Comparing the effects of these substances on molecular signaling in the prenatal and adult brain may provide insights into nicotinic and broader tobacco consequences that are developmental-stage specific or age-independent. Pregnant mice were administered nicotine or exposed to chronic cigarette smoke, and RNA-sequencing was performed on frontal cortices of postnatal day 0 pups born to these mice, as well as on frontal cortices and blood of the adult dams. We identified 1,010 and 4,165 differentially expressed genes (DEGs) in nicotine and smoking-exposed pup brains, respectively (FDR<0.05, Ns = 19 nicotine-exposed vs 23 vehicle-exposed; 46 smoking-exposed vs 49 controls). Prenatal nicotine exposure (PNE) alone was related to dopaminergic synapses and long-term synaptic depression, whereas MSDP was associated with the SNARE complex and vesicle transport. Both substances affected SMN-Sm protein complexes and postsynaptic endosomes. Analyses at the transcript, exon, and exon-exon junction levels supported gene level results and revealed additional smoking-affected processes. No DEGs at FDR<0.05 were found in adult mouse brain for any substance (12 nicotine-administered vs 11 vehicle-administered; 12 smoking-exposed vs 12 controls), nor in adult blood (12 smoking-exposed vs 12 controls), and only 3% and 6.41% of the DEGs in smoking-exposed pup brain replicated in smoking-exposed blood and human prenatal brain, respectively. Together, these results demonstrate variable but overlapping molecular effects of PNE and MSDP on the developing brain, and attenuated effects of both smoking and nicotine on adult versus fetal brain.
Direct observation of secondary nucleation in huntingtin amyloid formation by High-Speed Atomic Force Microscopy
Authors: van Ewijk C. , Jain G. , Knelissen Y. K. , Maity S. , van der Wel P.C.A. , Roos W.H. 
Published: November 2024

AbstractAmyloid fibril formation is a hallmark of various neurodegenerative diseases such as Huntington’s (HD), Alzheimer’s and Parkinson’s disease. The protein aggregation process involves slow nucleation events followed by rapid growth and elongation of formed fibrils. Understanding the pathways of amyloid formation is key to development of novel therapeutic agents that can interfere with the pathogenic protein misfolding events. Recent studies of aggregation by polypeptides from Alzheimer’s and Huntington’s disease have identified the importance of a poorly understood secondary nucleation process that may even be the dominant source of protein aggregate formation. Here we focus on the polyglutamine-expansion disorder HD and employ mechanistic and structural studies to study different aspects of secondary nucleation in the aggregation of huntingtin Exon 1 (HttEx1). Notably, we apply high-speed atomic force microscopy (HS-AFM) to directly observe the process on the single-particle level and in real time. Our observations show unique features of the amyloid formation dynamics in real time, including secondary nucleation, elongation and the formation of large bundles of fibrils as a result of nucleated branching. We examine the role of HttEx1 flanking segments during the aggregation process, revealing that the N-terminal HttNTsegment exhibits a clear primary nucleation-aggregation-enhancing ability, however, that it does not seem to induce or affect the secondary nucleation process. The obtained results illuminate the complex aggregation process of HttEx1 and have implications for attempts to inhibit or modulate it for therapeutic purposes.