bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence for Stepwise Disruption ofE. coliRNA Polymerase - λPRPromoter Contacts and Bubble Collapse in Transcription Initiation
Authors: Rector Max , Li Renxi , Wang Hao-Che , Record M Thomas 
Published: November 2024

AbstractIn transcription initiation byE. coliRNA polymerase (RNAP), translocation of the RNA-DNA hybrid disrupts RNAP-promoter and σ70-core RNAP contacts, releasing σ70and allowing RNAP to escape. Previously, to investigate whether RNAP-promoter contacts break step-by-step as the hybrid lengthens or concertedly at escape, we determined rate constants and activation energies of nucleotide-incorporation steps involving translocation at the λPRpromoter. Trends in these quantities with hybrid length were inconsistent with concerted models and provided evidence for stepwise disruption of RNAP-promoter contacts and collapse of the upstream bubble (−1 to −11). Here we report kineticm-values quantifying urea and glycine betaine (GB) effects on rate constants of individual nucleotide-incorporation steps and compare withm-values predicted from structural and model compound information. GB is predicted to favor and urea disfavor binding the initiating NTPs and trigger-helix formation in 2-mer (pppApU) synthesis, largely because of burial of phosphate oxygens in NTP binding and amide oxygens in trigger-helix formation. Consistent with these predictions, GB accelerates and urea retards steps synthesizing 3-mer and 4-mer. However, both solutes retard mid-initiation steps (5-mer to 9-mer synthesis) where −10 contacts are proposed to break, exposing DNA phosphates and allowing the upstream initiation bubble to collapse. Strikingly, urea greatly accelerates while GB retards the 11-mer synthesis step, where −35-contacts are proposed to break, σ70is released and RNAP escapes. Urea and GB kineticm-values and activation energies of these steps are inconsistent with concerted models and support a stepwise model of contact disruption and bubble collapse in initiation.
CNV-Profile Regression: A New Approach for Copy Number Variant Association Analysis in Whole Genome Sequencing Data
Authors: Si Yaqin , Lu Wenbin , Holloway Shannon , Wang Hui , Tucci Albert A. , Brucker Amanda , Cheng Yuhuan , Wang Li-San , Schellenberger Gerard , Lee Wan-Ping , Tzeng Jung-Ying 
Published: November 2024

AbstractCopy number variants (CNVs) are DNA gains or losses involving >50 base pairs. Assessing CNV effects on disease risk requires consideration of several factors. First, there are no natural definitions for CNV loci. Second, CNV effects can depend on dosage and length. Third, CNV effects can be more accurately estimated when all CNV events in a genomic region are analyzed together to assess their joint effects. We propose a new framework for association analysis that directly models an individual’s entire CNV profile within a genomic region. This framework represents an individual’s CNVs using a CNV profile curve to capture variations in CNV length and dosage and to bypass the need to predefine CNV loci. CNV effects are estimated at each genome position, making the results comparable across different studies. To jointly estimate the effects of all CNVs, we use a Lasso penalty to select CNVs associated with the trait and integrate a weighted L2-fusion penalty to encourage similar effects of adjacent CNVs when supported by the data. Simulations show that the proposed model can more effectively identify causal CNVs while maintaining false positive rates comparable to baseline methods and yield more precise effect-size estimates across different settings. When applied to CNV derived from whole genome sequencing data of the Alzheimer’s Disease Sequencing Project, the proposed methods identify additional CNVs associated with Alzheimer’s Disease (AD). These identified CNVs overlap with several known AD-risk genes and are significantly enriched by biological processes related to neuron structures and functions crucial in AD development.
Anti Alzheimer’s effects of Neem oil nanoemulsion against Amyloid β1-40induced in human neuroblastoma SH-SY5Y cell line
Authors: Govindaswamy Balaji 
Published: November 2024

AbstractAlzheimer’s disease is characterized by cognitive decline associated with neurodegeneration and accumulation of amyloid beta. Here, experimenting with in vitro models of AD using SHSY5Y cell line after exposing them with Aβ1-40. The natural derivatives like neem oil, plays a potential role in neuroprotection and reduction of tau aggregates in animal models, eventually becoming a alternative treatment beneficiaries in neurodegenerative disease. Formulating the neem oil into a nanoemulsion with Tween 80® and Brij 30™ for stabilization, can be a beneficial drug development for AD. The optimization of this neem oil nanoemulsion was performed with Response Surface Methodology - Box Behnken Design, showed the S mix 10, Sonication power 55%, and Sonication time 15%; as the optimized value to synthesize a stable neem oil nanoemulsion. Secondly, they subjected to thermodynamic stability analysis for estimating the shelf life duration with different temperature conditions. Amyloid beta induced SHSY5Y tend to show severe cell oxidative stress, to estimate that, the neem oil and neem oil nanoemulsion were subjected to DPPH assay. Further, the neem oil and neem oil nanoemulsion analyzed with the Aβ1-40induced SHSY5Y, resulted in good cytotoxicity, where the nanoemulsion exhibited more reduction in cell viability than neem oil. This trend shows that synthesizing the natural oils into nanocarriers can potentially increase the drug release rate, enhanced stability and good biomedical application
Non-viral derivation of induced pluripotent stem cells from the canine umbilical cord
Authors: Yamazaki Atsushi , Shiozawa Seiji , Koushige Yuko , Kondo Hirotaka , Shibuya Hisashi , Edamura Kazuya 
Published: November 2024

AbstractIn our previous study, canine induced pluripotent stem cells (iPSCs) were successfully generated from skin-derived fibroblasts, without the use of viral vectors. However, for clinical application of canine iPSCs in veterinary regenerative medicine, iPSCs generated from less invasive cell sources would be desirable. Therefore, the purpose of this study was to generate iPSCs from canine umbilical cords discarded at fetal birth. Canine umbilical cords were cut into small pieces and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Episomal vectors carrying 10 reprogramming gene sets were introduced into fibroblasts obtained from the umbilical cord using electroporation. When putative iPSCs colonies emerged, constitutive cell characterization was performed to evaluate cell morphology, proliferative potential, alkaline phosphatase staining, expression of stem cell markers, and the ability to differentiate into a trilineage following embryoid body and teratoma formation. Multiple putative iPSC colonies formed when reprogramming gene sets were introduced into fibroblasts obtained from the canine umbilical cord. The resulting colony cells stained positive for alkaline phosphatase, and showed expression of OCT4, SOX2, NANOG, SSEA1, and SSEA3 on fluorescent immunostaining for stem cell markers. Furthermore, the mRNA expression of canine endogenousOCT4,SOX2, andNANOGsignificantly increased, confirming the multi-differentiation potential of cells after embryoid and teratoma formation. In this study, iPSCs were successfully generated from canine umbilical cord without the use of a viral vector. Furthermore, canine umbilical cord-derived iPSCs were successfully cultured in a feeder-free manner. This study contributes to the development of veterinary regenerative medicine by using canine iPSCs.
Comparative analysis of iPSC-derived human kidney organoid and tubuloid models
Authors: Nunez-Nescolarde Ana B. , Santos Leilani L. , Kong Lingyun , Ekinci Elif , Moore Paddy , Dimitriadis Evdokia , Nikolic-Paterson David J. , Combes Alexander N. 
Published: November 2024

ABSTRACTBackgroundEpithelial kidney organoids (tubuloids) made from kidney biopsies, urine, or iPSC-derived kidney organoids offer new opportunities in experimental and clinical nephrology. Yet, we have limited knowledge of how tubuloid models differ from each other, from iPSC-derived kidney organoids and from the human kidney. New insight is required to guide model selection for studies in kidney physiology and disease.MethodsTubuloids were generated from adult nephrectomy samples (adult tubuloids n=3), iPSC-derived kidney organoids (iTubuloids n=3), and for the first time, from human fetal kidneys (fetal tubuloids n=3). Kidney organoid and tubuloid models were compared to each other and to adult human kidney using bulk RNA sequencing. Tubuloids were subject to hypoxic insults over three passages and profiled with RNA sequencing to assess cumulative injury and repair.ResultsExpression signatures of proximal and distal tubules were stronger in adult kidneys than any organoid or tubuloid model. Comparative analysis between the models revealed that iPSC-derived kidney organoids had the highest expression of proximal tubule markers, even though adult tubuloids were derived from mature kidneys. Collecting duct signatures were enriched in adult and fetal tubuloids. Adult tubuloids showed stronger signatures of ageing and inflammation, while fetal tubuloids had enhanced ureteric tip progenitor signatures. Over 80 genes linked to inherited disorders were expressed in all tubuloid cultures, while a further 54 were expressed at higher levels in either adult, fetal or iTubuloids. Tubuloids subject to a single hypoxic injury effectively recovered by the end of the passage, while iTubuloids exposed to hypoxia over three passages expressed markers of maladaptive repair.ConclusionsThis study provides new transcriptome-wide reference data to aid in the selection and optimization of disease modelling for the human kidney. It defines common and unique opportunities to model inherited disorders in adult, fetal and iTubuloid models and illustrates new potential to model repetitive injury in long-lived tubuloid cultures.
Mitochondrial respiratory capacity in kidney podocytes is high, age-dependent, and sexually dimorphic
Authors: Campbell Matthew D. , Sanchez-Contreras Monica , Sibley Britta D. , Keiser Phoebe , Ruiz Sanchez Carla , Mann Carolyn N. , Marcinek David J. , Najafian Behzad , Sweetwyne Mariya T. 
Published: November 2024

ABSTRACTWhether and how podocytes depend on mitochondria across their long post-mitotic lifespan is yet unclear. With limited cell numbers and broad kidney distribution, isolation of podocyte mitochondria typically requires first isolating podocytes themselves. Disassociation of podocytes from their basement membrane, however, recapitulates an injured state that may stress mitochondria. To address this, we crossed floxed hemagglutinin (HA) -mitochondria tagged (MITO-Tag) mice with those expressing Cre in either podocytes (NPHS2) or distal tubule and collecting duct (CDH16), thus allowing for rapid, kidney cell-specific, isolation of mitochondria via immunoprecipitation. Mitochondrial respiration in fresh isolates from young (4-7 mo) and aged (22-26 mo) mice of both sexes demonstrated several previously unreported significant differences between podocyte and tubule mitochondria. First, although podocytes contain fewer mitochondria than do tubule cells, mitochondria isolated from podocytes averaged twice the respiratory capacity of tubule mitochondria when normalized to mitochondrial content by citrate synthase (CS) levels. Second, age-related decline in respiration was detected only in podocyte mitochondria and only in aged male mice. Finally, disassociating podocytes for cell culture initiates functional decline in mitochondria as those from cultured primary podocytes have half the respiratory capacity, but twice the hydrogen peroxide production of podocyte mitochondria isolated directly from fresh kidneys. Thus, podocytes maintain sexually dimorphic mitochondria with greater oxidative phosphorylation capacity than mitochondria-dependent tubules per organelle. Previous studies may not have detected these differences due to reliance on podocyte cell culture conditions, which results in artifactual suppression of mitochondrial function.
The hyaluronan-binding activity of aggrecan is important, but not essential, for its specific insertion into perineuronal nets
Authors: Otsuka Matthew Y. , Essel Leslie B. , Sinha Ashis , Nickerson Gabrielle , Mejia Seth M. , Matthews Russell T. , Bouyain Samuel 
Published: November 2024

ABSTRACTAggrecan (ACAN) is a large, secreted chondroitin sulfate proteoglycan that includes three globular regions named G1, G2, G3, and is decorated with multiple glycosaminoglycan attachments between its G2 and G3 domains. The N-terminal G1 region interacts with the glycosaminoglycan hyaluronan (HA), which is an essential component of the vertebrate extracellular matrix. In the central nervous system, ACAN is found in perineuronal nets (PNNs), honeycomb-like structures that are enriched on parvalbumin-positive neurons in specific neural circuits. PNNs regulate the plasticity of the central nervous system, and it is believed that association between ACAN and HA is a foundational event in the assembly of these reticular structures. Here, we report the co-crystal structure of the G1 region of ACAN in the absence and presence of an HA decasaccharide and analyze the importance of the HA-binding activity of ACAN for its integration into PNNs. We demonstrate that the single immunoglobulin domain and the two Link modules that comprise the G1 region form a single structural unit, and that HA is clamped inside a groove that spans the length of the tandem Link domains. Introduction of point mutations in the glycosaminoglycan-binding site eliminates HA-binding activity in ACAN, but, surprisingly, only decreases the integration of ACAN into PNNs. Thus, these results suggest that the HA-binding activity of ACAN is important for its recruitment to PNNs, but it does not appear to be essential.
Anatomical Region-Specific Transcriptomic Signatures and the Role of Epithelial Cells in Pterygium Inflammation: A Multi-Omics Analysis
Authors: Song Chaerim , Myung Seokho , Cho Hanseul , Kim Tae Gi , Chun Soohyun , Seo Minju , Yu Hyunmin , Kim Seoyeon , Kim Ye-Ah , Kim Junghyun , Shin Jaeyong , Kang Jaheon , Lee Yoonsung , Kang Min Seok , Kim Man S 
Published: November 2024

ABSTRACTBackgroundThe anatomy of pterygium consists of the head, neck, and body. Prior studies have examined the three parts collectively, yet the treatment methods and their success rates differ based on the anatomical characteristics. In this study, we divide the pterygium tissue into Main (head and neck) and Acc (body) to investigate its pathogenesis across regions.PurposesThis study aims to understand the pathogenesis and identify potential therapeutic targets of pterygium. Through a robust multi-omics analysis of bulk and single-cell RNA sequencing (RNA-seq) data from pterygium patients, we focus on the expression of inflammatory and mitochondrial energy pathways and identify potential genes responsible for the upregulated pathways in pterygium.MethodsWe collected bulk RNA-seq data from six pterygium patients and single-cell RNA-seq data from two pterygium patients. We then investigated the pathway enrichment, pathway correlation, differential gene expressions, protein-protein interactions, and cell-cell communications of pterygium.ResultsFrom the analysis of bulk RNA-seq data, the distribution of sample points from the principal component analysis plot and the pathways enriched from the Gene Ontology analysis showed distinct expression patterns in the Acc group compared to the Main and control groups. This suggested the need to separate the Main and Acc regions within pterygium samples and utilize single-cell RNA-seq data to understand the differences between the Main and control groups that the bulk data could not capture. The annotation of integrated single-cell data revealed a cluster of epithelial cells containing only pterygium samples. Cells from this cluster exhibited significant contributions to the ANGPTL, IL1, and KLK signaling networks in the cell-cell communication analysis. We also observed significant upregulation of the cells in the inflammatory pathways related to integrated stress response and the renin-angiotensin-aldosterone system, both of which showed high correlations with energy metabolism pathways. Significant changes in the expression of multiple pro-inflammatory, antioxidant, and immune-related genes were also identified.ConclusionThe different expression patterns between the Main and Acc groups suggest the need to consider different anatomical regions separately in future studies of pterygium. Additionally, the significant role epithelial cells from the Main group play in the inflammation of pterygium presents a potential clinical approach to the disease.
Old age variably impacts chimpanzee engagement and efficiency in stone tool use
Authors: Howard-Spink E , Matsuzawa T , Carvalho S , Hobaiter C , Almeida-Warren K , Gruber T , Biro D 
Published: November 2024

AbstractWe know vanishingly little about how long-lived apes experience senescence in the wild, particularly with respect to their foraging behaviors, which are essential for survival. Some wild apes use tools during foraging and, given the additional cognitive and physical challenges presented by tool use, we predict that such behaviors are at a heightened risk of senescence. However, until the present, longitudinal analysis of the effects of progressive aging on wild ape tool-use behaviors has not been possible due to a lack of available data. In response to this research gap, we sampled data from a longitudinal video archive that contained footage of wild chimpanzees engaging in one of their most complex forms of tool use - the cracking of hard-shelled nuts with hammers and anvil stones, termednut cracking- at an ‘outdoor laboratory’ at Bossou, Guinea. By sampling data over a 17-year period, we describe how progressive old age influences the extent to which wild chimpanzees engage with – and efficiently perform – nut cracking. With increasingly old age, chimpanzees began attending experimental nut cracking sites less frequently than younger individuals. Several elderly chimpanzees exhibited reductions in efficiency across multiple components of nut cracking, including taking more time to select stone tools prior to use, and taking longer to crack open nuts and consume the associated pieces of kernel. Two chimpanzees also began using less streamlined behavioral sequences to crack nuts, including a greater number of actions (such as more numerous strikes of the hammer stone). Most notably, we report interindividual variability in the extent to which tool-use behaviors senesced during aging – ranging from small to profound reductions in tool engagement and efficiency – as well as differences in the specific aspects of nut cracking behaviors which senesced for each individual. We discuss the possible causes of these changes with reference to research into senescence in captive primates, and provide future directions for research of primate aging in both captive and wild settings.
Timing of delamination of inner ear neurons specifies their topography and target innervation
Authors: Saini Surjit Singh , Ladher Raj K. 
Published: November 2024

ABSTRACTThe neurons of the inner ear delaminate from a sensorineurogenic epithelium in the ventral part of the otocyst. Delaminated neuroblasts then condense to form the acoustico-vestibular ganglion (AVG). As they differentiate, the neurons connect mechanosensory hair cells (HCs) of the inner ear with their targets in the hindbrain in a precise topographical manner. However, it is unclear how or when positional identities of neurons are specified within the acoustico- vestibular ganglion (AVG), such that topographical information from HCs is maintained into the auditory centres of the brain. Here we find that the time of delamination from the otocyst correlates with neuroblast position in the ganglion. Using markers for neuronal differentiation, we find that the ganglion differentiates from a proximal to distal wave. Neurons that differentiate first innervate the vestibular apparatus, including the lagena and the proximal regions of the BP. Using sequential somatic cell labelling, we find that the central projection also follows a similar dependency on delamination order. Our studies show that the time of delamination of otic neuroblasts presage their target innervation choice and fibre positions within the developing eighth cranial nerve. We suggest that temporal information specifies the spatial identities during early inner ear neuron development.
Engineering de novo binder CAR-T cell therapies with generative AI
Authors: Mergen Markus , Abele Daniela , Koleci Naile , Schmahl Fernandez Alba , Sugden Maya , Holzleitner Noah , Carr Andreas , Rieger Leonie , Leone Valentina , Reichert Maximilian , Laugwitz Karl-Ludwig , Bassermann Florian , Busch Dirk H. , Grünewald Julian , Schmidts Andrea 
Published: November 2024

AbstractChimeric antigen receptor T cell (CAR-T) therapies have revolutionized cancer treatment, with six CAR-T products currently in clinical use1–4. Despite their success, high resistance rates due to antigen escape remain a major challenge5,6. In silico design of de novo binders (DNBs) has the potential to accelerate the development of new binding domains for CAR-T, possibly enabling personalized therapies for cancer resistance7,8. Here, we show that DNBs can be used for CAR-T, targeting clinically relevant cancer antigens. Using a DNB against the epidermal growth factor receptor (EGFR), we demonstrate comparable cytotoxicity, cytokine secretion, long-term proliferation, and lysis of primary patient-derived cancer organoids with single-chain variable fragment (scFv)-based and DNB-based CAR-T cells. Moreover, we use generative artificial intelligence (AI) guided binder design with RFdiffusion9to target the B cell maturation antigen (BCMA), a key antigen in multiple myeloma treatment10–17. We confirmed the activity of our AI-designed BCMA CAR-T in short- and long-term effector readouts, including a xenograft mouse model of multiple myeloma. Notably, our AI-guided CAR-T approach also successfully targets a mutated BCMA protein variant resistant to the clinically used bispecific antibody teclistamab. In sum, we demonstrate a proof-of-concept for engineering new, bespoke cellular immunotherapies targeting cancer resistance with the help of generative AI. This approach may further accelerate the development of new CAR-T therapies addressing cancer resistance.