bluuebunny/crossref_metadata_2025_split huggingface dataset:
Forskolin-induced swelling of intestinal organoids predicts long-term cystic fibrosis disease progression
Authors: Muilwijk D. , de Poel E. , van Mourik P. , Suen S.W.F. , Vonk A.M. , Brunsveld J.E. , Kruisselbrink E. , Oppelaar H. , Hagemeijer M.C. , Berkers G. , de Winter-de Groot K.M. , Heida-Michel S. , Jans S.R. , van Panhuis H. , van der Eerden M.M. , van der Meer R. , Roukema J. , Dompeling E. , Weersink E.J.M. , Koppelman G.H. , Vries R. , Zomer-van Ommen D.D. , Eijkemans M.J.C. , van der Ent C.K. , Beekman J.M. 
Published: February 2021

ABSTRACTPatient-derived organoids hold great potential as predictive biomarker for disease expression or therapeutic response. Here, we used intestinal organoids to estimate individual cystic fibrosis transmembrane conductance regulator (CFTR) function of people with cystic fibrosis, a monogenic life-shortening disease associated with more than 2000 CFTR mutations and highly variable disease progression. In vitro CFTR function in CF intestinal organoids of 176 individuals with diverse CFTR mutations was quantified by forskolin induced swelling and was strongly associated with longitudinal changes of lung function and development of pancreatic insufficiency, CF-related liver disease and diabetes. This association was not observed when the commonly used biomarker of CFTR function sweat chloride concentration was used. The data strongly exemplifies the value of an organoid-based biomarker in a clinical disease setting and supports the prognostic value of forskolin induced swelling of intestinal organoids, especially for people with CF who have rare CFTR genotypes with unclear clinical consequences.
Discovery of 17 conserved structural RNAs in fungi
Authors: Gao William , Jones Thomas A. , Rivas Elena 
Published: February 2021

AbstractMany non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes(Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus,andSchizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include 4 H/ACA box small nucleolar RNAs, 4 intergenic RNAs, and 9 RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3′ UTRs of the metabolic genesGLY1andMET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.
Machine Learning Differentiates Enzymatic and Non-Enzymatic Metals in Proteins
Authors: Feehan Ryan , Franklin Meghan W. , Slusky Joanna S.G. 
Published: February 2021

AbstractMetalloenzymes are 40% of all enzymes and can perform all seven classes of enzyme reactions. Because of the physicochemical similarities between the active sites of metalloenzymes and inactive metal binding sites, it is challenging to differentiate between them. Yet distinguishing these two classes is critical for the identification of both native and designed enzymes. Because of similarities between these two types of metal binding sites, finding physicochemical features that distinguish active and inactive metal sites can indicate aspects that are critical to enzyme function. In this work, we develop the largest structural dataset of enzymatic and non-enzymatic metalloprotein sites to date. We then use a decision-tree ensemble machine learning model to classify metals bound to proteins as enzymatic or non-enzymatic with 92.2% precision and 90.1% recall. Our model scores electrostatic and pocket lining features as more important than pocket volume, despite the fact that volume is the most quantitatively different feature between enzyme and non-enzymatic sites. Finally, we find our model has overall better performance in a side-to-side comparison against other methods that differentiate enzymatic from non-enzymatic sequences. We anticipate that our model’s ability to correctly identify which metal sites are responsible for enzymatic activity could enable identification of new enzymatic mechanisms andde novoenzyme design.
Potential Early Rabies Infection Detected in Two Raccoon Cases by LN34 pan-lyssavirus real-time RT-PCR Assay in Pennsylvania
Authors: Dettinger Lisa , Gigante Crystal M. , Strohecker Maria , Seiders Melanie , Patel Puja , Orciari Lillian A. , Yager Pamela , Lute James , Regec Annette , Li Yu , Xia Dongxiang 
Published: February 2021

AbstractDuring 2017 – 2019, the Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6,855 animal samples for rabies using both the gold standard direct fluorescent antibody (DFA) test and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR). Two samples (0.03 %) were identified as LN34 RT-qPCR positive after failure to detect rabies virus antigen during initial DFA testing: an adult raccoon collected in 2017 and a juvenile raccoon collected in 2019. After the positive PCR result, additional tissues were collected and re-tested by DFA, where very sparse, disperse antigen was observed. Tissues from both animals were submitted to the Centers for Disease Control and Prevention (CDC) for confirmatory testing, and were confirmed positive. At both PABOL and CDC, rabies virus antigen and RNA levels were much lower than for a typical rabies case. In addition, rabies virus antigen and RNA levels were higher in brain stem and rostral spinal cord than cerebellum, hippocampus and cortex. Cross-contamination was ruled out in the case of the 2019 juvenile raccoon by sequencing, as nucleoprotein and glycoprotein gene sequences displayed >1% nucleotide differences to sequences from all positive samples processed at PABOL within two weeks of the juvenile raccoon. Taken together, the low level of rabies virus in the central nervous system combined with presence in more caudal brain structures suggest the possibility of an early infection in both cases. These two cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR in rabies diagnostics for the identification of low positive cases.
HeterozygousKCNH2variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology
Authors: Ng Chai-Ann , Farr Jessica , Young Paul , Windley Monique J. , Perry Matthew D. , Hill Adam P , Vandenberg Jamie I 
Published: February 2021

AbstractKCNH2is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reportedKCNH2variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of anyKCNH2missense variant. The key components of the assay include quick and cost-effective generation of a bicistronic vector to co-express WT and anyKCNH2variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch-clamp electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 hours of channel expression. This high throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of anyKCNH2variant.
Synergistic Therapy of Doxorubicin with Cationic Anticancer Peptide L-K6 Reverses Multidrug Resistance in Cancer Cells in vitro via P-glycoprotein Inhibition
Authors: Wang Che , Huang Lili , Li Ruojin , Wang Ying , Wu Xiaoxue , Shang Dejing 
Published: February 2021

AbstractMultidrug resistance (MDR) is one of the major obstacles to efficient chemotherapy against cancers, resulting from the overexpression of drug efflux transporters such as P-glycoprotein (P-gP). In the present study, we aimed to evaluate the MDR reversal activity and synergistic therapeutic potential of cationic anticancer peptide L-K6 with doxorubicin (DOX) on P-gP-overexpressing and DOX-resistant MCF-7/Adr human breast cancer cells. Flow cytometry and confocal laser scanning microscopy were used to determine the intracellular accumulation of DOX and another P-gP substrate, Rho123. P-gP-Glo assay, Western blot and Biacore analysis were further performed to evaluate the P-gP function and expression. The cytotoxicity in MCF-7 or MCF-7/Adr cells was measured by MTT assay. Flow cytometry assay and confocal laser scanning microscopy observation clearly revealed an increased intracellular accumulation of DOX and Rho123 in MCF-7/Adr cells treated with L-K6, suggesting a P-gP inhibiting potential. Biacore analysis, P-gP-Glo assay and Western blot further confirmed that L-K6 could directly interact with P-gP, inhibit P-gP function and decrease P-gP expression in MCF-7/Adr cells. In addition, as expected, the data from MTT assay indicated that L-K6 restored the sensitivity of MCF-7/Adr cells to DOX, indicating a MDR reversal potential and a promising synergistic anticancer activity. All these findings may provide experimental evidence to support the promising applications and synergistic therapeutic potential of peptidic P-gP inhibitors against MDR cancer.
Functionalized extracellular matrix scaffolds loaded with endothelial progenitor cells promote neovascularization and diabetic wound healing
Authors: He Siqi , Walimbe Tanaya , Chen Hongyuan , Gao Kewa , Kumar Priyadarsini , Wei Yifan , Hao Dake , Liu Ruiwu , Farmer Diana L , S.Lam Kit , Zhou Jianda , Panitch Alyssa , Wang Aijun 
Published: February 2021

AbstractDiabetic ischemic wound treatment remains a critical clinical challenge. Strategies that enhance angiogenesis and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic condition. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates VEGF receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a pro-angiogenic molecule LXW7-DS-SILY by conjugating LXW7 to a collagen-binding proteoglycan mimetic DS-SILY and further employed this novel bifunctional ligand to functionalize extracellular matrix (ECM) scaffolds, promote neovascularization and accelerate ischemic wound healing. We established a Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model and found the wounds treated by LXW7-DS-SILY-functionalized ECM scaffolds, with or without EPCs, significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis. These functionalized ECM scaffolds also significantly promoted EPC attachment, growth and survival in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.
Computational insights into differential interaction of mamalian ACE2 with the SARS-CoV-2 spike receptor binding domain
Authors: Lupala Cecylia S. , Kumar Vikash , Su Xiao-dong , Wu Chun , Liu Haiguang 
Published: February 2021

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causing agent of the COVID-19 pandemic, has spread globally. Angiotensin-converting enzyme 2 (ACE2) has been identified as the host cell receptor that binds to receptor-binding domain (RBD) of the SARS-COV-2 spike protein and mediates cell entry. Because the ACE2 proteins are widely available in mammals, it is important to investigate the interactions between the RBD and the ACE2 of other mammals. Here we analyzed the sequences of ACE2 proteins from 16 mammals and predicted the structures of ACE2-RBD complexes. Analyses on sequence, structure, and dynamics synergistically provide valuable insights into the interactions between ACE2 and RBD. The comparison results suggest that the ACE2 of bovine, cat and panda form strong binding with RBD, while in the cases of rat, least horseshoe bat, horse, pig, mouse and civet, the ACE2 proteins interact weakly with RBD.
A Bayesian method to cluster single-cell RNA sequencing data using Copy Number Alterations
Authors: Milite Salvatore , Bergamin Riccardo , Patruno Lucrezia , Calonaci Nicola , Caravagna Giulio 
Published: February 2021

AbstractMotivationCancers are composed by several heterogeneous subpopulations, each one harbouring different genetic and epigenetic somatic alterations that contribute to disease onset and therapy response. In recent years, copy number alterations leading to tumour aneuploidy have been identified as potential key drivers of such populations, but the definition of the precise makeup of cancer subclones from sequencing assays remains challenging. In the end, little is known about the mapping between complex copy number alterations and their effect on cancer phenotypes.ResultsWe introduce CONGAS, a Bayesian probabilistic method to phase bulk DNA and single-cell RNA measurements from independent assays. CONGAS jointly identifies clusters of single cells with subclonal copy number alterations, and differences in RNA expression. The model builds statistical priors leveraging bulk DNA sequencing data, does not require a normal reference and scales fast thanks to a GPU backend and variational inference. We test CONGAS on both simulated and real data, and find that it can determine the tumour subclonal composition at the single-cell level together with clone-specific RNA phenotypes in tumour data generated from both 10x and Smart-Seq assays.AvailabilityCONGAS is available as 2 packages: CONGAS (https://github.com/caravagnalab/congas), which implements the model in Python, and RCONGAS (https://caravagnalab.github.io/rcongas/), which provides R functions to process inputs, outputs, and run CONGAS fits. The analysis of real data and scripts to generate figures of this paper are available via RCONGAS; code associated to simulations is available at https://github.com/caravagnalab/rcongas_test.Contactgcaravagna@units.itSupplementary informationSupplementary data are available at Bioinformatics online.
RNA-Sequencing of Long-Term Label-Retaining Colon Cancer Stem Cells Identifies Novel Regulators of Quiescence
Authors: Regan Joseph L. , Schumacher Dirk , Staudte Stephanie , Steffen Andreas , Lesche Ralf , Toedling Joern , Jourdan Thibaud , Haybaeck Johannes , Mumberg Dominik , Henderson David , Győrffy Balázs , Regenbrecht Christian R.A. , Keilholz Ulrich , Schäfer Reinhold , Lange Martin 
Published: February 2021

SUMMARYRecent data suggests that colon tumors contain a subpopulation of therapy resistant quiescent cancer stem cells (qCSCs) that are the source of relapse following treatment. Here, using colon cancer patient-derived organoids (PDOs) and xenograft (PDX) models, we identify a rare population of long-term label-retaining (PKH26Positive) qCSCs that can re-enter the cell cycle to generate new tumors. RNA-sequencing analyses demonstrated that these cells are enriched for stem cell associated gene sets such as Wnt and hedgehog signaling, epithelial-to-mesenchymal transition (EMT), embryonic development, tissue development and p53 pathway but have downregulated expression of genes associated with cell cycle, transcription, biosynthesis and metabolism. Furthermore, qCSCs are enriched for p53 interacting negative regulators of cell cycle, includingAKAP12, CD82, CDKN1A, FHL2, GPX3, KIAA0247, LCN2, TFF2, UNC5BandZMAT3, that we show are indicators of poor prognosis and may be targeted for qCSC abolition. Interestingly, CD82, KIAA0247 and UNC5B proteins localize to the cell surface and may therefore be potential markers for the prospective isolation of qCSCs. These data support the temporal inhibition of p53 signaling for the elimination of qCSCs and prevention of relapse in colorectal cancer.
Characterisation of tumour microenvironment remodelling following oncogene inhibition in preclinical studies with imaging mass cytometry
Authors: van Maldegem Febe , Valand Karishma , Cole Megan , Patel Harshil , Angelova Mihaela , Rana Sareena , Colliver Emma , Enfield Katey , Bah Nourdine , Kelly Gavin , Tsang Victoria Siu Kwan , Mugarza Edurne , Moore Christopher , Hobson Philip , Levi Dina , Molina-Arcas Miriam , Swanton Charles , Downward Julian 
Published: February 2021

AbstractMouse models are critical in pre-clinical studies of cancer therapy, allowing dissection of mechanisms through chemical and genetic manipulations that are not feasible in the clinical setting. In studies of the tumour microenvironment (TME), multiplexed imaging methods can provide a rich source of information. However, the application of such technologies in mouse tissues is still in its infancy. Here we present a workflow for studying the TME using imaging mass cytometry with a panel of 27 antibodies on frozen mouse tissues. We optimise and validate image segmentation strategies and automate the process in a Nextflow-based pipeline (imcyto) that is scalable and portable, allowing for parallelised segmentation of large multi-image datasets. With these methods we interrogate the remodelling of the TME induced by a KRAS G12C inhibitor in an immune competent mouse orthotopic lung cancer model, highlighting the infiltration and activation of antigen presenting cells and effector cells.
Accurate and robust inference of microbial growth dynamics from metagenomic sequencing
Authors: Joseph Tyler A. , Chlenski Philippe , Korem Tal , Pe’er Itsik 
Published: February 2021

AbstractPatterns of sequencing coverage along a bacterial genome—summarized by a peak-to-trough ratio (PTR)—have been shown to accurately reflect microbial growth rates, revealing a new facet of microbial dynamics and host-microbe interactions. Here, we introduce CoPTR (Compute PTR): a tool for computing PTRs from complete reference genomes and assemblies. We show that CoPTR is more accurate than the current state-of-the-art, while also providing more PTR estimates overall. We further develop theory formalizing a biological interpretation for PTRs. Using a reference database of 2935 species, we applied CoPTR to a case-control study of 1304 metagenomic samples from 106 individuals with irritable bowel disease. We show that PTRs have high inter-individual variation, are only loosely correlated with relative abundances, and are associated with disease status. We conclude by demonstrating how PTRs can be combined with relative abundances and metabolomics to investigate their effect on the microbiome.AvailabilityCoPTR is available fromhttps://github.com/tyjo/coptr, with documentation onhttps://coptr.readthedocs.io.
Increased energy expenditure and protection from diet-induced obesity in mice lacking the cGMP-specific phosphodiesterase, PDE9
Authors: Ceddia Ryan P. , Liu Dianxin , Shi Fubiao , Mishra Sumita , Kass David A. , Collins Sheila 
Published: February 2021

AbstractObesity is a central component to cardiometabolic diseases, predisposing patients to both heart failure and diabetes. As therapeutics targeting caloric intake have limited long-term efficacy, greater interest has been on increasing thermogenic energy expenditure. Cyclic nucleotides, cAMP and cGMP, are important second messengers that are critical for the regulation of adaptive thermogenesis. These are regulated not only by their synthesis but also by their degradation. Pharmacological inhibitors of the cGMP-specific phosphodiesterase 9 (PDE9) increased PKG signaling and UCP1 expression in adipocytes. To elucidate the role of PDE9 on energy balance and glucose homeostasisin vivo, mice carrying a targeted disruption of the PDE9 gene,Pde9a, were fed a nutrient matched high-fat diet (HFD) or low-fat diet (LFD).Pde9a-/-mice were resistant to obesity induced by a HFD.Pde9a-/-mice exhibited a global increase in energy expenditure while the brown adipose tissue had elevated expression ofUcp1and other thermogenic genes. The reduced adiposity of HFD-fedPde9a-/-mice was associated with improvements in glucose handling and hepatic steatosis. These findings support the conclusion that PDE9 is a critical regulator of energy metabolism and suggest that inhibiting this enzyme may be an important avenue to explore for combating metabolic disease.
Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia
Authors: Garcia-Bermudez Javier , Prasad Sheela , Baudrier Lou , Badgley Michael A. , Liu Yuyang , La Konnor , Soula Mariluz , Williams Robert T. , Yamaguchi Norihiro , Hwang Rosa F. , Taylor Laura J. , Stanchina Elisa De , Rostandy Bety , Alwaseem Hanan , Molina Henrik , Bar-Sagi Dafna , Birsoy Kıvanç 
Published: February 2021

ABSTRACTStress-adaptive mechanisms enable tumor cells to overcome metabolic constraints in nutrient and oxygen poor tumors. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumor growth are poorly understood. Here, using a combination of metabolomics and CRISPR-based genetic screens, we identify GOT2-catalyzed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumor cells under hypoxic culture conditions. In contrast, GOT2-catalyzed aspartate synthesis is dispensable for pancreatic tumor formationin vivo. The dependence of pancreatic tumor cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRas-dependent, and is mediated by hypoxia inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9) through the cooption of the bicarbonate-macropinocytosis signaling axis. Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRas tumors can overcome nutrient deprivation under hypoxic conditions.