bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Retinal Ganglion Cell Types and Brain Nuclei expressing the transcription factor Brn3c/Pou4f3 using a Cre recombinase knock-in allele
Authors: Parmhans Nadia , Fuller Anne Drury , Nguyen Eileen , Chuang Katherine , Swygart David , Wienbar Sophia Rose , Lin Tyger , Kozmik Zbynek , Dong Lijin , Schwartz Gregory William , Badea Tudor Constantin 
Published: June 2020

AbstractMembers of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell types (RGCs), the projection sensory neuron conveying visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing Alkaline Phosphatase (AP) and intersectional genetics had identified three types of Pou4f3/Brn3c positive (Brn3c+) RGCs. Here, we describe a novel Brn3cCremouse allele generated by serial Dre to Cre recombination. We use this allele to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus properties of Brn3c+RGC types. Furthermore, we explore Brn3c-expressing brain nuclei. Our analysis reveals a much larger number of Brn3c+RGCs and more diverse set of RGC types than previously reported. The majority of RGCs having expressed Brn3c during development are still Brn3c positive in the adult, and all of them express Brn3a while only about half express Brn3b. Intersection of Brn3b and Brn3c expression highlights an area of increased RGC density, similar to an area centralis, corresponding to part of the binocular field of view of the mouse. Brn3c+neurons and projections are present in multiple brain nuclei. Brn3c+RGC projections can be detected in the Lateral Geniculate Nucleus (LGN), Pretectal Area (PTA) and Superior Colliculus (SC) but also in the thalamic reticular nucleus (TRN), a visual circuit station that was not previously described to receive retinal input. Most Brn3c+neurons of the brain are confined to the pretectum and the dorsal midbrain. Amongst theses we identify a previously unknown Brn3c+subdivision of the deep mesencephalic nucleus (DpMe). Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity and midbrain cytoarchitectonic, and opens the avenue for specific characterization and manipulation of these structures.
Crystal structures of peanut lectin in the presence of synthetic β-N- and β-S-galactosides disclose evidences for the recognition of different glycomimetic ligands
Authors: Cagnoni Alejandro J. , Primo Emiliano D. , Klinke Sebastián , Cano María E. , Giordano Walter , Mariño Karina V. , Kovensky José , Goldbaum Fernando A. , Uhrig María Laura , Otero Lisandro H. 
Published: June 2020

AbstractCarbohydrate−lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation, and tumor metastasis. Hence, the structural studies of lectin-synthetic glycan complexes are essential for understanding the lectin recognition processes and the further design of promising chemotherapeutics that interfere with sugar-lectin interactions.Plant lectins are excellent models for the study of the molecular recognition process. Among them, peanut lectin (PNA) is highly relevant in the glycobiology field, because of its specificity for β-galactosides, showing high affinity towards the Thomsen-Friedenreich (TF) antigen, a well-known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of β-galactoside and β-lactoside ligands. Herein, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable β-N- and β-S-galactosides. These complexes, along with computational simulations, disclosed key molecular binding interactions of the different sugars to PNA at the atomic level, revealing the role of specific water molecules in the protein–ligand recognition. Furthermore, binding affinity studies measured by isothermal titration calorimetry showed dissociation constant values in the micromolar range, as well as a positive glycoside cluster effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides qualitative structural rationale for the upcoming synthesis of optimized glycoclusters, designed for the study of lectin-mediated biological processes. The understanding of the recognition of β-N- and β-S-galactosides with PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands not belonging to the family of O-linked glycosides.
Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast
Authors: Hepowit Nathaniel L. , Pereira Kevin N. , Tumolo Jessica M. , Chazin Walter J. , MacGurn Jason A. 
Published: June 2020

ABSTRACTUbiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) phosphorylation of ubiquitin functions in stress responses inSaccharomyces cerevisiae, including the oxidative stress response. We also identify and characterize the first known Ser57 ubiquitin kinases in yeast and human cells, and we report that two Ser57 ubiquitin kinases regulate the oxidative stress response in yeast. These studies implicate ubiquitin phosphorylation at the Ser57 position as an important modifier of ubiquitin function, particularly in response to proteotoxic stress.
Modelling CAR T-cell Therapy with Patient Preconditioning
Authors: Owens Katherine , Bozic Ivana 
Published: June 2020

AbstractThe Federal Drug Administration (FDA) approved the first Chimeric Antigen Receptor T-cell (CAR T-cell) therapies for the treatment of several blood cancers in 2017, and efforts are underway to broaden CAR T technology to address other cancer types. Standard treatment protocols incorporate a preconditioning regimen of lymphodepleting chemotherapy prior to CAR T-cell infusion. However, the connection between preconditioning regimens and patient outcomes is still not fully understood. Optimizing patient preconditioning plans and reducing the CAR T-cell dose necessary for achieving remission could make therapy safer. In this paper, we test treatment regimens consisting of sequential administration of chemotherapy and CAR T-cell therapy on a system of differential equations that models the tumor-immune interaction. We use numerical simulations of treatment plans from within the scope of current medical practice to assess the effect of preconditioning plans on the success of CAR T-cell therapy. Model results affirm clinical observations that preconditioning can be crucial for some patients, not just to reduce side effects, but to even achieve remission at all. We demonstrate that preconditioning plans using the same CAR T-cell dose and the same total concentration of chemotherapy can lead to different patient outcomes due to different delivery schedules. Results from sensitivity analysis of the model parameters suggest that making small improvements in the effectiveness of CAR T-cells in attacking cancer cells, rather than targeting the recruitment and longevity of CAR T-cells, will significantly reduce the minimum dose required for successful treatment. Our modeling framework represents a starting point for evaluating the efficacy of patient preconditioning in the context of CAR T-cell therapy.
Machine learning assistive rapid, label-free molecular phenotyping of blood with two-dimensional NMR correlational spectroscopy
Authors: Peng Weng Kung , Ng Tian-Tsong , Loh Tze Ping 
Published: June 2020

AbstractTranslation of the findings in basic science and clinical research into routine practice is hampered by large variations in human phenotype. Developments in genotyping and phenotyping, such as proteomics and lipidomics, are beginning to address these limitations. In this work, we developed a new methodology for rapid, label-free molecular phenotyping of biological fluids (e.g., blood) by exploiting the recent advances in fast and highly efficient multidimensional inverse Laplace decomposition technique. We demonstrated that using two-dimensional T1-T2correlational spectroscopy on a single drop of blood (<5 μL), highly time– and patient–specific ‘molecular fingerprint’ can be obtained in minutes. Machine learning techniques were introduced to transform the NMR correlational map into user-friendly information for point-of-care disease diagnostic. The clinical utilities of this technique were demonstrated through the direct analysis of human whole blood in various physiological (e.g., oxygenated/deoxygenated states) and pathological (e.g., blood oxidation, hemoglobinopathies) conditions.
Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities
Authors: Clarke Ryan , Terry Alexander R. , Pennington Hannah , MacDougall Matthew S. , Regan Maureen , Merrill Bradley J. 
Published: June 2020

SUMMARYGenetic manipulation of mammalian cells is instrumental to modern biomedical research but is currently limited by poor capabilities of sequentially controlling multiple manipulations in cells. Currently, either highly multiplexed manipulations can be delivered to populations of cells all at one time, or gene regulatory sequences can be engineered to conditionally activate a few manipulations within individual cells. Here, we provide proof-of-principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of theS. pyogenesCas9 RNA-guided nuclease and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single guide RNA (sgRNA) - like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of genetic manipulations without the need for engineering cell type specific promoters or gene regulatory sequences. With the expanding diversity of available tools that utilize spCas9 to edit, repress or activate genes, this sgRNA-based system provides multiple levels for interfacing with host cell biology. In addition, ability of the system to progress through multiple modules from episomal plasmid DNA makes it suitable for applications sensitive to the presence of heterologous genomic DNA sequences and broadly applicable to biomedical research and mammalian cell engineering.
Transcriptional response of signalling pathways to SARS-CoV-2 infection in normal human bronchial epithelial cells
Authors: Enes Ak , Pir Pınar 
Published: June 2020

ABSTRACTSARS-CoV-2 virus, the pathogen that causes Covid-19 disease, emerged in Wuhan region in China in 2019, infected more than 4M people and is responsible for death of at least 300K patients globally as of May 2020. Identification of the cellular response mechanisms to viral infection by SARS-CoV-2 may shed light on progress of the disease, indicate potential drug targets, and make design of new test methods possible.In this study, we analysed transcriptomic response of normal human bronchial epithelial cells (NHBE) to SARS-CoV-2 infection and compared the response to H1N1 infection. Comparison of transcriptome of NHBE cells 24 hours after mock-infection and SARS-CoV-2 infection demonstrated that most genes that respond to infection were upregulated (320 genes) rather than being downregulated (115 genes).While upregulated genes were enriched in signalling pathways related to virus response, downregulated genes are related to kidney development. We mapped the upregulated genes on KEGG pathways to identify the mechanisms that mediate the response. We identified canonical NFκB, TNF and IL-17 pathways to be significantly upregulated and to converge to NFκB pathway via positive feedback loops. Although virus entry protein ACE2 has low expression in NHBE cells, pathogen response pathways are strongly activated within 24 hours of infection. Our results also indicate that immune response system is activated at the early stage of the infection and orchestrated by a crosstalk of signalling pathways. Finally, we compared transcriptomic SARS-CoV-2 response to H1N1 response in NHBE cells to elucidate the virus specificity of the response and virus specific extracellular proteins expressed by NHBE cells.
Engineered human mesenchymal stem cells as new vaccine platform for COVID-19
Authors: Liu Junhua , Jiao Huping , Yin Xiushan 
Published: June 2020

AbstractRecently, there are several routes for COVID-19 vaccine research, yet their weaknesses lie in low efficiency, tolerability, immune adaptability and safety. We describe a new approach to COVID-19 based on engineered human mesenchymal stem cells(hu-MSC), which is like a small protein antigen response device, but will be gradually cleared and degraded by body’s immune system among recognization process. The antibody response results show that this is effective and fast. Furthermore, after several antibody response tests, we obtained an injection of a set of cocktail-like modified human mesenchymal stem cell line. This strategy opened a new avenue for vaccine design against COVID-19.
UNC-45A Weakens and Breaks MT Lattice Independent of its Effect on Non-Muscle Myosin II
Authors: Habicht Juri , Mooneyham Ashley , Hoshino Asumi , Shetty Mihir , Zhang Xiaonan , Emmings Edith , Yang Qing , Coombes Courtney , Gardner Melissa K. , Bazzaro Martina 
Published: June 2020

AbstractIn invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only and UNC-45A, expressed in all cells and implicated in regulating both Non-Muscle Myosin II (NMII)- and microtubule (MT)-associated functions. Here we show for the first time that:a) in vitroUNC-45A binds to the MT lattice and weakens its integrity leading to MT bending, breakage and depolymerization,b)in cells, UNC-45A overexpression causes loss of MT mass and increase in MT breakages,c)bothin vitroand in cells, UNC-45A destabilizes MTs independent of its NMII C-terminal binding domain and destabilization occurs even in presence of the NMII inhibitor blebbistatin. These findings are consistent with a not mutually exclusive but rather dual role of UNC-45A in regulating NMII activity and MT stability.Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability our findings have profound implications in both, the biology of MTs as well as the biology of human diseases and possible therapeutic implications for their treatment.
Activated regulatory T-cells, dysfunctional and senescent T-cells dominate the microenvironment of pancreatic cancer
Authors: Sivakumar Shivan , Abu-Shah Enas , Ahern David J , Arbe-Barnes Edward H , Mangal Nagina , Reddy Srikanth , Rendek Aniko , Easton Alistair , Kurz Elke , Silva Michael , Soonawalla Zahir , Heij Lara R , Bashford- Rogers Rachael , Middleton Mark R , Dustin Michael L 
Published: June 2020

AbstractPancreatic cancer has the worst prognosis of any human malignancy and leukocyte infiltration is a major prognostic marker of the disease. As current immunotherapies confer negligible survival benefits, there is a need to better characterise leukocytes in pancreatic cancer to identify better therapeutic strategies.In this study, we analysed 32 human pancreatic cancer patients from two independent cohorts. A multi-parameter mass-cytometry analysis was performed on 32,000 T-cells from eight patients. Single-cell RNA sequencing dataset analysis was performed on a cohort of 24 patients. Multiplex immunohistochemistry imaging and spatial analysis were performed to map immune infiltration into the tumour microenvironment.Regulatory T-cell populations demonstrated highly immunosuppressive states with high TIGIT, ICOS and CD39 expression. CD8+T-cells were found to be either in senescence or an exhausted state. The exhausted CD8 T-cells had low PD-1 expression but high TIGIT and CD39 expression. These findings were corroborated in an independent pancreatic cancer single-cell RNA dataset from additional 24 patients.These data suggest that T-cells are major players in the suppressive microenvironment of pancreatic cancer. Our work identifies novel therapeutic targets that should form the basis for rational design of a new generation of clinical trials in pancreatic ductal adenocarcinoma.
Dufour’s gland analysis reveals caste and physiology specific signals inBombus impatiens
Authors: Derstine Nathan T. , Villar Gabriel , Orlova Margarita , Hefetz Abraham , Millar Jocelyn , Amsalem Etya 
Published: June 2020

AbstractReproductive division of labor in insect societies is regulated through multiple concurrent mechanisms, primarily chemical and behavioral. Here, we examined if the Dufour’s gland secretion in the primitively eusocial bumble beeBombus impatienssignals information about caste, social condition, and reproductive status. We chemically analyzed Dufour’s gland contents across castes, age groups, social and reproductive conditions, and examined worker behavioral and antennal responses to gland extracts.We found that workers and queens each possess caste-specific compounds in their Dufour’s glands. Queens and gynes differed from workers based on the presence of diterpene compounds which were absent in workers, whereas four esters were exclusive to workers. These esters, as well as the total amounts of hydrocarbons in the gland, provided a separation between castes and also between fertile and sterile workers. Olfactometer bioassays demonstrated attraction of workers to Dufour’s gland extracts that did not represent a reproductive conflict, while electroantennogram recordings showed higher overall antennal sensitivity in queenless workers. Our results demonstrate that compounds in the Dufour’s gland act as caste- and physiology-specific signals and are used by workers to discriminate between workers of different social and reproductive status.
Unique K-mer sequences for validating cancer-related substitution, insertion and deletion mutations
Authors: Lee HoJoon , Shuaibi Ahmed , Bell John M. , Pavlichin Dmitri S. , Ji Hanlee P. 
Published: June 2020

ABSTRACTThe cancer genome sequencing has led to important discoveries such as identifying cancer gene. However, challenges remain in the analysis of cancer genome sequencing. One significant issue is that mutations identified by multiple variant callers are frequently discordant even when using the same genome sequencing data. For insertion and deletion mutations, oftentimes there is no agreement among different callers. Identifying somatic mutations involves read mapping and variant calling, a complicated process that uses many parameters and model tuning. To validate the identification of true mutations, we developed a method using k-mer sequences. First, we characterized the landscape of unique versus non-unique k-mers in the human genome. Second, we developed a software package, KmerVC, to validate the given somatic mutations from sequencing data. Our program validates the occurrence of a mutation based on statistically significant difference in frequency of k-mers with and without a mutation from matched normal and tumor sequences. Third, we tested our method on both simulated and cancer genome sequencing data. Counting k-mer involving mutations effectively validated true positive mutations including insertions and deletions across different individual samples in a reproducible manner. Thus, we demonstrated a straightforward approach for rapidly validating mutations from cancer genome sequencing data.
Individual cell-based modeling of tumor cell plasticity-induced immune escape after CAR-T therapy
Authors: Zhang Can , Shao Changrong , Jiao Xiaopei , Bai Yue , Li Miao , Shi Hanping , Lei Jinzhi , Zhong Xiaosong 
Published: June 2020

AbstractChimeric antigen receptor (CAR) therapy targeting CD19 is an effective treatment for refractory B cell malignancies, especially B cell acute lymphoblastic leukemia (B-ALL). The majority of patients achieve a complete response following a single infusion of CD19-targeted CAR-modified T cells (CAR-19 T cells); however, many patients suffer relapse after therapy, and the underlying mechanism remains unclear. To better understand the mechanism of tumor relapse, we developed an individual cell based computational model for tumor cell plasticity and the heterogeneous responses to the CAR-T treatment. Model simulations reproduced the process of tumor relapse, and predicted that CAR-T stress-induced cell plasticity can lead to tumor relapse in B-ALL. Model predictions were verified by applying the second-generation CAR-T cells to mice injected with NALM-6-GL leukemic cells, in which 60% of the mice relapse within 3 months, and relapsed tumors retained CD19 expression but exhibited a subpopulation of cells with CD34 transcription. These findings lead to a mechanism of tumor replace by which CAR-T treatment induced tumor cells to transition to hematopoietic stem-like cells (HSLCs) and myeloid-like cells and hence escape of CAR-T targeting. The computational model framework was successfully developed to recapitulate the individual evolutionary dynamics, which could predict clinical survival outcomes in B-ALL patients after CAR-T therapy.
A combined characterization of coelomic fluid cell types in the spiny starfishMarthasterias glacialis– inputs from flow cytometry and imaging
Authors: Oliveira Bárbara , Guatelli Silvia , Martinez Pedro , Simões Beatriz , Bispo Claúdia , Andrade Claúdia , Ferrario Cinzia , Bonasoro Francesco , Rino José , Sugni Michela , Gardner Rui , Zilhão Rita , Coelho Ana Varela 
Published: June 2020

ABSTRACTCoelomocytes is a generic name for a collection of cellular morphotypes, present in many coelomate animals, that has been reported as highly variable across echinoderm classes. The roles attributed to the major types of the free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. The main aim of the present study is the thorough characterization of coelomocytes present in the coelomic fluid ofMarthasterias glacialis(class Asteroidea) through the combined use of flow cytometry (FC) and fluorescence plus transmission electron microscopy. Two coelomocyte populations (here named P1 and P2) were identified by flow cytometry and subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells showed two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the small P1 haploid cellular population was characterized by a low mitotic activity, relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. These last cells resemble stem-cell types present in other animals. P1 and P2 cells differ also in cell viability and cell cycle profiles. Additionally, two other morphotypes were only detected by fluorescence microscopy and a third one when using a combined microscopy/FC approach.