bluuebunny/crossref_metadata_2025_split huggingface dataset:
CREB-mediated transcriptional activation of NRMT1 drives muscle differentiation
Authors: Tooley John G. , Catlin James P. , Schaner Tooley Christine E. 
Published: May 2021

AbstractThe N-terminal methyltransferase NRMT1 is an important regulator of protein-DNA interactions and plays a role in many cellular processes, including mitosis, cell cycle progression, chromatin organization, DNA damage repair, and transcriptional regulation. Accordingly, loss of NRMT1 results in both developmental pathologies and oncogenic phenotypes. Though NRMT1 plays such important and diverse roles in the cell, little is known about its own regulation. To better understand the mechanisms governing NRMT1 expression, we first identified its predominant transcriptional start site and minimal promoter region with predicted transcription factor motifs. We then used a combination of luciferase and binding assays to confirm CREB1 as the major regulator of NRMT1 transcription. We tested which conditions known to activate CREB1 also activated NRMT1 transcription, and found CREB1-mediated NRMT1 expression was increased during recovery from serum starvation and muscle cell differentiation. To determine how NRMT1 expression affects myoblast differentiation, we used CRISPR/Cas9 technology to knock out NRMT1 expression in immortalized C2C12 mouse myoblasts. C2C12 cells depleted of NRMT1 lacked Pax7 expression and were unable to proceed down the muscle differentiation pathway. Instead, they took on characteristics of C2C12 cells that have transdifferentiated into osteoblasts, including increased alkaline phosphatase and type I collagen expression and decreased proliferation. These data implicate NRMT1 as an important downstream target of CREB1 during muscle cell differentiation.
A rapid and reliable multiplexed LC-MS/MS method for simultaneous analysis of six monoamines from brain tissues
Authors: Davla Sejal , Daly Edward , Nedow Jenn , Gritsas Ari , Curran Laura , Taylor Lorne , van Meyel Donald J. 
Published: May 2021

AbstractMonoamines are a class of neuromodulators that are crucial for a variety of brain functions, including control of mood, movement, sleep and cognition. From mammals to insects, the nervous system is enriched in monoamines such as dopamine, serotonin and melatonin, analytes which range from being highly polar to non-polar. Here we developed a method using liquid chromatography coupled with mass spectrometry (LC-MS) to quantify in a single run the amounts of six distinct monoamines in extracts from dissected Drosophila and mouse brain tissues. The measured monoamines were dopamine (DA), serotonin (also known as 5-hydroxytryptamine (5-HT)), octopamine (OA, an insect equivalent of norepinephrine), tyramine (TA), melatonin (MT) and N-acetyl-hydroxy-serotonin (NAS). The analytical range of these monoamines was between 0.25 to 5.0 ng/mL.
HIV-1 Nef and CycK:CDK13 antagonize SERINC5 for optimal viral infectivity
Authors: Chai Qingqing , Li Sunan , Collins Morgan K. , Li Rongrong , Ahmad Iqbal , Johnson Silas F. , Frabutt Dylan A. , Yang Zhichang , Shen Xiaojing , Sun Liangliang , Hu Jian , Hultquist Judd F. , Matija Peterlin B. , Zheng Yong-Hui 
Published: May 2021

SummaryHIV-1 Nef antagonizes SERINC5 by redirecting this potent restriction factor to the endosomes and lysosomes for degradation. However, the precise mechanism remains unclear. Using affinity purification/mass spectrometry, we identified cyclin K and cyclin-dependent kinase 13 (CycK:CDK13) as a new Nef-associated kinase complex. CycK:CDK13 phosphorylates the serine at position 360 (S360) in SERINC5, which is required for Nef downregulation of SERINC5 from the cell surface and its counter activity of the SERINC5 antiviral activity. To understand the role of S360 phosphorylation, we created chimeric proteins between CD8 and SERINC5. Nef not only downregulates, but importantly, also binds to this chimera in a S360-dependent manner. Thus, S360 phosphorylation increases interactions between Nef and SERINC5 and initiates the destruction of SERINC5 by the endocytic machinery.
Cancer Prognosis According to Parthanatos Features
Authors: Messikommer Alessandra , Maru Bruktawit , Seipel Katja , Valk Peter J. M. , Theocharides Alexandre P.A. , Pabst Thomas , McKeague Maureen , Luedtke Nathan W. 
Published: May 2021

SUMMARYFor nearly 50 years, translational research studies aimed at improving chemotherapy-induced killing of cancer cells have focused on the induction of apoptosis. Here we show that a PARP-1-mediated programmed cell death mechanism “parthanatos” is associated with the successful, front-line treatment of a common cancer. Peripheral blood mononuclear cells (PBMCs) from healthy human donors (10 of 10 tested), as well as primary cancer cells from approximately 50% of acute myeloid leukemia (AML) patients (n = 18 of 39 tested, French-American-British (FAB) subtypes M4 and M5) exhibited two distinctive features of parthanatos upon treatment with a front-line drug combination of cytarabine and an anthracycline. Statistically significant improvements in survival rates were observed in the parthanatos positive versus parthanatos negative AML patient groups (HR = 0.22 – 0.38, p = 0.002 – 0.05). Near-median expression of PARP1 mRNA was associated with a 50% longer survival time (HR = 0.66, p = 0.01), and the poly [ADP-ribose] polymerase (PARP) inhibitor Olaparib exhibited antagonistic activities against ara-C and idarubicin in primary blood monocytes from healthy donors as well as primary cancer isolates from ~50% of AML patients. Together these results suggest that PARP activity is a prognostic biomarker for AML subtypes M4 and M5 and support the relevance of parthanatos in curative chemotherapy of AML.In BriefMessikommer and co-workers report that PARP-1-mediated programmed cell death is associated with successful, front-line treatment of acute myeloid leukemia (AML).HighlightsThe first-line cancer drug cytarabine (ara-C) induces parthanatos or apoptosis, depending on the specific AML cell line being treated.OCI-AML3 cells undergo parthanatos or apoptosis, depending on the specific drug being added.The presence of two parthanatos features in primary cancer cells from AML patients (n = 18 of 39 tested) having French-American-British (FAB) subclassifications M4 or M5 is associated with four-fold improved survival (HR = 0.23, p = 0.01) following curative chemotherapy with ara-C and an anthracycline.The poly [ADP-ribose] polymerase (PARP) inhibitor Olaparib exhibits antagonistic activities against ara-C and idarubicin in primary blood monocytes from healthy donors as well as primary cancer isolates from ~50% of AML patients.Near-median expression of PARP1 mRNA is associated with a 50% increase in survival time (HR = 0.66, p = 0.01) of AML patients following chemotherapy with ara-C and idarubicin.GRAPHICAL ABSTRACT
The RNA chaperone protein CspA stimulates translation during cold acclimation by promoting the progression of the ribosomes
Authors: Giuliodori Anna Maria , Belardinelli Riccardo , Duval Melodie , Garofalo Raffaella , Schenckbecher Emma , Hauryliuk Vasili , Ennifar Eric , Marzi Stefano 
Published: May 2021

SUMMARYCspA is an RNA binding protein expressed during cold-shock in Escherichia coli, capable of stimulating translation of several mRNAs – including its own – at low temperature. We used reconstituted translation systems to monitor the effects of CspA on the different steps of the translation process and probing experiments to analyze the interactions with its target mRNAs. We specifically focused on cspA mRNA which adopts a cold-induced secondary structure at temperatures below 20°C and a more closed conformation at 37°C. We show that at low temperature CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome (37°C form). CspA interacts with its mRNA without inducing large structural rearrangement, does not bind the ribosomal subunits and is not able to stimulate the formation of the translation initiation complexes. On the other hand, CspA promotes the progression of the ribosomes during translation of its mRNA at low temperature and this stimulation is mRNA structure-dependent. A similar structure-dependent mechanism may be responsible for the CspA- dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.
Estimating the Dynamic Range of Quantitative Single-Molecule Localization Microscopy
Authors: Nino Daniel F. , Milstein Joshua N. 
Published: May 2021

AbstractIn recent years, there have been significant advances in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events from different fluorophores becomes progressively more difficult, affecting the accuracy of such measurements. Although essential to the design of quantitative experiments, the dynamic range of SMLM counting techniques has not yet been studied in detail. Here we provide a working definition of the dynamic range for quantitative SMLM in terms of the relative number of missed localizations or blinks, and explore the photophysical and experimental parameters that affect it. We begin with a simple two-state model of blinking fluorophores, then extend the model to incorporate photobleaching and temporal binning by the detection camera. From these models, we first show that our estimates of the dynamic range agree with realistic simulations of the photoswitching. We find that the dynamic range scales inversely with the duty cycle when counting both blinks and localizations. Finally, we validate our theoretical approach on dSTORM datasets of photo-switching Alexa647 dyes. Our results should help guide researchers in designing and implementing SMLM-based molecular counting experiments.
The molecular mechanism of load adaptation by branched actin networks
Authors: Li Tai-De , Bieling Peter , Weichsel Julian , Mullins R. Dyche , Fletcher Daniel A. 
Published: May 2021

AbstractBranched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes. Load forces slow the growth and increase the density of these networks, but the molecular mechanisms governing this force response are not well understood. Here we use single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Unexpectedly, force slows the rate of filament nucleation by promoting the interaction of nucleation promoting factors with actin filament ends, limiting branch formation. This inhibition is countered by an even larger force-induced drop in the rate of filament capping, resulting in a shift in the balance between nucleation and capping that increases network density. Remarkably, the force dependence of capping is identical to that of filament elongation because they require the same size gap to appear between the filament and load for insertion. These results provide direct evidence that Brownian Ratchets generate force and govern the load adaptation of branched actin networks.
A Neural Model of RNA Splicing: Learning Motif Distances with Self-Attention and Toeplitz Max Pooling
Authors: Quinn Thomas P. , Nguyen Dang , Gupta Sunil , Venkatesh Svetha 
Published: May 2021

AbstractAlternative RNA splicing is an important regulator of tissue development and specificity, and a relevant mechanism in cancer progression. There exists a strong motivation to disentangle the rules that govern RNA splicing, in part because this knowledge may one day yield new clinically relevant diagnostic tools and therapeutics. It is no easy to task to reverse engineer how the splicesome machinery choreographs the removal and addition of RNA elements following transcription. Here, we propose an interpretable neural network called the Toeplitz ATtention Architecture (TATA), which learns distance-dependent motif interactions through a novel Toeplitz max pool layer that captures the relative distance between interacting CNN filters. TATA is a completely transparent “clear-box” solution: every model parameter is human-interpretable. We validate TATA on simulated data, then apply it to real data to identify putative cis-regulatory elements that interact with primary RNA splice sites.
Structural basis for DNA targeting by the Tn7 transposon
Authors: Shen Yao , Gomez-Blanco Josue , Petassi Michael T. , Peters Joseph E. , Ortega Joaquin , Guarné Alba 
Published: May 2021

AbstractTn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7-family utilize a AAA+ adaptor (TnsC) to coordinates target-site selection with transposase activation and prevent insertions at sites already containing a Tn7 element. Due to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. We find that TnsC forms an asymmetric ring on target DNA that segregates target-site selection and transposase recruitment to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP-hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.
Disc Large Homolog 1 is critical for early microcluster formation and activation in human T cells
Authors: June Guha , Raj Chari 
Published: May 2021

AbstractT cell activation by antigen involves multiple sequential steps, including TCR-microcluster (MC) formation, immunological synapse formation and phosphorylation of mediators downstream of the TCR. The adaptor protein Disc Large Homolog 1 (DLG1) is known to regulate proximal TCR signaling and, in turn, T cell activation, acting as a molecular chaperone that organizes specific kinases downstream of antigen recognition. Here, we report using knockdown and knockout studies in human T cells that DLG1 functions even earlier to regulate T cell activation by promoting TCR-MC formation. Moreover, we found that DLG1 can act as a bridge between the TCR-ζ chain and ZAP70 while inhibiting binding of the phosphatase SHP1 to TCR-ζ. Together, these effects drive dysregulation of T cell activation in DLG1-deficient T cells.
Phase variable expression of pdcB, a phosphodiesterase influences sporulation in Clostridioides difficile
Authors: Dhungel Babita Adhikari , Govind Revathi 
Published: May 2021

AbstractClostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to hypervirulence strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovered that PdcB, a phosphodiesterase to influence sporulation in C. difficile UK1 strain positively. Through genetic and biochemical assays, we have shown that phase variable expression of pdcB results in hypo- and hyper-sporulation phenotype. In the “ON” orientation, the identified promotor is the right orientation to drive the expression of pdcB. Production of PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP concentration, resulting in hyper-sporulation phenotype. The OFF orientation of pdcB switch or mutating pdcB results in increased cyclic-di-GMP and hypo-sporulating phenotype. Additionally, we demonstrated that CodY binds to the upstream region of pdcB to represses its expression, and CodY mediated repression is relieved by the DNA inversion.
Statistical Analysis of Three-Dimensional Chromatin Packing Domains Determined by Chromatin Scanning Transmission Electron Microscopy (ChromSTEM)
Authors: Li Yue , Agrawal Vasundhara , Virk Ranya K. A. , Roth Eric , Li Wing Shun , Eshein Adam , Frederick Jane , Huang Kai , Almassalha Luay , Bleher Reiner , Carignano Marcelo A. , Szleifer Igal , Dravid Vinayak P. , Backman Vadim 
Published: May 2021

AbstractChromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression.
An hidden Markov model to estimate homozygous-by-descent probabilities associated with nested layers of ancestors
Authors: Druet Tom , Gautier Mathieu 
Published: May 2021

AbstractInbreeding results from the mating of related individuals and has negative consequences because it brings together deleterious variants in one individual. Genomic estimates of the inbreeding coefficients are preferred to pedigree-based estimators as they measure the realized inbreeding levels and they are more robust to pedigree errors. Several methods identifying homozygous-by-descent (HBD) segments with hidden Markov models (HMM) have been recently developed and are particularly valuable when the information is degraded or heterogeneous (e.g., low-fold sequencing, low marker density, heterogeneous genotype quality or variable marker spacing). We previously developed a multiple HBD class HMM where HBD segments are classified in different groups based on their length (e.g., recent versus old HBD segments) but we recently observed that for high inbreeding levels with many HBD segments, the estimated contributions might be biased towards more recent classes (i.e., associated with large HBD segments) although the overall estimated level of inbreeding remained unbiased. We herein propose a new model in which the HBD classification is modeled in successive nested levels with decreasing expected HBD segment lengths, the underlying exponential rates being directly related to the number of generations to the common ancestor. The non-HBD classes are now modeled as a mixture of HBD segments from later generations and shorter non-HBD segments (i.e., both with higher rates). The new model has improved statistical properties and performs better on simulated data compared to our previous version. We also show that the parameters of the model are easier to interpret and that the model is more robust to the choice of the number of classes. Overall, the new model results in an improved partitioning of inbreeding in different HBD classes and should be preferred.
Optimized liquid and gas phase fractionation increase HLA-peptidome coverage for primary cell and tissue samples
Authors: Klaeger Susan , Apffel Annie , Clauser Karl R. , Sarkizova Siranush , Oliveira Giacomo , Rachimi Suzanna , Le Phuong M. , Tarren Anna , Chea Vipheaviny , Abelin Jennifer G. , Braun David A. , Ott Patrick A. , Keshishian Hasmik , Hacohen Nir , Keskin Derin B. , Wu Catherine J. , Carr Steven A. 
Published: May 2021

AbstractMass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require many millions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 100 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens.
Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors
Authors: Carter Angela M. , Kumar Nilesh , Herring Brendon , Tan Chunfeng , Guenter Racheal , Telange Rahul , Howse Wayne , Viol Fabrice , Graham Chris , McCaw Tyler R. , Bickerton Hayden , Gillardon Frank , Woltering Eugene A. , Dhall Deepti , Totenhagen John , Banerjee Ronadip , Kurian Elizabeth , Reddy Sushanth , Chen Herbert , Schrader Joerg , Rose J. Bart , Mukhtar M. Shahid , Bibb James A. 
Published: May 2021

AbstractPancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bitransgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in β–islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. The utility of this model is enhanced by ability to form tumor-derived allografts. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.