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Mashup Score: 0Comprehensive Assessment of Force-Field Performance in Molecular Dynamics Simulations of DNA/RNA Hybrid Duplexes - 2 hour(s) ago
Mixed double helices formed by RNA and DNA strands, commonly referred to as hybrid duplexes or hybrids, are essential in biological processes like transcription and reverse transcription. They are also important for their applications in CRISPR gene editing and nanotechnology. Yet, despite their significance, the hybrids have been seldom modeled by atomistic molecular dynamics methodology, and there is no benchmark study systematically assessing the force-field performance. Here, we present an extensive benchmark study of the hybrids using contemporary and commonly utilized pairwise additive and polarizable nucleic acid force fields. Our findings indicate that none of the available force-field choices accurately reproduces all the characteristic structural details of the hybrids. The AMBER force fields are unable to populate the C3′-endo (north) pucker of the DNA strand and underestimate inclination. CHARMM force field accurately describes the C3′-endo pucker and inclination but shows
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Mashup Score: 0Compartmentalized CRISPR Reactions (CCR) for High-Throughput Screening of Guide RNA Potency and Specificity - 2 hour(s) ago
CRISPR ribonucleoproteins (RNPs) use a variable segment in their guide RNA (gRNA) called a spacer to determine the DNA sequence at which the effector protein will exhibit nuclease activity and generate target-specific genetic mutations. However, nuclease activity with different gRNAs can vary considerably, in a spacer sequence-dependent manner that can be difficult to predict. While computational tools are helpful in predicting a CRISPR effector’s activity and/or potential for off-target mutagenesis with different gRNAs, individual gRNAs must still be validated in vitro prior to their use. Here, we present compartmentalized CRISPR reactions (CCR) for screening large numbers of spacer/target/off-target combinations simultaneously in vitro for both CRISPR effector activity and specificity, by confining the complete CRISPR reaction of gRNA transcription, RNP formation, and CRISPR target cleavage within individual water-in-oil microemulsions. With CCR, large numbers of the candidate gRNAs
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Mashup Score: 1Local structural dynamics of Rad51 protomers revealed by cryo-electron microscopy of Rad51-ssDNA filaments - 2 hour(s) ago
Homologous recombination (HR) is a high-fidelity repair mechanism for double-strand breaks. Rad51 is the key enzyme that forms filaments on single-stranded DNA (ssDNA) to catalyze homology search and DNA strand exchange in recombinational DNA repair. In this study, we employed single-particle cryo-electron microscopy (cryo-EM) to ascertain the density map of the budding yeast Rad51-ssDNA filament bound to ADP-AlF3, achieving a resolution of 2.35 Angstrom without imposing helical symmetry. The model assigned 6 Rad51 protomers, 24 nt of DNA, and 6 bound ADP-AlF3. It shows 6-fold symmetry implying monomeric building blocks, unlike the structure of the Rad51-I345T mutant filament with three-fold symmetry implying dimeric building blocks, for which the structural comparisons provide a satisfying mechanistic explanation. This image analysis enables comprehensive comparisons of individual Rad51 protomers within the filament and reveals local conformational movements of amino acid side chains.
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Mashup Score: 1Positional 13C Enrichment Analysis of Aspartate by GC-MS to Determine PEPC Activity In Vivo - 2 hour(s) ago
Photoautotrophic organisms fix inorganic carbon (Ci) by two enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase (PEPC). RUBISCO assimilates Ci (CO2) into the 1-C position of 3-phosphoglycerate (3PGA). The Calvin-Benson-Basham (CBB) cycle redistributes fixed carbon atoms into 2,3-C2 of the same molecule. PEPC uses phosphoenolpyruvate (PEP) derived from 3PGA and assimilates Ci (HCO3-) into 4-C of oxaloacetate (OAA). 1,2,3-C3 of OAA and of its transaminase product aspartate originate directly from 1,2,3-C3 of 3PGA. Positional isotopologue analysis of aspartate, the main downstream metabolite of OAA in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), allows differentiation between PEPC, RUBISCO, and CBB cycle activities within one molecule. We explored in source fragmentation of gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) at nominal mass resolution and GC-atmospheric pressure chemical ioni
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Mashup Score: 0Structures of native SV2A reveal the binding mode for tetanus neurotoxin and anti-epileptic racetams - 2 hour(s) ago
The synaptic vesicle glycoprotein 2A (SV2A) is a bona fide synaptic vesicle (SV) constituent of controversial function with homology to the major facilitator superfamily (MFS) and essential in vertebrate neurotransmission. Despite its high medical relevance as the target of the anti-epileptic drug Levetiracetam (LEV) and as receptor for clostridial neurotoxins (CNTs), among them several botulinum neurotoxin (BoNT) serotypes and potentially tetanus neurotoxin (TeNT), we lack detailed insight about these molecular interactions. We purified native SV2A from brain and subjected it to a structural analysis to advance our understanding of drug-binding to this enigmatic protein and explore structurally uncharacterized toxin-SV2A interfaces. Our analysis uncovered that TeNT binds SV2 proteins strikingly different from BoNT/A and delivers visual evidence for the dual receptor hypothesis through structurally resolved, co-purified gangliosides in the complex. The structures provide compelling sup
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Mashup Score: 1
Sortases are cysteine transpeptidases located on the surface of Gram-positive bacteria. These critical enzymes facilitate the attachment of proteins to the cell wall, and are potential targets for novel antibiotic development, as well as versatile tools in protein engineering applications. Although there are six classes of sortases recognized, class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes recognize the canonical Cell Wall Sorting Signal (CWSS), LPXTG, where X=any amino acid, although work in recent years identified additional promiscuity in multiple positions of this recognition motif. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes have previously been shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantl
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Mashup Score: 0
BACKGROUND: The goal of the study is to use CT imaging in patients with aSAH to evaluate the anatomic distribution of hemorrhage and compartmentalization of subarachnoid space to investigate potential in vivo visualization of recently discovered layer named subarachnoid lymphatic like membrane (SLYM). METHODS: We conducted a retrospective cohort study of cases with aneurysmal SAH (aSAH) at our institution between January 2015 and June 2022. Subarachnoid hemorrhage distribution into superficial and deep subarachnoid spaces was classified based on proximity to the dural or pial surfaces, respectively, as seen on multiplanar CT head. RESULTS: A total of 97 patients with aSAH were included. Patients with lower modified Fisher score (MFS) of 1-2 were more likely to have SAH compartmentalizing in the (deep) pial-adjacent subarachnoid space. Patients with higher MFS of 3-4 were more likely to have SAH in both (superficial) and (deep) compartments along the brainstem. There is a significant as
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Mashup Score: 0Ih Block Reveals Separation of Timescales in Pyloric Rhythm Response to Temperature Changes in Cancer borealis - 7 hour(s) ago
Motor systems operate over a range of frequencies and relative timing (phase). We studied the contribution of the hyperpolarization-activated inward current (Ih) to frequency and phase in the pyloric rhythm of the stomatogastric ganglion (STG) of the crab, Cancer borealis as temperature was altered from 11 degrees C to 21 degrees C. Under control conditions, the frequency of the rhythm increased monotonically with temperature, while the phases of the pyloric dilator (PD), lateral pyloric (LP), and pyloric (PY) neurons remained constant. When we blocked Ih> with cesium (Cs+) PD offset, LP onset, and LP offset were all phase advanced in Cs+ at 11 degrees C, and the latter two further advanced as temperature increased. In Cs+ the steady state increase in pyloric frequency with temperature diminished and the Q10 of the pyloric frequency dropped from ~1.75 to ~1.35. Unexpectedly in Cs+, the frequency displayed non-monotonic dynamics during temperature transitions; the frequency initially dr
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Mashup Score: 2
Due to the increasing number of human leishmaniasis cases in Murcia, sandflies were sampled and 7.48% of female sandflies tested positive. The majority were Leishmania infantum, but Leishmania adleri was found in one female Sergentomyia minuta. The presence of L. adleri in sandflies raises concerns about zoonotic transmission. Further research is needed to investigate its spread and potential impact on public health. Surveillance of sandflies and potential hosts is essential to prevent the spread of the disease. ### Competing Interest Statement The authors have declared no competing interest.
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Mashup Score: 1A novel heuristic of rigid docking scores positively correlates with full-length nuclear receptor LRH-1 regulation. - 8 hour(s) ago
The nuclear receptor Liver Receptor Homolog-1 (LRH-1, NR5A2) is a ligand-regulated transcription factor and validated drug target for several human diseases. LRH-1 activation is regulated by small molecule ligands, which bind to the ligand binding domain (LBD) within the full-length LRH-1. We recently identified 57 compounds that bind LRH-1, and unexpectedly found these compounds regulated either the isolated LBD, or the full-length LRH-1 in cells, with little overlap. Here, we correlated compound binding energy from a single rigid-body scoring function with full-length LRH-1 activity in cells. Although docking scores of the 57 hit compounds did not correlate with LRH-1 regulation in wet lab assays, a subset of the compounds had large differences in binding energy docked to the isolated LBD vs. full-length LRH-1, which we used to empirically derive a new metric of the docking scores we call “ΔΔG”. Initial regressions, correlations and contingency analyses all suggest compounds with hig
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Comprehensive Assessment of Force-Field Performance in Molecular Dynamics Simulations of DNA/RNA Hybrid Duplexes https://t.co/eytnN53F0B #bioRxiv