1. Multinucleation in cleavage stage embryos - Oxford Academic
Multinucleation is a frequently observed phenomenon. It is associated with impaired cleavage and increased fragmentation and is compromising the ongoing ...
2. Enhancement of trans-cleavage activity of Cas12a with engineered ...
30 sep 2020 · In this study, we engineer crRNAs with high sensitivity and specificity for detecting nucleic acids using CRISPR-Cas12a.
The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3′- or 5′-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40–60 min. CRISPR-Cas12a based detection systems can be sensitive down to the picomolar range. Here the authors modify the 3′- and 5′-ends o...
3. Time-lapse imaging of cleavage divisions in embryo quality ...
In the current review, we focus on biological significance of morphokinetic parameters and show how they can be used to predict a reproductive outcome.
In vitro fertilization (IVF) is one of the most important procedures for treating infertility. As several embryos are usually produced in a single IVF cycle, it is crucial to select only the most viable ones for transfer to the patient. Morphokinetics, i.e. analysis of the dynamics of cleavage divisions and processes such as compaction and cavitation, has provided both biologists and clinicians with a new set of data regarding embryonic behaviour during preimplantation development and its association with embryo quality. In the current review, we focus on biological significance of morphokinetic parameters and show how they can be used to predict a reproductive outcome. We also explain the statistics behind the predictive algorithms and discuss the future perspectives of morphokinetics.
4. Multinucleation in cleavage stage embryos - Lirias - KU Leuven
Abstract: The aim was to analyse multinucleation in relation to its incidence in time and in the population, and its correlation with clinical variables, with ...
The aim was to analyse multinucleation in relation to its incidence in time and in the population, and its correlation with clinical variables, with other morphological characteristics and with the implantation rate of cleavage stage embryos.
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5. Prediction of HIV-1 protease cleavage site from octapeptide sequence ...
8 nov 2022 · In this work, we built and compared the performance of selected machine learning models for the prediction of HIV-1 protease cleavage site utilizing a hybrid ...
In most parts of the world, especially in underdeveloped countries, acquired immunodeficiency syndrome (AIDS) still remains a major cause of death, disability, and unfavorable economic outcomes. This has necessitated intensive research to develop effective therapeutic agents for the treatment of human immunodeficiency virus (HIV) infection, which is responsible for AIDS. Peptide cleavage by HIV-1 protease is an essential step in the replication of HIV-1. Thus, correct and timely prediction of the cleavage site of HIV-1 protease can significantly speed up and optimize the drug discovery process of novel HIV-1 protease inhibitors. In this work, we built and compared the performance of selected machine learning models for the prediction of HIV-1 protease cleavage site utilizing a hybrid of octapeptide sequence information comprising bond composition, amino acid binary profile (AABP), and physicochemical properties as numerical descriptors serving as input variables for some selected machine learning algorithms. Our work differs from antecedent studies exploring the same subject in the combination of octapeptide descriptors and method used. Instead of using various subsets of the dataset for training and testing the models, we combined the dataset, applied a 3-way data split, and then used a "stratified" 10-fold cross-validation technique alongside the testing set to evaluate the models. Among the 8 models evaluated in the “stratified” 10-fold CV experiment, logistic regression,...
6. Endopeptidase Cleavage of Anti-Glomerular Basement ...
... Eric Daugas, Arnaud Lionet, Nassim Kamar, Cedric Rafat, Marek Myslivecek, Vladimir Tesar, Anders Fernstrom, Christian Kjellman, Charlotte Elfving, Stephen ...
Background: The prognosis for kidney survival is poor in patients presenting with circulating anti–glomerular basement membrane (GBM) antibodies and severe kidney injury. It is unknown if treatment with an endopeptidase that cleaves circulating and kidney bound IgG can alter the prognosis.
7. "Mutations Affecting Cleavage at the p10-capsid Protease Cleavage Site ...
... Eric Barklis · Jonathan Leis. Document Type. Article. Publication Date. 2005 ... rate of cleavage of the junction. The effects of these substitutions on p10 ...
A series of amino acid substitutions (M239F, M239G, P240F, V241G) were placed in the p10-CA protease cleavage site (VVAM*PVVI) to change the rate of cleavage of the junction. The effects of these substitutions on p10-CA cleavage by RSV PR were confirmed by measuring the kinetics of cleavage of model peptide substrates containing the wild type and mutant p10-CA sites. The effects of these substitutions on processing of the Gag polyprotein were determined by labeling Gag transfected COS-1 cells with 35S-Met and -Cys, and immunoprecipitation of Gag and its cleavage products from the media and lysate fractions. All substitutions except M239F caused decreases in detectable Gag processing and subsequent release from cells. Several of the mutants also caused defects in production of the three CA proteins. The p10-CA mutations were subcloned into an RSV proviral vector (RCAN) and introduced into a chick embryo fibroblast cell line (DF-1). All of the mutations except M239F blocked RSV replication. In addition, the effects of the M239F and M239G substitutions on the morphology of released virus particles were examined by electron microscopy. While the M239F particles appeared similar to wild type particles, M239G particles contained cores that were large and misshapen. These results suggest that mutations affecting cleavage at the p10-CA protease cleavage site block RSV replication and can have a negative impact on virus particle morphology.
8. Thio-assisted reductive electrolytic cleavage of lignin β-O-4 models ...
... cleavage of lignin β-O-4 models and authentic lignin†. Check for updates. Zhen Fang, ORCID logo ab Michael G. Flynn,a James E. Jackson · ORCID logo *c and Eric ...
Avoiding the use of expensive catalysts and harsh conditions such as elevated temperatures and high pressures is a critical goal in lignin depolymerization and valorization. In this study, we present a thio-assisted electrocatalytic reductive approach using inexpensive reticulated vitreous carbon (RVC) as th
9. The different role of high- and low-affinity metal ions in cleavage ...
Natalia Kisseleva, Anastasia Khvorova, Eric Westhof, Olav Schiemann, Alexey D. ... The tsHHRz cleavage rate is up to 20-fold faster than that of the mHHRz ...
The aim of this study was to investigate the dependence of the observed cleavage rates (k(obs)) of a tertiary stabilized hammerhead ribozyme (tsHHRz) and of a minimal hammerhead ribozyme (mHHRz), both derived from tobacco ringspot virus, on the type and concentration of divalent metal ions in order to interpret the functional role of high-affinity ions detected by electron paramagnetic resonance (EPR). To measure the fast cleavage of the cis tsHHRz, a new method using chemically synthesized fluorescent-labeled RNAs has been developed. The tsHHRz cleavage rate is up to 20-fold faster than that of the mHHRz under similar conditions. The presence of Mn2+ ions leads to a 60-fold faster cleavage than in the presence of Mg2+ ions. The functional role of the high-affinity ion was evaluated using neomycin B inhibition studies. Neomycin B reduces the cleavage activity of both ribozymes but the inhibitory effect on tsHHRz is much weaker than that on the mHHRz. EPR data had shown that neomycin B displaces both low-affinity and high-affinity Mn2+ ions from the mHHRz, but only low-affinity ions from tsHHRz. Inhibition of the tsHHRz activity may be due to the displacement of weakly bound Me2+ ions required for the local folding leading to cleavage, whereas both the high-affinity ion required for folding and the weakly bound ions are replaced in the mHHRz. The high-affinity metal ion is required for the stabilization of the global HHRz structure, but is not involved in catalysis or stabiliz...
10. Unravelling key enzymatic steps in C-ring cleavage during angucycline ...
18 dec 2023 · ... cleavage of their tetracyclic backbone. While the enzymes ... In this regard, full scan MS spectra (m/z 100–1700, scan rate 10 Hz ...
Angucyclines are type II polyketide natural products, often characterized by unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone. While the enzymes involved in B-ring cleavage have been extensively studied, little is known of the enzymes leading to C-ring cleavage. Here, we unravel the function of the oxygenases involved in the biosynthesis of lugdunomycin, a highly rearranged C-ring cleaved angucycline derivative. Targeted deletion of the oxygenase genes, in combination with molecular networking and structural elucidation, showed that LugOI is essential for C12 oxidation and maintaining a keto group at C6 that is reduced by LugOII, resulting in a key intermediate towards C-ring cleavage. An epoxide group is then inserted by LugOIII, and stabilized by the novel enzyme LugOV for the subsequent cleavage. Thus, for the first time we describe the oxidative enzymatic steps that form the basis for a wide range of rearranged angucycline natural products. Angucyclines are a class of natural products that harbor unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone, however, the enzymes leading to C-ring cleavage remain poorly understood. Here, the authors use targeted gene deletion and complementation as well as metabolomics to study the function of putative oxygenases involved in lugdunomycin biosynthesis, and reveal their potential roles towards C-ring cleavage.
11. Reaction Mechanisms and Rate Constants of Auto-Catalytic ...
For the non-catalytic urethane formation and cleavage reactions, the dominating reaction mechanism has long been debated. To our knowledge, the reaction ...
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12. FASTKD5 processes mitochondrial pre-mRNAs at non-canonical ...
18 jul 2024 · Hillen, View ORCID ProfileEric A. Shoubridge. doi: https://doi ... For the cleavage assays, the RNA cleavage assay buffer and FASTKD5 ...
The regulation of mammalian mitochondrial gene expression is largely post-transcriptional and the first step in translating the 13 polypeptides encoded in mtDNA is endonucleolytic cleavage of the primary polycistronic transcripts. As the rRNAs and most of the mRNAs in mtDNA are flanked by tRNAs, the release of the mature RNAs occurs mostly by excision of the tRNAs. Processing the non-canonical mRNAs, not flanked by tRNAs, requires FASTKD5, but the molecular mechanism remains unknown. To investigate this, we created and characterized a knockout cell line to use as an assay system. The absence of FASTKD5 resulted in a severe combined OXPHOS assembly defect due to the inability to translate mRNAs with unprocessed 5’-UTRs. Analysis of RNA processing of FASTKD5 variants allowed us to map amino acid residues essential for function. Remarkably, this map was RNA substrate-specific, arguing against a one size fits all model. A reconstituted in vitro system with purified FASTKD5 protein and synthetic RNA substrates showed that FASTKD5 on its own was able to cleave client substrates correctly, but not non-specific RNA sequences. These results establish FASTKD5 as the missing piece of the biochemical machinery required to completely process the primary mitochondrial transcript. ### Competing Interest Statement The authors have declared no competing interest.
13. Fresh MESA improved embryo fertilization, cleavage, blastula ...
Rovner, Eric Gonzalez, David Weiner ... Live Birth Rate 25%. Blastulation 25%. Blastulation Rate 25%. Prospective Observational Trial 25%. Cleavage Rate 25%.
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