Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the ...prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3' region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3' terminus of pegRNAs that enhance their stability and prevent degradation of the 3' extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3-4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3' structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3' motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Most genetic variants that contribute to disease
are challenging to correct efficiently and without excess byproducts
. Here we describe prime editing, a versatile and precise genome editing method ...that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The future trend in achieving precision medicine involves the development of non-invasive cancer biomarker sensors that offer high accuracy, low cost, and time-saving benefits for risk clarification, ...early detection, disease detection, and therapeutic monitoring. A facile approach for the synthesis of MoO3 nanosheets was developed by thermally oxidizing MoS2 nanosheets in air followed by thermal annealing. Subsequently, Au@MnO2 nanocomposites were prepared using a combined hydrothermal process and in situ chemical synthesis. In this study, we present a novel immunosensor design strategy involving the immobilization of antiHSP70 antibodies on Au@MnO2/MoO3 nanocomposites modified on a screen-printed electrode (SPE) using EDC/NHS chemistry. This study establishes HSP70 as a potential biomarker for monitoring therapeutic response during anticancer therapy. Impedance measurements of HSP70 on the Au@MnO2/MoO3/SPE immunosensor using EIS showed an increase in impedance with an increase in HSP70 concentration. The electrochemical immunosensor demonstrated a good linear response in the range of 0.001 to 1000 ng mL−1 with a detection limit of 0.17 pg mL−1 under optimal conditions. Moreover, the immunosensor was effective in detecting HSP70 at low concentrations in a lung adenocarcinoma cell line following Paclitaxel treatment, indicating its potential for early detection of the HSP70 biomarker in organ-on-a-chip and clinical applications.
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IJS, KILJ, NUK, UL, UM, UPUK
Using 31P nuclear magnetic resonance (NMR) spectroscopy, we monitor the competition between tri-n-butylphosphine (Bu3P) and various amine and phosphine ligands for the surface of chloride terminated ...CdSe nanocrystals. Distinct 31P NMR signals for free and bound phosphine ligands allow the surface ligand coverage to be measured in phosphine solution. Ligands with a small steric profile achieve higher surface coverages (Bu3P = 0.5 nm–2, Me2P-n-octyl = 2.0 nm–2, NH2Bu = >3 nm–2) and have greater relative binding affinity for the nanocrystal (binding affinity: Me3P > Me2P–n-octyl ∼ Me2P–n-octadecyl > Et3P > Bu3P). Among phosphines, only Bu3P and Me2P–n-octyl support a colloidal dispersion, allowing a relative surface binding affinity (K rel) to be estimated in that case (K rel = 3.1). The affinity of the amine ligands is measured by the extent to which they displace Bu3P from the nanocrystals (K rel: H2NBu ∼ N-n-butylimidazole > 4-ethylpyridine > Bu3P ∼ HNBu2 > Me2NBu > Bu3N). The affinity for the CdSe surface is greatest among soft, basic donors and depends on the number of each ligand that bind. Sterically unencumbered ligands such as imidazole, pyridine, and n-alkylamines can therefore outcompete stronger donors such as alkylphosphines. The influence of repulsive interactions between ligands on the binding affinity is a consequence of the high atom density of binary semiconductor surfaces. The observed behavior is distinct from the self-assembly of straight-chain surfactants on gold and silver where the ligands are commensurate with the underlying lattice and attractive interactions between aliphatic chains strengthen the binding.
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IJS, KILJ, NUK, PNG, UL, UM
This study assessed the expression of GATA3 in primary lung carcinomas and correlated it with tumor histology and immunostains routinely utilized in the work up of primary lung cancers. Tissue ...microarrays (TMAs) were constructed from a cohort of 184 non-small cell carcinomas, stained with GATA3, p40, TTF-1, and napsin A, and analyzed semi-quantitatively. All TMA cases with GATA3 expression were further analyzed using corresponding whole slide sections. Positive GATA3 staining was present in 16 cases (9%), including 7 squamous cell carcinomas (SqCCs) (4%), 4 adenocarcinomas (AdCs) (2%), 2 adenosquamous carcinomas (AdSqCs) (1%), 2 large cell carcinomas (LCCs) (1%), and 1 sarcomatoid carcinoma (SC) (<1%). Among tumor histotypes, SqCC was more likely to stain with GATA3 (7/49, 14%), while AdC was less likely (4/111, 4%) (p = 0.04). In GATA3-positive cases, high-level expression was observed in 9 cases (56%), including 5 p40-positive SqCCs (3 were nonkeratinizing), 1 p40-positive AdSqC (negative for TTF-1 and napsin A), and 1 AdC (solid), 1 LCC, and 1 SC, each negative for p40, TTF-1, and napsin A). Low-level GATA3 expression was found in 3 AdCs (1 was lepidic and 2 were acinar predominant), 2 SqCCs (keratinizing), 1 AdSqC, and 1 LCC. These findings indicate that GATA3 expression occurs in a minor but significant proportion of primary non-small cell lung carcinomas, most often involves SqCC, and tends to show increasing levels of expression in more poorly differentiated subtypes. Caution should be exercised when interpreting GATA3 expression, and a panel of immunostains should be utilized when assigning tumor origin.
•GATA3 is a sensitive biomarker for carcinomas of breast and urothelial origins, however its expression is not specific.•GATA3 expression is found in a relatively small but significant proportion of primary non-small cell lung carcinoma (NSCLCs), especially lung squamous cell carcinoma, which can potentially lead to a misdiagnosis of metastatic carcinoma.•Distinguishing primary NSCLCs from metastatic carcinoma is crucial for patient management.•Caution should be exercised when interpreting GATA3 expression in the setting of lung tumors, especially in the absence of clinical and imaging information.•A panel of immunostains should be utilized when assigning tumor origin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Existing methods detect the keypoints in a non-differentiable way, therefore they can not directly optimize the position of keypoints through back-propagation. To address this issue, we present a ...partially differentiable keypoint detection module, which outputs accurate sub-pixel keypoints. The reprojection loss is then proposed to directly optimize these sub-pixel keypoints, and the dispersity peak loss is presented for accurate keypoints regularization. We also extract the descriptors in a sub-pixel way, and they are trained with the stable neural reprojection error loss. Moreover, a lightweight network is designed for keypoint detection and descriptor extraction, which can run at 95 frames per second for <inline-formula><tex-math notation="LaTeX">640\times 480</tex-math></inline-formula> images on a commercial GPU. On homography estimation, camera pose estimation, and visual (re-)localization tasks, the proposed method achieves equivalent performance with the state-of-the-art approaches, while greatly reduces the inference time.
•Lentils are an excellent source of phenolic compounds.•21 phenolic compounds were identified.•Among them flavonols and flavanols contributed to the strong antioxidant activities.•Flavonols, not ...flavanols, were strong α-glucosidase and pancreatic lipase inhibitors.
Phenolic extracts from 20 Canadian lentil cultivars (Lens culinaris) were evaluated for total phenolic contents and composition, antioxidant activities (DPPH, FRAP, ORAC), and inhibitory properties against α-glucosidase and pancreatic lipase. Twenty one phenolic compounds were identified in the present study, with the majority being flavonoids, including kaempeferol glycosides, catechin/epicatechin glucosides and procyanidins. These phenolic compounds not only contributed significantly to the antioxidant activities, but they were also good inhibitors of α-glucosidase and lipase, two enzymes, respectively, associated with glucose and lipid digestion in the human intestine, thus contributing significantly to the control of blood glucose levels and obesity. More interestingly, it was the flavonols, not the flavanols, which showed the inhibitory activities against α-glucosidase and pancreatic lipase. Our result provides supporting information for developing lentil cultivars and functional foods with improved health benefits and suggests a potential role of lentil consumption in managing weight and control of blood glucose.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Understanding chemical bonding and conductivity at the electrode-molecule interface is key for the operation of single-molecule junctions. Here we apply the d-band theory that describes interfacial ...interactions between adsorbates and transition metal surfaces to study electron transport across these devices. We realized bimetallic Au electrodes modified with a monoatomic Ag adlayer to connect α,ω-alkanoic acids (HO
C(CH
)
CO
H). The force required to break the molecule-electrode binding and the contact conductance G
are 1.1 nN and 0.29 G
(the conductance quantum, 1 G
= 2e
/h ≈ 77.5 μS), which makes these junctions, respectively, 1.3-1.8 times stronger and 40-60-fold more conductive than junctions with bare Au or Ag electrodes. A similar performance was found for Au electrodes modified by Cu monolayers. By integrating the Newns-Anderson model with the Hammer-Nørskov d-band model, we explain how the surface d bands strengthen the adsorption and promote interfacial electron transport, which provides an alternative avenue for the optimization of molecular electronic devices.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Unextractable phenolics from plant foods and their role in health benefits have become increasingly important. Meal residues of three quinoa seeds free of fat and extractable phenolics were subjected ...to acid, alkaline, and enzymatic hydrolyses. The total and individual phenolic compounds released were analyzed, and 19 phenolics, predominantly phenolic acids and several flavonoids, were identified. The concentration of bound phenolics was highest in black quinoa followed by red and white, regardless of the hydrolysis method. Higher phenolic contents also showed stronger antioxidant activities and inhibition of α-glucosidase and pancreatic lipase activities. Carbohydrases, that is, pectinase, xylanase and feruloyl esterase, which effectively liberated bound phenolics are known to be secreted by colonic bacteria, suggesting potential antioxidant and anti-inflammatory effects by these compounds in the large intestine during colonic fermentation. These results can also be applied to treat foods high in bound phenolics to enhance bioaccessibility.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Herein, we report the employment of the MoMo quintuple bonded amidinate complex to stabilize Group 10 metal fragments {(Et3P)2M} (M=Pd, Pt) and give rise to the isolation of the unprecedented δ ...complexes. X‐ray analysis unambiguously revealed short contacts between Pd or Pt and two Mo atoms and a slight elongation of the MoMo quintuple bond in these two compounds. Computational studies show donation of the MoMo quintuple‐bond δ electrons to an empty σ orbital on Pd or Pt, and back‐donation from a filled Pd or Pt dπ orbital into the MoMo δ* level (LUMO), consistent with the Dewar–Chatt–Duncanson model.
Akin to alkenes and alkynes, which form metal π complexes upon coordinating to metal fragments, the MoMo quintuple‐bonded amidinate serves as a δ type of ligand binding Group 10 metal elements to give rise to the formation of the first examples of δ complexes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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