In the present paper, results of X-ray photoelectron studies of electroceramic thin films of barium strontium titanate, Ba1-xSrxTiO3 (BST), composition deposited on stainless-steel substrates are presented. The thin films were prepared by the sol-gel method. A spin-coating deposition of BST layers with different chemical compositions was utilized so the layer-type structure of (0-2) connectivity was formed. After the deposition, the thin-film samples were heated in air atmosphere at temperature T = 700 °C for 1 h.
The surfaces of BST thin films subjected to thermal treatment were studied by X-ray diffraction. X-ray diffraction measurements confirmed the perovskite-type phase for all grown thin-film samples. The oxidation states of the elements were examined by the X-ray photoelectron spectroscopy method. X-ray photoelectron spectroscopy survey spectra as well as high-resolution spectra (photo-peaks) of the main metallic elements, such as Ti, Ba, and Sr, were compared for the layer-type structures, differing in the deposition sequence of the barium strontium titanate layers constituting the BST thin film.
Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach
The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme.
- In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP.
- The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme.
- The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated.
- Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.
Foot-and-Mouth disease Virus (FMDV) is a highly infectious RNA virus that causes severe economic losses in cloven-hoofed animals. Early detection is needed to control epidemics, and loop-mediated isothermal amplification (LAMP) can be performed using inexpensive and commonly available equipment with a short processing time, but existing assays for FMDV still require an additional reverse transcriptase enzyme to convert RNA to cDNA prior to amplification. We sought to develop a novel RT-LAMP assay for FMDV with carboxamide and N-alkylcarboxamide additives to reduce non-specific amplification in combination with an improved commercially available polymerase (Bst 3.0) with efficient reverse transcriptase activity.
SYBR Green I dye was used for sensitive visual detection of amplification products from our LAMP assay within 15 minutes without the need for a colorimeter. In the presence of a carefully titrated mixture of carboxamide and N-alkylcarboxamide additives, longer reactions of up to 1 hour were also possible on both RNA and cDNA without the appearance of non-specific amplification products, thereby increasing the potential robustness of the assay by allowing a greater window of time in which to detect weak positives.
High-Throughput NanoBiT-Based Screening for Inhibitors of HIV-1 Vpu and Host BST-2 Protein Interaction
Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy.
Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.
Recombinant expression, purification, and characterization of full-length human BST-2 from Escherichia coli
HIV-1 virus
from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express pLysY E. coli culture.
Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner,HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.
A high-specificity flap probe-based isothermal nucleic acid amplification method based on recombinant FEN1-Bst DNA polymerase
The COVID-19 pandemic has unfortunately demonstrated how easily infectious diseases can spread and harm human life and society. As of writing, pandemic has now been on-going for more than one year. There is an urgent need for new nucleic acid-based methods that can be used to diagnose pathogens early, quickly, and accurately to effectively impede the spread of infections and gain control of epidemics.
We developed a flap probe-based isothermal nucleic acid amplification method that is triggered by recombinant FEN1-Bst DNA polymerase, which-through enzymatic engineering-has both DNA synthesis, strand displacement and cleavage functions. This novel method offers a simpler and more specific probe-primer pair than those of other isothermal amplifications.
Bst Nickase |
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| M1235-1000 | Biovision | each | 268.8 EUR |
Bst DNA Polymerase |
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| S600 | GeneOn | 2000 U | 128.4 EUR |
BST-1 Polyclonal Antibody |
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| 41847-100ul | SAB | 100ul | 302.4 EUR |
BST-1 Polyclonal Antibody |
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| 41847-50ul | SAB | 50ul | 224.4 EUR |
BST-2 Polyclonal Antibody |
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| 41977-100ul | SAB | 100ul | 302.4 EUR |
BST-2 Polyclonal Antibody |
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| 41977-50ul | SAB | 50ul | 224.4 EUR |
BST-1 Polyclonal Antibody |
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| ABP50797-003ml | Abbkine | 0.03ml | 189.6 EUR |
BST-1 Polyclonal Antibody |
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| ABP50797-01ml | Abbkine | 0.1ml | 346.8 EUR |
BST-1 Polyclonal Antibody |
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| ABP50797-02ml | Abbkine | 0.2ml | 496.8 EUR |
BST-2 Polyclonal Antibody |
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| ABP56318-003ml | Abbkine | 0.03ml | 189.6 EUR |
BST-2 Polyclonal Antibody |
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| ABP56318-01ml | Abbkine | 0.1ml | 346.8 EUR |
BST-2 Polyclonal Antibody |
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| ABP56318-02ml | Abbkine | 0.2ml | 496.8 EUR |
BST-1 Polyclonal Antibody |
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| ABP53207-003ml | Abbkine | 0.03ml | 189.6 EUR |
BST-1 Polyclonal Antibody |
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| ABP53207-01ml | Abbkine | 0.1ml | 346.8 EUR |
BST-1 Polyclonal Antibody |
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| ABP53207-02ml | Abbkine | 0.2ml | 496.8 EUR |
BST-2 Polyclonal Antibody |
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| ABP53343-003ml | Abbkine | 0.03ml | 189.6 EUR |
BST-2 Polyclonal Antibody |
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| ABP53343-01ml | Abbkine | 0.1ml | 346.8 EUR |
BST-2 Polyclonal Antibody |
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| ABP53343-02ml | Abbkine | 0.2ml | 496.8 EUR |
BST-1 Polyclonal Antibody |
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| ES1796-100ul | ELK Biotech | 100ul | 334.8 EUR |
BST-1 Polyclonal Antibody |
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| ES1796-50ul | ELK Biotech | 50ul | 248.4 EUR |
BST-1 Polyclonal Antibody |
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| ES4206-100ul | ELK Biotech | 100ul | 334.8 EUR |
BST-1 Polyclonal Antibody |
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| ES4206-50ul | ELK Biotech | 50ul | 248.4 EUR |
BST-2 Polyclonal Antibody |
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| ES4342-100ul | ELK Biotech | 100ul | 334.8 EUR |
BST-2 Polyclonal Antibody |
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| ES4342-50ul | ELK Biotech | 50ul | 248.4 EUR |
BST-2 Polyclonal Antibody |
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| ES7317-100ul | ELK Biotech | 100ul | 334.8 EUR |
BST-2 Polyclonal Antibody |
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| ES7317-50ul | ELK Biotech | 50ul | 248.4 EUR |
Anti-BST-1 antibody |
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| STJ91903 | St John's Laboratory | 200 µl | 236.4 EUR |
Anti-BST-2 antibody |
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| STJ91904 | St John's Laboratory | 200 µl | 236.4 EUR |
Anti-BST-1 antibody |
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| STJ96841 | St John's Laboratory | 200 µl | 236.4 EUR |
Anti-BST-2 antibody |
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| STJ97307 | St John's Laboratory | 200 µl | 236.4 EUR |
BST-1 Polyclonal Conjugated Antibody |
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| C41847 | SAB | 100ul | 476.4 EUR |
BST-2 Polyclonal Conjugated Antibody |
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| C41977 | SAB | 100ul | 476.4 EUR |
Bst DNA Polymerase Large Fragment |
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| P701-01 | Vazyme | 800 U | 146.4 EUR |
Bst DNA Polymerase Large Fragment |
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| P701-02 | Vazyme | 8000 U | 325.2 EUR |
Bst X I unit: 200 |
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| YRBSTX1 | Yeastern Biotech | 1 vial | Ask for price |
Bst DNA Polymerase, Large Fragment |
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| M1213-200 | Biovision | each | 346.8 EUR |
We tested the method’s ability to detect SARS-CoV-2 (both ORF1ab and N genes), rotavirus, and Chlamydia trachomatis. The limits of detection were 10 copies/μL for rotavirus, C. trachomatis, and SARS-CoV-2 N gene, and 100 copies/μL for SARS-CoV-2 ORF1ab gene. There were no cross-reactions among 11 other common pathogens with characteristics similar to those of the test target, and the method showed 100% sensitivity and 100% specificity in clinical comparisons with RT-PCR testing.
In addition to real-time detection, the endpoint could be displayed under a transilluminator, which is a convenient reporting method for point-of-care test settings. Therefore, this novel nucleic acid senor has great potential for use in clinical diagnostics, epidemic prevention, and epidemic control.