Journal: Veterinary Sciences

Article Title: Development and Evaluation of a Semi-Nested PCR Method Based on the 18S ribosomal RNA Gene for the Detection of Babesia aktasi Infections in Goats

doi: 10.3390/vetsci11100466

Figure Lengend Snippet: Specificity of semi-nested PCR. Agarose gel electrophoresis of semi-nested PCR products (438 bp) from Babesia - and Theileria -positive reference controls and field DNA samples using B. aktasi -specific primers. M, 100 bp marker; lines 1 and 2, negative controls (1, PCR-grade water; 2, genomic DNA obtained from a one-month-old goat not infected with Babesia , Theileria , or Anaplasma species); lines 3–11, standard positive-control DNA samples (3, B. aktasi ; 4, B. ovis ; 5, B. motasi ; 6, B. crassa ; 7, B. divergens ; 8, B. venatorum ; 9, B. capreoli ; 10, T. ovis ; 11, T. annulata ); lines 12–14, field DNA samples collected from apparently healthy goats infected with B. aktasi .

Article Snippet: Plasmid DNAs of B. venatorum and B. capreoli were kindly provided by Professor Martin Pfeffer from Epidemiologie Biochemie, Universitat Leipzig, Germany.

Techniques: Nested PCR, Agarose Gel Electrophoresis, Marker, Infection, Positive Control

Journal: Nature Communications

Article Title: Structural-profiling of low molecular weight RNAs by nanopore trapping/translocation using Mycobacterium smegmatis porin A

doi: 10.1038/s41467-021-23764-y

Figure Lengend Snippet: a A schematic diagram of hsa-miR-21 translocation through MspA. A single MspA is inserted in a lipid membrane separating the cis and the trans chambers. The cis chamber was filled with 1.5 M KCl buffer and the trans chamber with a 1.5 M KCl or a 1 M CaCl 2 buffer. Hsa-miR-21 was added to cis with a final concentration of 200 nM. A transmembrane potential of +150 mV was continuously applied. b Current–voltage ( I – V ) curves of MspA in the presence of 1.5 M KCl (black) or 1 M CaCl 2 (red) in trans . Different combinations of electrolyte buffers were applied and no analytes were added. c A representative trace containing successive hsa-miR-21 translocations. The measurement was performed with a 1.5 M KCl buffer in both cis and trans . Dashed box: a zoomed-in view of the section marked with a triangle on the trace. The open pore current ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${I}_{o}$$\end{document} I o ), blockage current ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${I}_{b}$$\end{document} I b ), dwell time ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t}_{{off}}$$\end{document} t o f f ) and inter-event duration ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t}_{{on}}$$\end{document} t o n ) are marked. d A representative trace containing successive hsa-miR-21 translocations. The measurement was performed with a 1.5 M KCl buffer in cis and a 1 M CaCl 2 buffer in trans . Dashed box: a zoomed-in view of the section marked with a triangle on the trace. In this condition, translocation events appear more frequently and are systematically retarded when c ompared with those in c . e Scatter plot of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\% {I}_{b}$$\end{document} % I b versus \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t}_{{off}}$$\end{document} t o f f for hsa-miR-21 translocations and corresponding histogram of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\% {I}_{b}$$\end{document} % I b . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\% {I}_{b}$$\end{document} % I b is defined as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$({{I}_{o}-I}_{b})/{I}_{o}$$\end{document} ( I o − I b ) / I o . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\% {I}_{b}$$\end{document} % I b is larger and more uniformly distributed when a 1 M CaCl 2 buffer in trans was applied (red). f The event histogram of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t}_{{off}}$$\end{document} t o f f for hsa-miR-21 translocations. g The event histogram of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t}_{{on}}$$\end{document} t o n for hsa-miR-21 translocations. The histogram in f and g was single exponential fitted according to the equation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y=a\ast\exp(-x/\tau)$$\end{document} y = a * exp ( − x / τ ) . The mean dwell time ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tau }_{{off}}$$\end{document} τ o f f ) or the mean inter-event interval ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\tau }_{{on}}$$\end{document} τ o n ) was respectively derived from the fitting results. Events with a dwell time <1 ms were ignored during the statistics.

Article Snippet: The plasmid DNAs encoding M1 or M2 MspA were custom synthesized by Genescript (New Jersey) and have been shared via https://www.molecularcloud.org/s/shuo-huang .

Techniques: Translocation Assay, Membrane, Concentration Assay, Derivative Assay

Journal: Nature Communications

Article Title: Structural-profiling of low molecular weight RNAs by nanopore trapping/translocation using Mycobacterium smegmatis porin A

doi: 10.1038/s41467-021-23764-y

Figure Lengend Snippet: a The scheme of nanopore trapping/translocation using MspA. Current fluctuations between trapping and translocation reveal the RNA identity. A tRNA was employed as an example. b Representative RNA molecules studied in this manuscript. Five types of RNAs, including miRNA (single stranded, 22 nt), overhanged siRNA (double-stranded, 21 bp), blunt siRNA (double-stranded, 21 bp), tRNA (L shaped, 76 nt), and 5 S rRNA (Y shaped, 120 nt) were investigated. The measurements were carried out as described in Methods. MiRNA (has-miR-21), overhanged siRNA (SiFoxA1), blunt siRNA (luciferase siRNA), or tRNA (tRNA phe ) were added to cis with a final concentration of 200 nM for each analyte. E.coli 5 S rRNA was added to cis with a final concentration of 10 nM. c Representative traces of successive translocations of miRNA (orange), overhanged siRNA (blue), blunt siRNA (green), tRNA (red), or 5 S rRNA (purple). The open pore current ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${I}_{o}$$\end{document} I o ) is marked with dashed lines. d Zoom-in views of representative translocation events from marked triangles of corresponding traces. Translocations of different RNA types result in highly distinguishable events features. MiRNA gives rise to fast spiky events. Overhanged siRNA produces two-step events. Blunt siRNA and tRNA both generate two types of events, termed type 1 and type 2. 5 S rRNA gives rise to three types of signals, which the most characteristic type is shown in the figure. The \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\% {I}_{b}$$\end{document} % I b refers to the first-level blockade amplitude which is defined as marked in d . e A scatter plot of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\% {I}_{b}$$\end{document} % I b versus \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${t}_{{off}}$$\end{document} t o f f for five RNA samples. Events from five types of RNAs are clearly distinguishable. f The corresponding event histogram of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\% {I}_{b}$$\end{document} % I b of different RNA types. Black lines are Gaussian fittings to the data. g A representative trace during simultaneous sensing of siRNA, tRNA, and 5 S rRNA. Different RNA types (overhanged siRNA: 25 nM; blunt siRNA: 10 nM; tRNA: 400 nM; 5 S rRNA: 30 nM) were simultaneously added to cis side. Characteristic events from different RNA types are clearly recognized from the trace, which are marked with blue, green red, or purple bars respectively.

Article Snippet: The plasmid DNAs encoding M1 or M2 MspA were custom synthesized by Genescript (New Jersey) and have been shared via https://www.molecularcloud.org/s/shuo-huang .

Techniques: Translocation Assay, Luciferase, Concentration Assay

Journal: Nature Communications

Article Title: Structural-profiling of low molecular weight RNAs by nanopore trapping/translocation using Mycobacterium smegmatis porin A

doi: 10.1038/s41467-021-23764-y

Figure Lengend Snippet: a–c Equilibrated structures of tRNA entering an MspA nanopore. The conformations are respectively termed as stem-down a , loop-down b , or arm-down c . The green sphere marked on each conformation referred to the leading nucleotide, which was used to characterize the position of tRNA in the following simulation results. d – f The z-coordinates of the leading nucleotide as a function of time for the simulations with the stem-down d , the loop-down e , and the arm-down f conformation. Seven trajectories were shown for each condition. The results show that the stem-down conformation has a higher tendency to translocate through the pore, while the other two conformations cannot reach the pore constriction to initiate a translocation. g Simulated cumulative ion currents through the pore at the open pore (green), the arm-down (gray), the stem-down (red), the loop-down (blue), and the tRNA translocation (purple) state. The external electric field was 0.09 V/10 nm, which corresponds to a voltage bias of ~+150 mV. The tRNA does not have obvious movement along the z -axis during the simulation timescale with such a low voltage. The slopes of the cumulative currents represent the ion current values. h The derived ionic currents for different states of sensing. All values were scaled so that the open pore current reports 1.

Article Snippet: The plasmid DNAs encoding M1 or M2 MspA were custom synthesized by Genescript (New Jersey) and have been shared via https://www.molecularcloud.org/s/shuo-huang .

Techniques: Translocation Assay, Derivative Assay

Journal: Nature Communications

Article Title: Structural-profiling of low molecular weight RNAs by nanopore trapping/translocation using Mycobacterium smegmatis porin A

doi: 10.1038/s41467-021-23764-y

Figure Lengend Snippet: All measurements were performed as described in Methods. Yeast tRNA was added to cis with a final concentration of 20 ng/μL. E.coli tRNA was added to cis with a final concentration of 2 ng/μL. Trace segmentation and event recognition were performed with the custom machine learning algorithm (Fig. ). a A representative trace containing successive yeast tRNA translocation through MspA. Two types of events, termed type 1 (blue triangle) and type 2 (red triangle), were observed, forming the majority of all events that were recorded. Dashed box: Zoomed-in views of representative type 1 and type 2 events, which are respectively marked with i and ii on the trace. The type 1 event has a single blockade level (level 1). The type 2 event contains two blockade levels (level 1 and level 2). b A representative trace containing successive E.coli tRNA translocation through MspA. Two types of events, termed type 1 (blue triangle) and type 2(red triangle) events, were also observed, forming the majority of all events that were recorded. Dashed box: Zoomed-in views of representative type 1 and type 2 events, which are respectively, marked with i and ii on the trace. c The event histogram of blockade amplitude of type 1 and type 2 events (gray: tRNA phe . red: yeast total tRNA. green: E.coli total tRNA). Please note that the current fluctuations between level 1 and 2 show slight variations between events. This variation of fluctuation is more clearly observed in measurements with total tRNAs than those with tRNA phe (Supplementary Fig. ). However, the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\% {I}_{b}$$\end{document} % I b of level 1 and 2 of type 2 events are much more conserved. d Comparison of percentage blockage ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${I}_{p}$$\end{document} I p ) of type 1 and type 2 events acquired from different tRNA samples. e The proportion of characteristic tRNA events from different sources of tRNAs. Error bars in d and e represent standard deviation, n = 3 independent replicates.

Article Snippet: The plasmid DNAs encoding M1 or M2 MspA were custom synthesized by Genescript (New Jersey) and have been shared via https://www.molecularcloud.org/s/shuo-huang .

Techniques: Concentration Assay, Translocation Assay, Comparison, Standard Deviation

Journal: Nature Communications

Article Title: Structural-profiling of low molecular weight RNAs by nanopore trapping/translocation using Mycobacterium smegmatis porin A

doi: 10.1038/s41467-021-23764-y

Figure Lengend Snippet: a Isolation of LMW RNA from E.coli . (I) E.coli pellets were lysed in the RNAiso buffer (Takara). (II) LMW RNA was extracted with chloroform and retained in the supernatant. (III) After centrifugation, the supernatant was collected and added with isopropanol to precipitate all LMW RNA. (IV) The precipitant was collected and washed with 75% ethanol. (V) The LMW RNA was dissolved in ribonuclease (RNase)-free water. b Denaturing urea polyacrylamide gel electrophoresis (Urea-PAGE) analysis of E.coli LMW RNA, extracted as described in a . L1: low range RNA ladder. L2-L4: E.coli LMW RNA. The band corresponding to tRNA is marked on the gel. The uncropped gel is provided in Supplementary Fig. . c A representative trace containing successive translocation of E.coli LMW RNA through MspA. Characteristic tRNA type 1 and type 2 events are respectively marked with blue and red triangles. d Zoomed-in views of representative translocation events in c , which were marked with i and ii on the trace. e The proportion of tRNA translocation events (purple: 5 S rRNA. red: tRNA. gray: others). 48% of all acquired events were recognized as either tRNA type 1 or tRNA type 2 events. Error bars represent standard deviation, n = 3 independent replicates. The measurements in c – e were performed as described in Methods. E.coli LMW RNA was added to cis with a final concentration of 40 ng/μL. Trace segmentation and event recognition were performed with the custom machine learning algorithm (Fig. ).

Article Snippet: The plasmid DNAs encoding M1 or M2 MspA were custom synthesized by Genescript (New Jersey) and have been shared via https://www.molecularcloud.org/s/shuo-huang .

Techniques: Isolation, Centrifugation, Polyacrylamide Gel Electrophoresis, Translocation Assay, Standard Deviation, Concentration Assay