Inactivating Activities and Mechanism of Imidazo[1,2‑<i>c</i>]pyrimidin-5(6<i>H</i>)‑one Nucleoside Derivatives Incorporating a Sulfonamide Scaffold

Twenty-eight imidazo[1,2-c]pyrimidin-5(6H)-one nucleoside derivatives incorporating a sulfonamide scaffold with preferable inactivating activities on pepper mild mottle virus (PMMoV) were designed and synthesized. Then, compound B29 with illustrious inactivating activity against PMMoV was received on the basis of the three-dimensional quantitative structure–activity relationship (3D-QSAR) model, with the EC50 of 11.4 μg/mL, which was superior to ningnanmycin (65.8 μg/mL) and template molecule B16 (15.3 μg/mL). Furthermore, (1) transmission electron microscopy (TEM) indicated that B29 could cause severe fracture of virions; (2) microscale thermophoresis (MST) and molecular docking further demonstrated that B29 had faintish binding affinities with PMMoV CPR62A (Kd = 202.84 μM), PMMoV CPL144A (Kd = 141.57 μM), and PMMoV CPR62A,L144A (Kd = 332.06 μM) compared to PMMoV CP (Kd = 4.76 μM); and (3) western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results of pCB-GFP-PMMoV CPR62A, pCB-GFP-PMMoV CPL144A, and pCB-GFP-PMMoV CPR62A,L144A were consistent with MST and confocal. In brief, the above results indicated that the amino acids at positions 62 and 144 of PMMoV CP might be the key amino acid sites of B29 acted on

Additional file 3 of Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China

Additional file 3: Table S3. Primers and reaction conditions used in the characterization of the SSU rRNA gene of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Blastocystis sp. and gp60 gene

Additional file 1 of Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China

Additional file 1: Table S1. Specimens from wildlife at six zoos in Henan, China examined in this study

Additional file 2 of Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China

Additional file 2: Table S2. Nucleotide substitutions and indels at the ITS region of CHPM1 and CHDW1 genotypes

Primers used for analysis.

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Confocal analysis of transiently expressed P1-GFP in <i>N</i>. <i>benthamiana</i>. Bars, 50 μm.

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MeJA treatment alleviated the susceptibility of <i>AOCs</i>-silenced plants to TuMV.

A. Phenotype in TRV:00 and TRV:AOCs treated plants at 12 dpi. B. Effect of MeJA treatment on TuMV-infection in plants inoculated with TRV:00 or TRV:AOCs at 7 dpi. Plants were photographed under UV light. Bars, 2 cm. C. Accumulation of viral CP protein quantified by WB. Ponceau S-stained RBCL was used as a loading control. Tests were performed independently three times with similar results. D. Quantification of viral RNA levels by qRT-PCR. Means ± SD values are from three independent plants per treatment. **, Pt-test. (TIF)</p

Confocal microscopy analysis showing the localization of AOC.2-GFP within chloroplasts in the context of P1-Myc expression (A) and TuMV infection (B) at 3 dpi.

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qRT-PCR analysis of <i>AOCs</i> transcripts in TuMV-infected plants within 14 dpi.

Means ± SD values are from three independent plants per treatment. (TIF)</p

Silencing of <i>cpSRP54</i> by RNAi construct in <i>N</i>. <i>benthamiana</i>.

A. Phenotype of leaves inoculated with GUS RNAi construct (as control) and cpSRP54 hairpin RNAi construct at 3 dpi. Silencing of cpSRP54 did not cause obvious chlorosis. B. cpSRP54 mRNA level analysis by qRT-PCR in cpSRP54-silenced plants compared to control plants at 3 dpi. Means ± SD values are from three independent plants per treatment and were normalized against NbActin. **, Pt-test. C. TuMV-GFP infection in plants pretreated with GUS RNAi construct and cpSRP54 RNAi construct. Plants were photographed under UV at 7 dpi. Viral CP accumulation in systemic leaves was determined by WB. Actin served as a loading control. This experiment was repeated at least three times, and one representative result is shown. The protein levels were quantified by ImageJ. D. Relative viral RNA levels quantified by qRT-PCR. Means ± SD values are from three independent plants per treatment. **, Pt-test. (TIF)</p