Journal: Advanced Science

Article Title: START: A Versatile Platform for Bacterial Ligand Sensing with Programmable Performances

doi: 10.1002/advs.202402029

Figure Lengend Snippet: Design and characterization of STARTs for tetracycline and MS2 coat protein. a) Detailed schematics of the tetracycline Apta‐trigger, Tetra_B3. The lower stem region of tetracycline aptamer subject to engineering is marked by a gray box. b) GFP fluorescence of toehold switch B and trigger RNA pairs in the absence (gray bars) and presence (green bars) of 12 µм tetracycline. Positive control (PC) used the toehold switch trigger B without the aptamer sequence, while negative control (NC) used the decoy RNA without any predicted interactions. c) GFP fluorescence output for switch B and Tetra_B3 pair for different concentrations of tetracycline inputs. d) Detailed schematics of the MS2 coat protein Apta‐trigger, MS2_C1. The lower stem region of MS2 aptamer subject to engineering is marked by a gray box. e) GFP fluorescence of toehold switch C and trigger RNA pairs without (gray bars) and with (green bars) MS2 induction, by 10 ng mL −1 anhydrotetracycline (aTc) treatment. PC used the toehold switch trigger C, and NC used the decoy RNA. f) GFP fluorescence output for switch C and MS2_C1 pair for different induction levels for MS2 expression. P‐values were determined by an unpaired t ‐test, with P > 0.05 designated by “ns”, P ≤ 0.05 designated by “*”, and P ≤ 0.0001 designated by “****”. Error bars are the SD from three biological replicates.

Article Snippet: The MS2 protein coding sequence was PCR amplified using the bacteriophage MS2 coat protein expression plasmid from Addgene (pHMM, #67717) as a template and inserted into a pACYCDuet plasmid with an aTc inducible promoter sequence via Gibson assembly.

Techniques: Fluorescence, Positive Control, Sequencing, Negative Control, Expressing

Journal: Advanced Science

Article Title: START: A Versatile Platform for Bacterial Ligand Sensing with Programmable Performances

doi: 10.1002/advs.202402029

Figure Lengend Snippet: Construction of Boolean logic circuits. a) Design of the two‐input OR gate for theophylline and MS2. b) GFP fluorescence output of the two‐input OR gate for different input combinations. c) Design of the two‐input AND gate for theophylline and MS2. d) GFP fluorescence output of the two‐input AND gate for different input combinations. e) Design of the NOT gate for theophylline using a 3WJ repressor. f) GFP fluorescence of the NOT gate in the absence and presence of theophylline input. Theophylline was treated at a concentration of 10 mм, and aTc was treated at a concentration of 10 ng mL −1 . Error bars represent the SD from three biological replicates.

Article Snippet: The MS2 protein coding sequence was PCR amplified using the bacteriophage MS2 coat protein expression plasmid from Addgene (pHMM, #67717) as a template and inserted into a pACYCDuet plasmid with an aTc inducible promoter sequence via Gibson assembly.

Techniques: Fluorescence, Concentration Assay

Journal: PLoS ONE

Article Title: Domain Dissection of AvrRxo1 for Suppressor, Avirulence and Cytotoxicity Functions

doi: 10.1371/journal.pone.0113875

Figure Lengend Snippet: (A) Symptoms caused by Xoo strains PXO99 A (wild type) (1), pHM1 (2), 2C10 (clone of RS105 genomic library) (3), 8E07 (clone of RS105 genomic library) (4), pHMavrRxo1 RS105 (5) and MgCl 2 (10 mM) (6) in Nicotiana benthamiana . Xoo strains [1×10 8 colony-forming units (cfu)/mL] were inoculated to N. benthamiana (4–6 weeks old) with a needleless syringe, and symptoms were measured at 2 days post-inoculation (dpi). (B) The phenotype of interactions between maize lines B73 and five AvrRxo1 clones: pHMavrRxo1 RS105 , pHMavrRxo1 RS85 , pHMavrRxo1 SDAU-1 , pHMavrRxo1 JSB2-24 and pHMavrRxo1 HNB8-47 . Xoo strains [1×10 8 cfu/mL] were infiltrated into B73 (4 weeks old) with a needleless syringe, and symptoms were measured at 2 dpi. Infiltration of B73 with PXO99 A containing avrRxo1 gene results in a hypersensitive response at 2 dpi. PXO99 A and PXO99 A (pHM1) produced no reaction. (C) Symptoms caused by five AvrRxo1 clones in Xoo strains PXO99 A (1) pHM1, (2) pHMavrRxo1 RS105 (3), pHMavrRxo1 RS85 (4), pHMavrRxo1 SDAU-1 (5), pHMavrRxo1 JSB2-24 (6), pHMavrRxo1 HNB8-47 (7), and PXO61 (8) in N. benthamiana . All experiments were repeated three times with similar results.

Article Snippet: The vector pHM1 was digested to completion with Bam HI (NEB, USA) and treated with calf intestinal alkaline phosphatase (NEB, USA).

Techniques: Clone Assay, Produced

Journal: PLoS ONE

Article Title: Domain Dissection of AvrRxo1 for Suppressor, Avirulence and Cytotoxicity Functions

doi: 10.1371/journal.pone.0113875

Figure Lengend Snippet: (A) The phenotype of interactions between maize lines B73 and deletion mutants of AvrRxo1. Infiltration of B73 with PXO99 A containing avrRxo1 gene results in HR at 2 dpi. PXO99 A (pHM1) as negative control. Fragments AvrRxo1(17–421), AvrRxo1(52–421), AvrRxo1(109–421), AvrRxo1(140–421), AvrRxo1(186–421), AvrRxo1(1–159), AvrRxo1(1–193), AvrRxo1(1–278), AvrRxo1(1–373), AvrRxo1(1–412), AvrRxo1(1–417), and AvrRxo1(1–418), abolished induce HR on B73, while fragments AvrRxo1(1–419) and AvrRxo1(1–420) produce HR on B73 at 2 dpi. (B) Suppressions of non-host HR by deletion mutants of AvrRxo1 in N. benthamiana . Infiltration of N. benthamiana with PXO99 A (pHM:avrRxo1) (1) and PXO99 A (pHM1) (2) as control. Fragments AvrRxo1(17–421) (3), AvrRxo1(52–421) (4), AvrRxo1(109–421) (5), AvrRxo1(140–421) (6), AvrRxo1(186–421) (7), AvrRxo1(1–412) (8), AvrRxo1(1–417) (9), AvrRxo1(1–418) (10), AvrRxo1(1–419) (11), AvrRxo1(1–420) (12) did not suppress the non-host HR caused by PXO99 A in N. benthamiana . RT-PCR was used to confirm the expression of avrRxo1 in inoculated leaves. All experiments were repeated three times with similar results.

Article Snippet: The vector pHM1 was digested to completion with Bam HI (NEB, USA) and treated with calf intestinal alkaline phosphatase (NEB, USA).

Techniques: Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: Domain Dissection of AvrRxo1 for Suppressor, Avirulence and Cytotoxicity Functions

doi: 10.1371/journal.pone.0113875

Figure Lengend Snippet: (A) The phenotype of interactions between maize lines B73 and four AvrRxo1 mutants: AvrRxo1(H71A), AvrRxo1(G165A), AvrRxo1(K166N), and AvrRxo1(nls). Mutants AvrRxo1(H71A) and AvrRxo1(G165A) induce HR at 2 dpi, however mutants AvrRxo1(K166N) and AvrRxo1(nls) are abolsihed to elicit HR on B73. (B) The phenotype of suppression of non-host HR in N. benthamiana caused by PXO99 A . PXO99 A (pHM1) was used as negative control and PXO99 A (pHM:avrRxo1) as positive control. PXO99 A containing AvrRxo1(H71A) (6) did not elicit the non-host HR in N. benthamiana , while PXO99 A containing the fragments AvrRxo1(G165A) (3), AvrRxo1(K166N) (4), and nls (5) can still elicit non-host HR. RT-PCR was used to confirm the expression of avrRxo1 in inoculated leaves. All experiments were repeated three times with similar results.

Article Snippet: The vector pHM1 was digested to completion with Bam HI (NEB, USA) and treated with calf intestinal alkaline phosphatase (NEB, USA).

Techniques: Negative Control, Positive Control, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: Domain Dissection of AvrRxo1 for Suppressor, Avirulence and Cytotoxicity Functions

doi: 10.1371/journal.pone.0113875

Figure Lengend Snippet: Strains and plasmids used in this study.

Article Snippet: The vector pHM1 was digested to completion with Bam HI (NEB, USA) and treated with calf intestinal alkaline phosphatase (NEB, USA).

Techniques: Isolation, Plasmid Preparation, Expressing

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: DEGs and PERP expression levels in Mkrn 2-KO cells and tissues. ( a ) Expression levels of DEGs for the Mkrn 2-KO and Mkrn 2-WT MEFs are analyzed by qRT-PCR. Data are presented and analyzed by the Student's t -test from three independent experiments performed in triplicate. ** Significant difference compared with WT group ( P < 0.01). 293T cells were co-transfected with an Mkrn2 shRNA (shMkrn2) or negative control (Mock) vector plus an MKRN2 expression vector or control vector. ( b ) qRT-PCR is used to analyze the mRNA expression of PERP . Data are presented as mean ± s.d. from three independent experiments performed in triplicate. ( c ) Western blot is used to analyze the protein expression of PERP, densitometric analysis of Western blot signals (right). ( b and c ) Results are presented and analyzed by the two-way ANOVA, *Significant difference at P < 0.05, **Significant difference at P < 0.01. ( d ) Correlation between MKRN 2 and PERP expression levels in GEO dataset GSE6872 (contains sperm samples), as analyzed by Pearson's correlation analysis. DEG: differential expression gene; Mkrn 2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to peripheral myelin protein 22 (PMP22); qRT-PCR: quantitative real-time polymerase chain reaction; s.d.: standard deviation; MEF: mouse embryonic fibroblast; GEO: Gene Expression Omnibus.

Article Snippet: The p53 expression vector (pHMT.wt p53) was a gift from James Sherley (catalog# 12139, Addgene plasmid, Boston, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, shRNA, Negative Control, Plasmid Preparation, Control, Western Blot, Quantitative Proteomics, Knock-Out, Real-time Polymerase Chain Reaction, Standard Deviation, Gene Expression

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: Correlation of PERP levels with spermatogenesis. ( a ) Relative expression levels of the Mkrn 2 in normal and OAT sperm samples from the GSE6872 dataset. Data are presented and analyzed by two-tailed, unpaired Student's t -test. ** Significant differences compared with WT group ( P < 0.01). ( b ) Enrichment and ranking of genes in the PERP -high and PERP -low expression groups of the GSE6872 dataset are assessed by GSEA analysis (C5: GO gene sets; BP: GO biological process; gene set: male gamete generation) and the corresponding clustering heatmap of genes that contribute most to enrichment score is on the right. ( c ) Enrichment and ranking of genes in the PERP -high and PERP -low expression groups of the GSE6872 dataset are assessed by GSEA analysis (H: hallmark gene set; gene set: spermatogenesis) and the corresponding clustering heatmap of genes that contribute most to enrichment score is on the right ( PERP- low datasets: GSM158476, GSE158477, and GSM158479 and PERP -high datasets: GSM158478, GSM158480, GSM158481, GSM158482, and GSM158483). ( d ) Correlations between spermatogenic signature scores and PERP expression levels are determined by Spearman's rank correlation analysis. Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; GSEA: gene set enrichment analysis; OAT: oligoasthenoteratozoospermia.

Article Snippet: The p53 expression vector (pHMT.wt p53) was a gift from James Sherley (catalog# 12139, Addgene plasmid, Boston, MA, USA).

Techniques: Expressing, Two Tailed Test, Knock-Out

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: Association of PERP and p53 expression levels in Mkrn 2-KO cells and tissues. ( a ) Expression levels of p53 are analyzed by GEO dataset GSE6872. Data are presented and analyzed by two-tailed, unpaired Student's t -test. * Significant difference compared with the normal group ( P < 0.05). ( b ) Expression levels of MKRN2, p53, and PERP in MEFs are analyzed by Western blot (left), densitometric analysis of Western blot signals (right). ( c ) Expression levels of p53 and PERP in MEFs transfected with a p53 vector or CON as indicated are analyzed by Western blot (left), densitometric analysis of Western blot signals (right). ( d ) PERP expression levels are determined by Western blot in 293T cells co-transfected with vector and control, Mkrn2 and CON, Mkrn2 and p53, respectively (left), densitometric analysis of Western blot signals (right). ( c and d ) Results are presented and analyzed by the two-way ANOVA from three independent experiments and each performed for three times. * Significant difference at P < 0.05, ** Significant difference at P < 0.01. CON: negative control vector; Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; GEO: Gene Expression Omnibus; MEF: mouse embryonic fibroblast.

Article Snippet: The p53 expression vector (pHMT.wt p53) was a gift from James Sherley (catalog# 12139, Addgene plasmid, Boston, MA, USA).

Techniques: Expressing, Two Tailed Test, Western Blot, Transfection, Plasmid Preparation, Control, Negative Control, Knock-Out, Gene Expression

Journal: Asian Journal of Andrology

Article Title: Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis

doi: 10.4103/aja.aja_76_19

Figure Lengend Snippet: p53/PERP expression levels in Mkrn 2-KO mouse testes. ( a ) TUNEL assay to assess the levels of apoptosis in Mkrn 2 KO versus WT mouse testis. Immunohistochemical staining of ( b ) p53 and ( c ) PERP expression in the testes of Mkrn 2-KO and Mkrn 2-WT mice. Scale bars=50 μm. ( d ) Western blot assay to test expression levels of MKRN2, p53, and PERP (left); and the different densitometric levels between two groups: Mkrn 2-KO and Mkrn 2-WT mouse testes are analyzed by the two-tailed, unpaired Student's t -test to compare the data of the two groups from three independent experiments (right). ** Significant difference at P < 0.01. Mkrn2: makorin-2; KO: knockout; WT: wild type; PERP: p53 apoptosis effector related to PMP22; MEF: mouse embryonic fibroblast; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: The p53 expression vector (pHMT.wt p53) was a gift from James Sherley (catalog# 12139, Addgene plasmid, Boston, MA, USA).

Techniques: Expressing, TUNEL Assay, Immunohistochemical staining, Staining, Western Blot, Two Tailed Test, Knock-Out