Roles of Ago1 isoforms in the shrimp immune response against WSSV infection.

<p>To characterize the roles of Ago1 isoforms in the antiviral immunity, shrimp were injected with WSSV and isoform-specific siRNAs. Shrimp were injected simultaneously with WSSV and the low or high concentration of Ago1A-siRNA (<b>A</b>), Ago1B-siRNA (<b>B</b>), Ago1A/B-siRNA (<b>C</b>) or Ago1C-siRNA (<b>D</b>), respectively. As control, WSSV+control siRNA and WSSV only were included in the injections. At 48 h post-inoculation, the shrimp from each treatment were subjected to quantitative real-time PCR to quantify the expressions of Ago1A, Ago1B, and Ago1C (left). The solutions used for injections were shown in the box. At the same time, the WSSV loads in shrimp were monitored by quantitative real-time PCR (right). The statistically significant differences between treatments were represented with asterisk (*P<0.05). Lane headings showed the solutions used for injections.</p

Southern blot and northern blot analysis of shrimp Ago1 isoforms.

<p>(<b>A</b>) Southern blot of shrimp genomic DNA with DIG-labeled Ago1-probe that could detect three Ago1 isoforms or Ago1-fragment 2-probe that was unique to Ago1A and Ago1B. (<b>B</b>) Northern blot of total RNAs extracted from shrimp gills. The probes used were shown on the top. The upper band likely consisted of co-migrated Ago1A and Ago1B transcripts, while the lower band potentially represented the Ago1C transcript.</p

Identification of shrimp Ago1 isoforms.

<p>Schematic diagram of three isoforms (Ago1A, Ago1B, and Ago1C) of shrimp Ago1 gene. The numbers show the sites of Ago1-fragment 1 and Ago1-fragment 2 in Ago1.</p

Expression profiles of Ago1 isoforms in shrimp.

<p>(<b>A</b>) Expression patterns of Ago1 isoforms in different tissues or organs of shrimp as revealed by quantitative real-time PCR. The shrimp β-actin was used as an internal standard. The relative expression levels of Ago1A, Ago1B, and Ago1C mRNAs were compared with that of Ago1A in lymphoid organ. Each column represented the mean of triplicate assays within 1% standard deviation. (<b>B</b>) The time-course of expression profiles of Ago1 isoforms in lymphoid organ of shrimp challenged with WSSV by quantitative real-time PCR. The relative expression levels of Ago1A, Ago1B, and Ago1C mRNAs at various times post-inoculation (0, 12, 24, 48, and 72 h) were compared with that of Ago1A at 0 h post-inoculation. The numbers indicated the time points post-inoculation with WSSV. Each column represented the mean of triplicate assays within 1% standard deviation. The statistically significant differences between treatments were represented with an asterisk (*P<0.05).</p

Specificities of siRNAs targeting Ago1 isoforms.

<p>S2 cells were transiently co-transfected with the Flag-tagged Ago1 isoform constructs and the isoform-specific siRNAs. At 48 h after transfection, cell lysates were analyzed using western blot with anti-FLAG antibody. The β-actin was used as a control. Lane headings showed the FLAG-tagged Ago1 isoforms and the isoform-specific siRNAs. The Ago1A/B-siRNA could specifically target both Ago1A and Ago1B. The antibodies used were indicated on the left.</p

Amino acid alignments of shrimp Ago1 isoforms and Ago homologs from other species.

<p>The conserved PAZ and PIWI domains were boxed. Amino acid differences between shrimp Ago1 isoforms were highlighted with asterisks. <i>Homo sapiens</i>, Hs Ago1 (GenBank accession no. NP_036331.1); <i>Mus musculus</i>, Mm Ago1 (AAI29916.1); <i>Tribolium castaneum</i>, Tc Ago1 (XP_971295.2); <i>Bombyx mori</i>, Bm Ago1 (NP_001095931.1); <i>Drosophila melanogaster</i>, Dm Ago1 (NP_725341.1); Dm Ago2 (NP_Q9VUQ5); <i>Apis mellifera</i> Am Ago1 (XP_624444.3); <i>Litopenaeus vannamei</i>, Lv Ago1 (NP_ADK25180.1); Lv Ago2 (NP_ADK25181.1); <i>Marsupenaeus japonicus</i>, Mj Ago1.</p

Contribution of the Argonaute-1 Isoforms to Invertebrate Antiviral Defense

<div><p>Argonaute (Ago) protein, the central component of the RNA interference (RNAi) pathway, plays important roles in host innate antiviral immunity. Most organisms harbor a large number of different Ago proteins and isoforms; however, the roles of Ago isoforms in immune defense against pathogens remain unclear. In the present study, three Argonaute-1 (Ago1) isoforms, termed Ago1A, Ago1B, and Ago1C, were found in <em>Marsupenaeus japonicus</em> shrimp. Quantitative real-time PCR (polymerase chain reaction) revealed that isoforms Ago1A and Ago1B containing an insertion sequence in the PIWI domain, were significantly up-regulated in lymphoid organ and hemolymph, and also upon white spot syndrome virus (WSSV) challenge, indicating the involvement of Ago1A and Ago1B in antiviral immunity. The results showed that silencing of Ago1A with a sequence-specific siRNA led to a significant increase of WSSV loads. It was revealed that knockdown of Ago1B mRNA by 37–70% resulted in higher virus loads in shrimp. However, upon silencing Ago1B by more than 85%, a two-fold increase in Ago1A mRNA was observed but viral load was the same as untreated controls challenged with WSSV, suggesting that the simultaneous up-regulation of Ago1A might compensate for the loss of Ago1B. These data indicated that Ago1A played more important roles in the antiviral immune response than Ago1B. The simultaneous inhibition of Ago1A and Ago1B resulted in a greater increase in viral loads than Ago1A or Ago1B alone, indicating that Ago1A and Ago1B isoforms were involved in shrimp antiviral immunity. It was revealed that Ago1C had no effect on virus infection. Therefore, the current study presented the first report on the contribution of Ago isoforms in the invertebrate defense against virus infection.</p> </div

The importance of all features derived from lightGBM in the validation set.

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The SHAP force plots.

The two representative SHAP force plots of a (A) dead and (B) survival patient. SHAP force plots are effective in interpreting the prediction value of the model in critical instances. The contribution of each feature to the output predicted value is shown with arrows with their force associated with the shapley values. Red arrows indicate features increasing the prediction results (i.e., yield values) to reach the predicted value (output value). Blue arrows show features decreasing the prediction values to reach the same output value. The arrows with positive and negative effects on yield values compensate on a point which is the prediction (output) value.</p

Performance of the prediction models using all features.

Performance of the prediction models using all features.</p