Journal: iScience

Article Title: Broad sarbecovirus neutralization by combined memory B cell antibodies to ancestral SARS-CoV-2

doi: 10.1016/j.isci.2024.110354

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Article Snippet: pUNO-1 SARS-CoV-2 Spike (BA.4/5)-dfur expression vector , InvivoGen , #p1-spike-v13-df.

Techniques: Virus, Recombinant, Electron Microscopy, Staining, Mutagenesis, Silver Staining, Plasmid Preparation, Expressing, Software, Membrane, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Imaging, Microscopy, Transmission Assay, Microplate Reader Absorbance Measurement

Journal: Cell Reports

Article Title: An Ancient Protein Phosphatase, SHLP1, Is Critical to Microneme Development in Plasmodium Ookinetes and Parasite Transmission

doi: 10.1016/j.celrep.2013.01.032

Figure Lengend Snippet: Phenotypic Analyses of WT and Δ shlp1 Ookinetes, Related to Figure 4 (A) Ookinete invasion assay in A. gambiae L3-5 mosquitoes. Microscopy images show melanized ookinetes (arrows) present in mid-guts of mosquitoes infected with either WT control and Δ shlp1 parasites but not with Δ ctrp parasites. Scale bar = 50 μm. The number of melanized Δ shlp1 ookinetes was not significantly reduced compared to the wild-type, but significantly higher compared to the Δ ctrp mutant (Mann-Whitney U-test, ∗∗∗ = p < 0.001). (B) Montage of longitudinal sections of the anterior apical end of WT (three left panels) and Δ shlp1 ookinetes (seven right panels) emphasizing the marked reduction or absence in apical micronemes, whereas all other structures at the apical end appear normal. Bar = 100 nm. (C) Normalized wild-type RNA expression of female-specific genes ( cith, dozi and nek4), shlp1 and microneme specific genes ( cht1, ctrp and soap ), relative to arginyl-tRNA synthetase and hsp70 used as endogenous controls. Each point is the mean of three biological replicates consisting of three technical replicates ±SD (normalized using qBasePlus). (D) Relative RNA expression of female-specific genes ( cith, dozi, nek4 ) and shlp1 (relative to arginyl-tRNA synthetase and hsp70) in Δ shlp1 mutant parasites compared to wild-type controls. Each point is the mean of three biological replicates consisting of three technical replicates ±SD (normalized using qBasePlus). Asexual blood = ASB; Activated gametocytes = AG; Ookinete = OO. ∗ = p < 0.05; ∗∗ = p < 0.01.

Article Snippet: Wild-type and relative expression were determined using qBasePlus v1.3 (Biogazelle) ( ; ), with relative quantification in mutant lines normalized against wild-type expression.

Techniques: Invasion Assay, Microscopy, Infection, Control, Mutagenesis, MANN-WHITNEY, RNA Expression

Journal: Cell Reports

Article Title: An Ancient Protein Phosphatase, SHLP1, Is Critical to Microneme Development in Plasmodium Ookinetes and Parasite Transmission

doi: 10.1016/j.celrep.2013.01.032

Figure Lengend Snippet: Microneme Reduction and Mislocalization in Δshlp1 Ookinetes (A) (i) Transmission electron micrograph (TEM) of a longitudinal section through a WT ookinete showing the conical apical end. The cytoplasm contains a number of apically located micronemes (M) and a more posteriorly located nucleus (N). Scale bar, 1 μm. (ii) Longitudinal TEM section through a Δ shlp1 ookinete showing substantial reduction of micronemes and mislocalization of remaining micronemes at the posterior end. Bar = 1 μm. (iii) Enlargement of the anterior of a WT ookinete showing the complex nature of the apical end consisting of a conical shaped electron dense collar (C) with a central aperture and an underlying less electron dense ring (R). Micronemes (M) are located within the cytoplasm. Scale bar, 100 nm. (iv and v). Detail of the anterior of Δshlp1 ookinetes showing reduction in micronemes (M). Scale bar, 100 nm. (B) Indirect immunofluorescence of a number of motor complex and micronemal proteins in WT and Δshlp1 ookinetes. Compared to WT, staining for micronemal proteins SOAP and CTRP was reduced in the Δ shlp1 mutant and spread throughout the cell body as opposed to the normal concentration at the apical end (arrow). No difference was observed between WT and mutant ookinetes for glideosomal GAP45 and MTIP. (C) Western blot analyses of ookinete micronemal proteins CTRP and SOAP. Expression levels were compared between WT and Δ shlp1 ookinetes and normalized to the GFP control using ImageJ software. CTRP expression was unaltered in the mutant whereas SOAP expression was reduced to 40%. (D) Relative RNA expression of microneme specific genes cht1 , ctrp , and soap in Δ shlp1 mutant parasites compared to WT controls. Each point is the mean of three biological replicates ±SD (normalized using qBasePlus). AG, activated gametocytes; OO, ookinete. ∗ p < 0.05; ∗∗ p < 0.01. See also Figure S3 .

Article Snippet: Wild-type and relative expression were determined using qBasePlus v1.3 (Biogazelle) ( ; ), with relative quantification in mutant lines normalized against wild-type expression.

Techniques: Transmission Assay, Immunofluorescence, Staining, Mutagenesis, Concentration Assay, Western Blot, Expressing, Control, Software, RNA Expression