Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 1. IGFBP3 expression inhibited tumor growth and created a hypoxic environment, which induced HIF-2α to overcome the oxygen stress. (A) Heterotransplantation of P4-pBIG2i and P4-pBIG2i-hIGFBP3 in SCID mice. Doxycycline was started to stimulate IGFBP3 at 8 d. Xenograft tumor sizes were measured, and the mice were sacrificed at 12, 15, 22, 28, and 36 d after the implantation. (Five mice per group in each time point. The error bars represent the SDs; ***P < 0.0005.) (B) Photographs of xenograft tumors. (C) Western blot analysis and signal quantitative detections of IGFBP3, HIF-1α, and HIF-2α expressions in xenograft tumors at 15 and 28 d. Cyclophilin A (CypA) as the loading control. The signals of Western blot were quantified and analyzed by Image Studio Lite version 5.2. Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Expressing, Western Blot, Control

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 2. Immunohistochemistry staining of xenografts on 12, 15, and 28 d after the tumor implantation. (A) Heterotransplantation tumors P4-pBIG2i and P4-pBIG2i-hIGFBP3 at 12, 15, and 28 d post-tumor implantation on IGFBP3 (100 ×), HIF-1α (40 × and 100 ×), HIF-2α (40 × and 100 ×), HO-1 (100 ×), and VHL (100 ×). P4-pBIG2i xenograft showed no IGFBP3 expressions, while P4-pBIG2i-hIGFBP3 showed a strong IGFBP3 expression after adding doxycycline (started on d 8). HIF-2α increased over time in P4-pBIG2i xenograft, but it only accumulated after 28 d in P4-pBIG2i-hIGFBP3 xenograft. Xenograft tumors without IGFBP3 expressed HIF-2α majorly, especially under a prolonged tumor growth in hypoxic environments. (B) Quantitative analysis of immunohistochemical staining. Images of IHC were taken by Motic Image Plus 3.0 software. The signals of IHC were quantified and analyzed by ImageJ 1.53 k software. Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Immunohistochemistry, Staining, Tumor Implantation, Expressing, Immunohistochemical staining, Software

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 3. Immunocytochemistry staining of A549, P0, and P4 cell cultures under normoxic and hypoxic conditions for 17 h. (A) Under normoxic conditions, P0 expressed more IGFBP3 than P4. P0 showed an increase in HIF-1α, while P4 showed a higher increase in HIF-2α under hypoxia than that under normoxia, indicating that tumor cells without IGFBP3 induced more HIF-2α under acute hypoxic conditions. The A549 cell culture that expressed HIF-2α under hypoxic conditions was used as the study control. (B) Quantitative analysis immunohistochemical staining. Images of ICC were taken by Motic Image Plus 3.0 software. The signals of IHC were quantified and analyzed by ImageJ software. Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Immunocytochemistry, Staining, Cell Culture, Control, Immunohistochemical staining, Software

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 4. Protein expression analysis of cell cultures under normoxic and hypoxic conditions. (A) Western blot analysis of IGFBP3, HIF-1α and HIF-2α, HO-1, VHL, and GAPDH expressions in P0, P4, P4 transfectants, and A549 under normoxic and hypoxic conditions. HSP90 was added as the control. (B) Western blot results were transformed into bar figures using Image Studio Lite version 5.2. Signal strengths were quantified and normalized with HSP90. P0 had a higher IGFBP3 expression than P4. In P0 and P4-I, IGFBP3 expression was increased under hypoxia. Accumulation of HIF-1α or HIF-2α correlated with IGFBP3 expressions: a higher IGFBP3 was with a stronger HIF-1α. Both HO-1 and VHL expressions were increased under hypoxia. Each sample was assayed in triplicates, and the experiment was repeated three times independently. (The error bars represent the SDs; **P < 0.001, ***P < 0.0005). Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Expressing, Western Blot, Control, Transformation Assay

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 5. IGFBP3 differentially regulated HIF-1α and HIF-2α synthesis in low and high invasion ability cells. Gene expression and regulation of IGFBP3, HIF-1α, and HIF-2α in cells cultured under normoxic and hypoxic conditions. (A) qPCR analyzed of IGFBP3, HIF-1α, and HIF-2α mRNA expressions in P0 and P4 cultured under normoxic and hypoxic conditions. GAPDH as the normalized control. P4 expressed higher HIF-1α and HIF-2α under normoxia, and P0 expressed lower HIF-2α compare with P4. (B) Luciferase promoter assays on the regulations of IGFBP3, HIF-1α, and HIF-2α promoters in P0 and P4 under normoxic and hypoxic conditions. The table below indicates the presence of endogenous or exogenous IGFBP3. In high-IGFBP3 expression P0, HIF-1α was more activated than HIF-2α at hypoxia. Both HIF-1α and HIF-2α were reduced after depletion of extracellular IGFBP3 by neutralizing antibodies (lanes 4 and 6). In low-IGFBP3 expression P4, both HIF-1α and HIF-2α activities increased under hypoxia. There were no changes in HIF-1α and HIF-2α expressions after adding recombinant hIGFBP3 (lanes 8 and 10). Each sample was assayed and analyzed repeated three times independently. (The error bars represent the SDs; **P < 0.001, ***P < 0.0005.). Excel 2016 was used to generate charts. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Gene Expression, Cell Culture, Control, Luciferase, Expressing, Recombinant

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 7. Clinical roles of HIF-1α and HIF-2α related to IGFBP3 expression in EOC. In an early stage of EOC, IGFBP3 expresses and inhibits proliferation and angiogenesis. The lack of blood vessels causes tumors to enter a hypoxic state, which stimulates cells to activate HIFs. HIF-1α is the major HIFs in this tumor stage. HIF-1α stimulates angiogenesis, proliferation, and induces IGFBP3 synthesis. The continuous expression of IGFBP3 prolongs the hypoxic state of the tumors by inhibiting tumor vasculogenesis. Prolonged hypoxia causes the promoter to be methylated and silence the expression of IGFBP3. As IGFBP3 decreases, the cells begin to proliferate and switch to activate HIF-2α majorly under hypoxic conditions. HIF-2α plays a significant role in cancer aggressiveness by regulating angiogenesis, invasion, and metastasis. Henceforth, the cancer cells mainly express HIF-2α under hypoxic stress. The decrease in IGFBP3 and the activation of HIF-2α instead of HIF-1α accelerate the progression in EOC. PowerPoint 2016 and Photoshop CS2 version 9 were used to generate figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Expressing, Methylation, Activation Assay

Journal: Scientific reports

Article Title: Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

doi: 10.1038/s41598-021-02400-1

Figure Lengend Snippet: Figure 6. Low IGFBP3 expression is associated with a high expression of angiogenic proteins related to HIF-2α under hypoxia. (A) Dot data of angiogenesis antibody array in P0, P4, P4-V, and P4-I lysates under normoxia and hypoxia. IL-8 showed a similar expression pattern as VEGF. Both IL-8 and VEGF were more expressed under hypoxia than in normoxia. (B) Expression signals were presented in categories by importance related to hypoxia and functions related to vasculogenesis. (IL-8 was negatively correlated with HIF-1α and positively correlated with HIF-2α. The proteins listed: IFNα to IL-4 are proteins with anti-angiogenic abilities; angiogenin to VEGF-R3 are known as angiogenic proteins.) The dot signals were analyzed with Image Studio Lite version 5.2, and the relative expression levels were compared between the groups. Red represents a higher signal performance, while green represents a lower signal performance than the comparison cell. Excel 2016 was used to generate chart. Photoshop CS2 version 9 was used to assemble the figure.

Article Snippet: The pGL3-IGFBP3, pGL3HIF-1α, pGL3-HIF-2α, or empty pGL3 plasmids were transiently transfected into P0 and P4 (500 ng plasmid per 5 × 104 cells) and incubated for 16 h. Recombinant IGFBP3 proteins (50 ng/mL; 675-B3; R&D Systems, Minneapolis, MN, USA) or IGFBP3 neutralizing antibodies (5 ng/mL; MAB305; R&D Systems, Minneapolis, MN, USA) were added and cultured in hypoxic or normoxic conditions for 16 h. FLUOstar Omega (BMG LABTECH, Offenburg, Germany) measured the luciferase activities, following the protocol of Luc-Pair Firefly Luciferase HS Assay Kit (Genecopoeia, Rockville, MD, USA).

Techniques: Expressing, Ab Array, Comparison

Journal: Pharmaceutics

Article Title: Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies

doi: 10.3390/pharmaceutics13091409

Figure Lengend Snippet: Mass spectrometric conditions for cyclobenzaprine, desmethyl cyclobenzaprine and cyclobenzaprine N-oxide. ESI: electrospray ionization, m/z: mass-to-charge ratio, msec: millisecond.

Article Snippet: The simultaneous quantification of cyclobenzaprine hydrochloride (≥98%, Hetero drugs Ltd., Hyderabad, India), its main metabolite desmethyl cyclobenzaprine hydrochloride (99.8%, Toronto Research Chemicals, Toronto, Canada) and cyclobenzaprine N-oxide (96%, Toronto Research Chemicals, Toronto, Canada) as its major degradation product was performed by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Shimadzu Prominence, Shimadzu Europe, Duisburg, Germany; AB Sciex API 2000, Darmstadt, Germany).

Techniques:

Journal: Pharmaceutics

Article Title: Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies

doi: 10.3390/pharmaceutics13091409

Figure Lengend Snippet: LC-MS/MS chromatogram of desmethyl cyclobenzaprine, cyclobenzaprine, cyclobenzaprine-d3 and cyclobenzaprine N-oxide with the respective structural formula.

Article Snippet: The simultaneous quantification of cyclobenzaprine hydrochloride (≥98%, Hetero drugs Ltd., Hyderabad, India), its main metabolite desmethyl cyclobenzaprine hydrochloride (99.8%, Toronto Research Chemicals, Toronto, Canada) and cyclobenzaprine N-oxide (96%, Toronto Research Chemicals, Toronto, Canada) as its major degradation product was performed by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Shimadzu Prominence, Shimadzu Europe, Duisburg, Germany; AB Sciex API 2000, Darmstadt, Germany).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Pharmaceutics

Article Title: Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies

doi: 10.3390/pharmaceutics13091409

Figure Lengend Snippet: Summary of accuracy and precision results for cyclobenzaprine, desmethyl cyclobenzaprine and cyclobenzaprine N-oxide (accuracy presented as mean relative error and precision as coefficient of variation, n = 5 per run). CV, coefficient of variation; LLOQ, Lower limit of quantification; QC, quality control.

Article Snippet: The simultaneous quantification of cyclobenzaprine hydrochloride (≥98%, Hetero drugs Ltd., Hyderabad, India), its main metabolite desmethyl cyclobenzaprine hydrochloride (99.8%, Toronto Research Chemicals, Toronto, Canada) and cyclobenzaprine N-oxide (96%, Toronto Research Chemicals, Toronto, Canada) as its major degradation product was performed by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Shimadzu Prominence, Shimadzu Europe, Duisburg, Germany; AB Sciex API 2000, Darmstadt, Germany).

Techniques: Control

Journal: Pharmaceutics

Article Title: Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies

doi: 10.3390/pharmaceutics13091409

Figure Lengend Snippet: Impact of excipient addition in preformulation and formulation development of cyclobenzaprine. A : Cumulative amount of permeated drug per cm 2 of the respective sublingual tablet (mean ± SEM; n ≥ 5). B : Cumulative amount of permeated drug per cm 2 of the respective solution (mean ± SEM; n ≥ 5) adapted from , Int. J. Pharm. 2021. C : Dissolution of the respective sublingual tablet (mean ± SEM; n = 3). D : Correlation of obtained cyclobenzaprine permeability with the added amount of dibasic phosphate (mean ± SEM). R 2 : determination coefficient, SEM: standard error of the mean, *: significant value (p < 0.05; unpaired t-test).

Article Snippet: The simultaneous quantification of cyclobenzaprine hydrochloride (≥98%, Hetero drugs Ltd., Hyderabad, India), its main metabolite desmethyl cyclobenzaprine hydrochloride (99.8%, Toronto Research Chemicals, Toronto, Canada) and cyclobenzaprine N-oxide (96%, Toronto Research Chemicals, Toronto, Canada) as its major degradation product was performed by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Shimadzu Prominence, Shimadzu Europe, Duisburg, Germany; AB Sciex API 2000, Darmstadt, Germany).

Techniques: Formulation, Dissolution, Permeability

Journal: Pharmaceutics

Article Title: Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies

doi: 10.3390/pharmaceutics13091409

Figure Lengend Snippet: Disintegration of cyclobenzaprine sublingual tablets after addition of 150 µL freshly collected human saliva in dependence on time.

Article Snippet: The simultaneous quantification of cyclobenzaprine hydrochloride (≥98%, Hetero drugs Ltd., Hyderabad, India), its main metabolite desmethyl cyclobenzaprine hydrochloride (99.8%, Toronto Research Chemicals, Toronto, Canada) and cyclobenzaprine N-oxide (96%, Toronto Research Chemicals, Toronto, Canada) as its major degradation product was performed by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Shimadzu Prominence, Shimadzu Europe, Duisburg, Germany; AB Sciex API 2000, Darmstadt, Germany).

Techniques:

Journal: Pharmaceutics

Article Title: Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies

doi: 10.3390/pharmaceutics13091409

Figure Lengend Snippet: Cytochrome P450 metabolism of cyclobenzaprine. A : Scheme of cyclobenzaprine demethylation by CYP isoenzymes. B : Cumulative amount of permeated desmethyl cyclobenzaprine per cm 2 (mean ± SEM; n = 8). C : Formation of desmethyl cyclobenzaprine by different mucosae and approaches (mean ± SEM; n ≥ 2). D : Formation of desmethyl cyclobenzaprine by human liver microsomes per time (mean ± SEM; n = 3). HLM: human liver microsomes, LLOQ: lower limit of quantification .

Article Snippet: The simultaneous quantification of cyclobenzaprine hydrochloride (≥98%, Hetero drugs Ltd., Hyderabad, India), its main metabolite desmethyl cyclobenzaprine hydrochloride (99.8%, Toronto Research Chemicals, Toronto, Canada) and cyclobenzaprine N-oxide (96%, Toronto Research Chemicals, Toronto, Canada) as its major degradation product was performed by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Shimadzu Prominence, Shimadzu Europe, Duisburg, Germany; AB Sciex API 2000, Darmstadt, Germany).

Techniques:

Journal: Pharmaceutics

Article Title: Formulation Development of Sublingual Cyclobenzaprine Tablets Empowered by Standardized and Physiologically Relevant Ex Vivo Permeation Studies

doi: 10.3390/pharmaceutics13091409

Figure Lengend Snippet: Alteration of cyclobenzaprine sublingual tablets under ambient and stress conditions. ( A ): Cumulative amount of permeated drug per cm 2 of the respective sublingual tablet stored (mean ± SEM; n ≥ 4). ( B ): Dissolution of the respective sublingual tablet stored (mean ± SEM; n = 3). ( C , D ): Visual and microscopic inspection of the primary packaging material and the tablet surface after storage under ambient conditions and stress conditions, respectively. ( E , F ): TOF-MS scan of the rinsed residuals from packaging material after storage under ambient conditions and stress conditions, respectively. m/z: mass-to-charge ratio, SEM: standard error of the mean , *: significant value (p < 0.05; unpaired t-test).

Article Snippet: The simultaneous quantification of cyclobenzaprine hydrochloride (≥98%, Hetero drugs Ltd., Hyderabad, India), its main metabolite desmethyl cyclobenzaprine hydrochloride (99.8%, Toronto Research Chemicals, Toronto, Canada) and cyclobenzaprine N-oxide (96%, Toronto Research Chemicals, Toronto, Canada) as its major degradation product was performed by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (Shimadzu Prominence, Shimadzu Europe, Duisburg, Germany; AB Sciex API 2000, Darmstadt, Germany).

Techniques: Dissolution

Journal: Nature Communications

Article Title: The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

doi: 10.1038/ncomms11659

Figure Lengend Snippet: ( a ) Survival of C57B/6 mice infected with mutant and wt parasites ( n =9 mice per group, P< 0.001, logrank test from Kaplan–Meier survival curve). ( b ) The course of parasitemia (mean±s.d.) in C57B/6 mice ( n =15 mice per group) infected with 10 5 mutant or wt parasites. Significant differences in parasitemia between wt and both mutants at day 3, 6, 7 ( P< 0.001) and day 5 ( P= 0.005) (two-tailed unpaired t -test). ( c ) Significant increase in number of mutant schizonts in the peripheral blood circulation compared with wt. Schizonts were quantified in Giemsa-stained smears of tail blood obtained 22 h after establishing synchronised infections (ratio of the number of mutant to wt schizonts, n =12 mice per group, P= 0.01; two-tailed unpaired t -test). ( d ) Representative distribution of sequestered schizonts in mice with synchronised infections 22 h post infection of mutant and wt parasites that express luciferase as shown by measuring luciferase activity (photons per s per cm 2 ; shown as radiance, y ; n =15 mice per group). ( e ) Quantification of parasite loads in isolated organs after perfusion 22 h after synchronised infections with mutant and wt parasites expressing luciferase. Parasite loads were determined by measuring luciferase activity (photons per s per cm 2 ) and expressed relative to wt ( n =15 mice per group). ( f ) Representative intravital images (spleen, fat) and ex vivo (lungs) confocal images of tissues from UBC-GFP mice 22 h after establishing synchronized infections of mutant and wt parasites that express mCherry. Insets in the images of fat tissue are enlargements of the yellow boxes. Arrowheads indicate parasites. Size bars, 10μm. ( g ) Spleens of mutant-infected mice are significantly larger (spleen-to-body weight ratio) than spleens of wt-infected mice ( n =9 mice per group, * P< 0.0001; unpaired Student's t - test; infections were done with 10 6 parasites per mouse). Images of representative spleens at day 4 after infection are shown to the right. ( h ) In vitro binding of schizonts of mutant and wt parasites to CD36-GFP-expressing CHO-745 cells (left) and to CD36 immobilized on plates (right). Binding is expressed as % of wt ( n =6, * P< 0.0001; two-tailed unpaired Student's t -test). ( i ) Quantitative FACS analysis of surface reactivity of RBCs infected with wt and gene-deletion mutant schizonts cultured ex vivo to mouse hyperimmune serum. Both deletion mutant schizonts exhibit significantly reduced levels of surface labelling with P. berghei sera compared to wt parasites. The geometric mean fluorescence intensity (MFI) relative to wt is shown (%). (*** P , 0.001, unpaired t -test).

Article Snippet: For in vitro measurement of the adhesion of mutant and wt P. berghei parasites to CD36-coated dishes recombinant mouse CD36/Fc chimera (purchased from R&D) was used.

Techniques: Infection, Mutagenesis, Two Tailed Test, Staining, Luciferase, Activity Assay, Isolation, Expressing, Ex Vivo, In Vitro, Binding Assay, Cell Culture, Fluorescence

Journal: Nature Communications

Article Title: The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

doi: 10.1038/ncomms11659

Figure Lengend Snippet: ( a ) The matched complemented mutants showed significantly increased binding to purified CD36 compared with the gene-deletion mutants (binding indicated as % of wt; * P< 0.0001, one-way ANOVA, Tukeys post hoc test.; n =3). ( b ) Survival of C57B/6 mice infected with matched complemented mutants was significantly increased in comparison with the mutants and the mismatched complemented mutants ( n =9 mice per group, P< 0.001, logrank test from Kaplan–Meier survival curve). ( c ) Significant decrease in number of schizonts in the peripheral blood circulation in matched complemented mutants compared with the gene-deletion mutants and to mismatched complemented mutants. Schizonts were quantified in Giemsa-stained smears of tail blood obtained 22 h after establishing synchronised infections ( n =9 mice per group; * P< 0.0001, one-way ANOVA, Tukeys post hoc test).

Article Snippet: For in vitro measurement of the adhesion of mutant and wt P. berghei parasites to CD36-coated dishes recombinant mouse CD36/Fc chimera (purchased from R&D) was used.

Techniques: Binding Assay, Purification, Infection, Staining