Journal: PLoS Biology

Article Title: C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

doi: 10.1371/journal.pbio.3001515

Figure Lengend Snippet: (A, B) CTL4-gRNA virgin females (RFP, red fluorescence) were crossed with Vasa-Cas9 males (YFP, yellow/green fluorescence) to produce CTL4 gene knockouts (CTL4/Cas9) expressing both red and green fluorescence in the larval ( A ) and adult ( B ) stages. Progeny that did not inherit CTL4-gRNA (gRNA) expresses the Vasa-Cas9 green fluorescent marker only (Cas9). ( C) PCR validation of CTL4 gene deletion, compared to X1, Cas9, and gRNA controls (364 bp). Image of original gel is shown in . ( D) Agarose gel image of the qRT-PCR products confirms CTL4 mRNA deletion (CTL4 null ) compared to the X1 control in different tissues (whole mosquito, midgut and fat body) (297 bp); expression levels were normalized to S7 (149 bp). Image of original gel is shown in . ( E) Western blotting confirms CTL4 protein (15–20 kDa) deletion (CTL4 null ) as compared to the X1 in whole mosquitoes; expression levels were normalized to ß-actin (41 kDa). Images of original blots are shown in . gRNA, guide RNA; qRT-PCR, quantitative reverse transcription-PCR.

Article Snippet: Polyclonal CTL4 and TEP1 antibodies were generated by Boster Biological Technology in rabbit targeting antigens corresponding to peptide sequences of the 2 genes (cDNA sequences used are available in and Files).

Techniques: Fluorescence, Expressing, Marker, Biomarker Discovery, Agarose Gel Electrophoresis, Quantitative RT-PCR, Control, Western Blot, Reverse Transcription

Journal: PLoS Biology

Article Title: C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

doi: 10.1371/journal.pbio.3001515

Figure Lengend Snippet: (A, B) P . berghei infection intensity and prevalence in control and CTL4 null A . gambiae females fed on a mouse with low ( A ) or high ( B ) gametocytemia and measured at 10 dpi. ( C) Images illustrate P . berghei -infected (high gametocytemia) A . gambiae control (upper left) and CTL4 null (upper right) midguts showing live parasites and 100% melanized parasites, respectively. Melanized P . falciparum ookinete (lower). ( D, E, F) P . falciparum infection intensity and prevalence in control and CTL4 null A . gambiae females fed on blood with a low ( D, E ) or a high ( F) gametocytemia and measured in the midgut at 8 dpi ( D, F ) or in the sg at 14 dpi ( E ). displays the underlying data. Dots and inverted triangles indicate the number of parasites in an individual midgut or salivary gland, respectively, and horizontal red bars indicate the median. Two-tailed p -values by Mann–Whitney test were used to compare the live parasites (L); M, melanized parasites (M), and all (L+M) parasites (A). Bars show the percentage of mosquitoes harboring at least one oocyst; the Fisher exact test was used to calculate p -values. Significance of parasite numbers: * *** : p < 0.0001; horizontal black lines alone: not significant. dpi, days postinfection; sg, salivary glands.

Article Snippet: Polyclonal CTL4 and TEP1 antibodies were generated by Boster Biological Technology in rabbit targeting antigens corresponding to peptide sequences of the 2 genes (cDNA sequences used are available in and Files).

Techniques: Infection, Control, Two Tailed Test, MANN-WHITNEY

Journal: PLoS Biology

Article Title: C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

doi: 10.1371/journal.pbio.3001515

Figure Lengend Snippet: (A, B) P . berghei ( A ) and P . falciparum ( B ) ookinete loads in the midgut lumen of A . gambiae females at 19 hpi were not significantly different between the control and the CTL4 null groups (two-tailed Mann–Whitney test). displays the underlying data. ( C) Confocal microscopy images illustrate P . falciparum -infected (top panels) and P . berghei -infected (bottom panels) immunostained sections of the A . gambiae CTL4 null and control midgut epithelium at 24–25 hpi. Images are representative of 3 independent experiments with at least 10 midguts per replicate. Red indicates parasites probed with α-CTL4 antibody; green indicates live parasites probed with α-Pfs25 (for Pf ) or α-Pbs28 (for Pb ) antibodies; yellow indicates colocalization of CTL4 and the parasite; and blue indicates DAPI-stained epithelial cells nuclei. Black dots (CTL4 null ) represent melanized ookinetes. Scale bars 10 μm. hpi, hours postinfection.

Article Snippet: Polyclonal CTL4 and TEP1 antibodies were generated by Boster Biological Technology in rabbit targeting antigens corresponding to peptide sequences of the 2 genes (cDNA sequences used are available in and Files).

Techniques: Control, Two Tailed Test, MANN-WHITNEY, Confocal Microscopy, Infection, Staining

Journal: PLoS Biology

Article Title: C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

doi: 10.1371/journal.pbio.3001515

Figure Lengend Snippet: (A, B) P . berghei infection in antibiotics-treated control and CTL4 null A . gambiae midguts at 10 dpi, compared to nonantibiotics treated ( A ) or after injection with dsCaspar or control dsGFP ( B ). ( C, D) P . falciparum infection in control and CTL4 null A . gambiae females measured at 8 dpi, after RNAi-mediated silencing of IMD ( C ) and Caspar ( D ). displays the underlying data. Dots indicate the number of parasites in an individual midgut (L, live; M, melanized), and horizontal red bars indicate the median, compared by two-tailed p -values by Mann–Whitney test. Bars show the percentage of mosquitoes harboring at least one oocyst, and the Fisher exact test was used to calculate p -values. Significance of parasite numbers: *: p < 0.05, * *** : p < 0.0001; horizontal black lines alone: not significant. dpi, days postinfection; RNAi, RNA interference.

Article Snippet: Polyclonal CTL4 and TEP1 antibodies were generated by Boster Biological Technology in rabbit targeting antigens corresponding to peptide sequences of the 2 genes (cDNA sequences used are available in and Files).

Techniques: Infection, Control, Injection, Two Tailed Test, MANN-WHITNEY

Journal: PLoS Biology

Article Title: C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

doi: 10.1371/journal.pbio.3001515

Figure Lengend Snippet: (A) P . falciparum ookinetes in the lumen of control A . gambiae females at 24 and 40 hpi at 19 °C and 24 hpi at 27 °C, and oocysts at 13 dpi at 19 °C and 8 dpi at 27 °C. ( B) P . falciparum ookinetes in the midgut epithelium of control A . gambiae females at 24 and 40 hpi at 19 °C and at 27 °C, and oocysts at 13 dpi at 19 °C and 8 dpi at 27 °C. ( C) P . falciparum infection in control and CTL4 null A . gambiae females measured at 8 dpi at 19 °C or 27 °C. displays the underlying data. Dots indicate the number of parasites in an individual midgut (L, live; M, melanized), and horizontal red bars indicate the median, compared by two-tailed p -values by Mann–Whitney test. Bars show the percentage of mosquitoes harboring at least one oocyst, and the Fisher exact test was used to calculate p -values. Significance of parasite numbers: *: p < 0.05, * * : p < 0.01; * ** : p < 0.001; * *** : p < 0.0001; horizontal black lines alone: not significant. dpi, days postinfection; hpi, hours postinfection.

Article Snippet: Polyclonal CTL4 and TEP1 antibodies were generated by Boster Biological Technology in rabbit targeting antigens corresponding to peptide sequences of the 2 genes (cDNA sequences used are available in and Files).

Techniques: Control, Infection, Two Tailed Test, MANN-WHITNEY

Journal: PLoS Biology

Article Title: C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

doi: 10.1371/journal.pbio.3001515

Figure Lengend Snippet: (A, B) Midgut microbial flora of control and CTL4 null A . gambiae females was compared using two-tailed p -values by Mann–Whitney ( A ) and ANOVA followed by a Tukey test ( B ). ( C) Control and CTL4 null A . gambiae females were injected with either E . coli (350,000 CFU), S . aureus (420,000 CFU), and B . subtilis (62,000 CFU), or PBS as a control, and longevity was analyzed up to 8 dpi. Kaplan–Meier survival analysis with a log-rank test was used to determine the p -values, and SEs of replicates are indicated. ( D) Abdomens were dissected to check bacterial melanization 3 d following E . coli and S . aureus challenge by injection, and the Fisher exact test was used to calculate the difference between control and CTL4 null A . gambiae females. (E) Fungi spores were efficiently melanized (white arrows) in the CTL4 null abdomens 12 h and 48 h following injection of B . bassiana (2.15 × 10 6 spores/ml), and the number of live spores (green arrows) was visibly higher in the control abdomens. ( F) Melanized and live fungi were measured in midguts tissues from 24 h to 7 d after mosquitoes fed on a sucrose solution containing 2.15 × 10 8 B . bassiana spores/ml, and the Fisher exact test was used to calculate the difference between control and CTL4 null A . gambiae females. ( G) Melanized fungi were dectected on the midgut tissue of CTL null females. ( H) Survival of control and CTL4 null A . gambiae females after dipping (surface exposure) in B . bassiana spores (1 × 10 9 spores/ml), compared by the Kaplan–Meier survival analysis with a log-rank test; SE of replicates are indicated. Significance: * *** : p < 0.0001. displays the underlying data. CFU, colony-forming unit; dpi, days postinfection; SE, standard error.

Article Snippet: Polyclonal CTL4 and TEP1 antibodies were generated by Boster Biological Technology in rabbit targeting antigens corresponding to peptide sequences of the 2 genes (cDNA sequences used are available in and Files).

Techniques: Control, Two Tailed Test, MANN-WHITNEY, Injection

Journal: PLoS Biology

Article Title: C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

doi: 10.1371/journal.pbio.3001515

Figure Lengend Snippet: (A, B) P . falciparum infection intensity and prevalence in control and CTL4 null A . gambiae females measured at 8 dpi, after RNAi-mediated silencing of CTLMA2 ( A ) and LRIM1 ( B ). ( C, D) P . falciparum infection intensity and prevalence ( C ) measured at 8 dpi were compared to P . berghei ( D ) infection intensity and prevalence measured at 10 dpi, following RNAi-mediated silencing of TEP1 in control and CTL4 null A . gambiae females. ( E, F) P . falciparum infection intensity and prevalence in control and CTL4 null A . gambiae females measured at 8 dpi, after RNAi-mediated silencing of CLIPA2 ( E ) and CLIPA14 ( F ). displays the underlying data. Dots indicate the number of parasites (L, live; M, melanized) in an individual midgut, and horizontal red bars indicate the median, compared by two-tailed p -values by Mann–Whitney test. Bars show the percentage of mosquitoes harboring at least one oocyst, and the Fisher exact test was used to calculate p -values. Significance of parasite numbers: *: p < 0.05; * * : p < 0.01; * *** : p < 0.0001; horizontal black lines alone: not significant. ( G) Confocal microscopy images illustrate P . falciparum -infected (left panels) and P . berghei -infected (right panels) immunostained sections of the A . gambiae CTL4 null and control midgut epithelium at 24–25 hpi. Images are representative of 3 independent experiments with at least 10 midguts per replicate. Red indicates parasites probed with α-TEP1 antibody; green indicates live parasites probed with α-Pfs25 (for Pf ) or α-Pbs28 (for Pb ) antibodies; yellow indicates colocalization of TEP1 and the parasite; and blue indicates DAPI-stained epithelial cells nuclei. Black dots (CTL4 null ) represent melanized ookinetes. Scale bars 10 μm. ( H) Schematic representation of the influence of CTL4 and complement-like immune-related factors (those addressed/related to this study) on P . berghei and P . falciparum . From left to right: P . falciparum ookinetes are lysed by the action of the Imd pathway and TEP1 and are protected from being melanized by the CTL4/CTLMA2 complex and CLIPA14 in wild-type mosquitoes. When TEP1 and the Imd pathway are silenced, P . falciparum can develop into oocysts. P . berghei ookinetes are lysed by the action of TEP1, APL1C, and LRIM1, and they are protected from being lysed by CLIPA2 and CLIPA14 and are protected from being melanized by the CTL4/CTLMA2 complex, CLIPA2 and CLIPA14 in wild-type mosquitoes. When TEP1, APL1C, and LRIM1 are silenced, the P . berghei ookinetes can develop into oocysts. When CLIPA2 and CLIPA14 are silenced, the P . berghei ookinetes are lysed. In CTL4 null mosquitoes, the Imd pathway is promoting lysis of P . falciparum ookinetes, while silencing of CLIPA2 will enhance melanization of P . falciparum ookinetes. TEP1, APL1C, and LRIM1 are promoting melanization of P . berghei ookinetes in CTL4 null mosquitoes, and silencing of CLIPA2 and CLIPA14 will also promote melanization of P . berghei ookinetes in CTL4 null mosquitoes. dpi, days postinfection; hpi, hours postinfection; Imd, immune deficiency; RNAi, RNA interference.

Article Snippet: Polyclonal CTL4 and TEP1 antibodies were generated by Boster Biological Technology in rabbit targeting antigens corresponding to peptide sequences of the 2 genes (cDNA sequences used are available in and Files).

Techniques: Infection, Control, Two Tailed Test, MANN-WHITNEY, Confocal Microscopy, Staining, Lysis

Journal: Cancer Biology & Therapy

Article Title: LOX and ACSL5 as potential relapse markers for pancreatic cancer patients

doi: 10.1080/15384047.2018.1564565

Figure Lengend Snippet: Primers designed for the qRT-PCR validation of candidate gene mRNA levels and β-actin.

Article Snippet: The sections were then incubated overnight at 4°C with primary antibodies [rabbit polyclonal anti-LOX (1:200, Novus Biological, NB100–2527); mouse monoclonal anti-ACSL5 (1:50, ab104892); rabbit polyclonal anti-SLC44A4 (1:200, Novus Biological, NBP2-33707); rabbit polyclonal anti-TOX3 (1:50, Abgent, Cat# AH11330-100)].

Techniques:

Journal: Cancer Biology & Therapy

Article Title: LOX and ACSL5 as potential relapse markers for pancreatic cancer patients

doi: 10.1080/15384047.2018.1564565

Figure Lengend Snippet: The relationship between clinicopathological characteristics and LOX/ACSL5/SLC44A4/TOX3 protein expression levels.

Article Snippet: The sections were then incubated overnight at 4°C with primary antibodies [rabbit polyclonal anti-LOX (1:200, Novus Biological, NB100–2527); mouse monoclonal anti-ACSL5 (1:50, ab104892); rabbit polyclonal anti-SLC44A4 (1:200, Novus Biological, NBP2-33707); rabbit polyclonal anti-TOX3 (1:50, Abgent, Cat# AH11330-100)].

Techniques: Expressing

Journal: Cancer Biology & Therapy

Article Title: LOX and ACSL5 as potential relapse markers for pancreatic cancer patients

doi: 10.1080/15384047.2018.1564565

Figure Lengend Snippet: Multivariable analyses of factors associated with OS and PFS.

Article Snippet: The sections were then incubated overnight at 4°C with primary antibodies [rabbit polyclonal anti-LOX (1:200, Novus Biological, NB100–2527); mouse monoclonal anti-ACSL5 (1:50, ab104892); rabbit polyclonal anti-SLC44A4 (1:200, Novus Biological, NBP2-33707); rabbit polyclonal anti-TOX3 (1:50, Abgent, Cat# AH11330-100)].

Techniques: