Additional file 2: of How does the dengue vector mosquito Aedes albopictus respond to global warming?

Regression analysis of the warming effects. (DOCX 14 kb

Additional file 1: of How does the dengue vector mosquito Aedes albopictus respond to global warming?

Two-season (SW2) and three-season (SW3) warming patterns. (DOCX 16 kb

Alginic Acid-Coated Chitosan Nanoparticles Loaded with Legumain DNA Vaccine: Effect against Breast Cancer in Mice

<div><p>Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments <i>in vitro</i>. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated <i>Salmonella typhimurium</i> and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated <i>Salmonella typhimurium</i> as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl₂, which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3⁺/CD8⁺/CD25⁺) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.</p> </div

Diagram shows the protective effect of A.C.NPs on DNA against enzymatic and acidic degradation.

<p>(A) Schematic of A.C.NPs-legumain preparation. (B) Schematic representation of A.C.NPs-legumain passing through the acidic gastrointestinal track and taken up by antigen-presenting cells in the intestinal Peyer’s patches.</p

Characteristics of C.NPs-legumain and A.C.NPs- legumain in an acidic environment.

<p>(A) Nanoparticles were treated in different acidity levels (pH 1.8∼12) for 2 hours. C.NPs-legumain and A.C.NPs-legumain particle diameter and zeta potential measurements at 37°C. (B) Representative images of A.C.NPs at pH 1.5 (left, scale bar = 1µm) and pH 7.0 (right, scale bar = 100 nm). (C) FTIR spectra of A.C.NPs-legumain at pH 1.5 and pH 7.0.</p

A.C.NPs loaded with DNA pass through the acidic gastric barrier and are taken up by macrophages and dendritic cells in the intestinal Peyer’s patches.

<p>Naked EGFP DNA plasmids, C.NPs-EGFP, and A.C.NPs-EGFP were separately given to BALB/c mice (n = 5) via intragastric gavage at a daily dose equivalent to 30 µg plasmid DNA per mouse for three consecutive days. Peyer’s patches were isolated and analyzed by flow cytometry. PE-conjugated F4/80 and APC-conjugated CD11c antibodies were used to stain (A) the macrophages and (B) dendritic cells, respectively. Histograms of the percentage of EGFP-positive (C) macrophages and (D) dendritic cells. (E) Histograms of the ratio of F4/80- or CD11c- positive cells to total EGFP-positive cells. Data are presented as mean ± SD of three independent experiments (**P<0.01; n = 5).</p

Illustration of A.C.NPs-legumain synthesis.

<p>Illustration of A.C.NPs-legumain synthesis.</p

A.C.NPs protect DNA against degradation at low pH.

<p>Naked, full-length legumain DNA plasmids, C.NPs-legumain, and A.C.NPs-legumain were each incubated with artificial gastric fluid (pH 1.5) for 0, 0.5, 1, 2 or 4 hours. Naked plasmid DNA was incubated in solution of pH 7.0 for the same time points to serve as a positive control. (A) A representative image of the agarose gel electrophoresis. A lane is observed in the A.C.NPs even after incubation at pH 1.5 for 4 h. (B) Graphical representation of relative OD values. Data are presented as mean ± SD of three independent experiments (**P<0.01).</p

Oral vaccination via A.C.NPs-legumain induces autoimmune CTLs that target legumain.

<p>Activated CTLs were accessed by flow cytometry. (A) Representative confocal microscopy images of immnofluorescence staining of 4T1 cells treated with or without CoCl₂ for 6 h. E-cadherin was used as a cell membrane marker. Significant translocation expression of legumain to the membrane after CoCl₂ stimulation was observed. (B) Representative flow cytometry histogram of splenocytes co-cultured with 4T1 cells treated with or without CoCl₂. (C) Histogram of the percentages of CD8 and CD25 double positive cells. Data are presented as mean ± SD of five independent experiments (*P<0.01).</p

“Triple-Punch” Strategy Exosome-Mimetic Nanovesicles for Triple Negative Breast Cancer Therapy

Triple-negative breast cancer (TNBC) is the most malignant breast cancer, with high rates of relapse and metastasis. Because of the nonspecific targeting of chemotherapy and insurmountable aggressiveness, TNBC therapy lacks an effective strategy. Exosomes have been reported as an efficient drug delivery system (DDS). CD82 is a tumor metastasis inhibitory molecule that is enriched in exosomes. Aptamer AS1411 specifically targets TNBC cells due to its high expression of nucleolin. We generated a “triple-punch” cell membrane-derived exosome-mimetic nanovesicle system that integrated with CD82 overexpression, AS1411 conjugation, and doxorubicin (DOX) delivery. CD82 enrichment effectively inhibits the migration of TNBC cells. AS1411 conjugation specifically targets TNBC cells. DOX loading effectively inhibits proliferation and induces apoptosis of TNBC cells. Our results demonstrate a system of exosome-mimetic nanovesicles with “triple-punch” that may facilitate TNBC therapeutics