Journal: Journal of Translational Medicine

Article Title: HMGB1/TREM2 positive feedback loop drives the development of radioresistance and immune escape of glioblastoma by regulating TLR4/Akt signaling

doi: 10.1186/s12967-024-05489-w

Figure Lengend Snippet: Schematic illustration of mechanism of TREM2 inhibition reversed the radioresistance and immune escape of GBM. A2-CM-NP/siTREM2/spam1 was successfully constructed with the following characteristics: passive targeting (less than 200 nm), active targeting (modified with A2), homologous targeting (coated with GBM CM vesicles), simultaneously penetrated BBB (mediated by the interaction of A2 and low-density lipoprotein related protein-1 expressed on the brain capillary endothelial cells) and degraded extracellular matrix (mediated by the co-loaded spam1 plasmid), GBM region-specific release of siTREM2 (coupled with disulfide bond). After TREM2 inhibition, the radioresistance and immune escape of GBM could be reversed by the following mechanisms: (1) directly interfering with DNA damage repair pathway; (2) inhibiting the HMGB1/TREM2 positive feedback loop; (3) restraining the secretion of immunosuppressive cytokines, the repolarization of macrophages from M1-type to M2-type, and the infiltration of pro-tumor T cells. (This figure was drawn by Figdraw.com)

Article Snippet: In order to responsively release and efficiently concentrate the loaded siTREM2 in GBM region, GBM CM-biomimetic, GSH-responsive, A2-modificatory NPs named A2-CM-NP/siTREM2/spam1 were designed and synthesized according to the synthesis process shown in Fig. A, and the co-loaded spam1 plasmid could express hyaluronidase to degrade extracellular matrix.

Techniques: Inhibition, Construct, Modification, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: HMGB1/TREM2 positive feedback loop drives the development of radioresistance and immune escape of glioblastoma by regulating TLR4/Akt signaling

doi: 10.1186/s12967-024-05489-w

Figure Lengend Snippet: A2-CM-NP/siTREM2/spam1 was successfully constructed with superior active targeting, homologous targeting and in vivo biodistribution. ( A ) Schematic illustration of the synthesis process of A2-CM-NP/siTREM2/spam1; ( B ) Agarose gel eletrophoresis assay of the binding capacity of siTREM2 and spam1 plasmid to A2-NP at various N/P ratios; ( C ) SDS-PAGE analysis of proteins extracted from GBM cells (lanes 1, 4 and 7), GBM CM vesicles (lanes 2, 5 and 8), and A2-CM-NP/siTREM2/spam1 (lanes 3, 6 and 9) by Coomassie blue staining; The particle size distribution ( D ), statistical charts of particle sizes ( E ) and zeta-potentials ( F ) of A2-NP, A2-NP/siTREM2/spam1, and A2-CM-NP/siTREM2/spam1 were detected by dynamic light scattering, data are presented as mean ± SD, n = 3; ( G ) Up: representative fluorescent microscopy images and statistical chart of the red fluorescence intensity in GL261 cells cultured with cy3-siRNA, A2-NP/cy3-siRNA and A2-CM-NP/cy3-siRNA; down: GL261, G422, U87MG cells cultured with A2-CM-NP/cy3-siRNA synthesized with GL261 CM vesicles; red: cy3-siRNA, green: EGFP expressed in GBM cells, blue: cell nucleus stained with DAPI, scale bar = 20 μm. ( H ) In vivo bioluminescence imaging of mice after implanting luc-labeled GL261 cells for 7 days and ex vivo fluorescence imaging of the organs including brain, heart, liver, spleen, lungs and kidneys 24 h after the injection of PBS, A2-NP/cy3-siRNA, A2-CM-NP/cy3-siRNA, and A2-CM-NP/cy3-siRNA/spam1 via tail vein

Article Snippet: In order to responsively release and efficiently concentrate the loaded siTREM2 in GBM region, GBM CM-biomimetic, GSH-responsive, A2-modificatory NPs named A2-CM-NP/siTREM2/spam1 were designed and synthesized according to the synthesis process shown in Fig. A, and the co-loaded spam1 plasmid could express hyaluronidase to degrade extracellular matrix.

Techniques: Construct, In Vivo, Agarose Gel Electrophoresis, Binding Assay, Plasmid Preparation, SDS Page, Staining, Microscopy, Fluorescence, Cell Culture, Synthesized, Imaging, Labeling, Ex Vivo, Injection

Journal: Journal of Translational Medicine

Article Title: HMGB1/TREM2 positive feedback loop drives the development of radioresistance and immune escape of glioblastoma by regulating TLR4/Akt signaling

doi: 10.1186/s12967-024-05489-w

Figure Lengend Snippet: A2-CM-NP/siTREM2/spam1 prominently improved the therapeutic effect of radiotherapy and PD-1 inhibitor in intracranial GBM models. ( A ) Schematic illustration of the in vivo experimental workflow; ( B ) In vivo bioluminescent imaging of luc-labeled GL261-bearing mice and luc-labeled G422-bearing mice from each treatment group at different time; The short-term efficacy evaluation ( C ) and Kaplan − Meier survival curves ( D ) of orthotopic luc-labeled GL261-bearing mice and luc-labeled G422-bearing mice in all groups, n = 5; ( E ) The apoptosis rates in GL261 tissues and G422 tissues from different treatment groups were detected by TUNEL assay, and cell nucleus were stained with DAPI, scale bar = 50 μm

Article Snippet: In order to responsively release and efficiently concentrate the loaded siTREM2 in GBM region, GBM CM-biomimetic, GSH-responsive, A2-modificatory NPs named A2-CM-NP/siTREM2/spam1 were designed and synthesized according to the synthesis process shown in Fig. A, and the co-loaded spam1 plasmid could express hyaluronidase to degrade extracellular matrix.

Techniques: In Vivo, Imaging, Labeling, TUNEL Assay, Staining

Journal: Reproductive biology and endocrinology : RB&E

Article Title: Spam1-associated transmission ratio distortion in mice: Elucidating the mechanism

doi: 10.1186/1477-7827-3-32

Figure Lengend Snippet: Transmission frequencies of Spam1-Hyal5 BAC transgenes in hemizygotes of three lines, Tg8, Tg9, and Tg11, reveal a distortion only for Tg8/+. a) Histograms showing the rate of production of transgenic and non-transgenic progeny analyzed at weaning on Day 21. Tg8-A represents the progeny of the founder and 3 F 1 hemizygous males, while progeny from F 2 -F 4 hemizygous males are seen in Tg8-B. Tg8-T represents the total progeny analyzed for the Tg8 line. There is a significant deviation from 1:1 for Tg8-A (χ 2 = 27.30; P < 0.001), Tg8-B (χ 2 = 17.47; P < 0.001), and Tg8-T (χ 2 = 41.8; P < 0.001) all represented by an asterisk; while there was no difference in 1:1 ratios (P > 0.05) for Tg9 and Tg11, represented by the diamond. b) The TRD for Tg8/+ mice does not result from post-zygotic selection against transgene-bearing zygotes, as revealed by the average litter sizes of transgenic lines. In addition to the BAC transgenic lines we included a Spam1 cDNA transgenic line, Cinn (179), which like Tg9 and Tg11 also had a 1:1 ratio in the progeny. The means for the litters ranged from 6.68 to 8.50 and are shown at the top of the histograms with their SDs at the sides. The highest mean, 8.50 ± 1.51, was seen for Tg8A which had the highest TRD, 2.8:1.

Article Snippet: After washing and blocking in 2% BSA in PBS they were stained using the rabbit antipeptide mouse Spam1 antiserum generated from a C-terminal 15-mer (#381 – 395) oligopeptide (custom made by Zymed, San Francisco, CA) (diluted 1:400) specific for Spam1 [ , ].

Techniques: Transmission Assay, Transgenic Assay, Selection

Journal: Reproductive biology and endocrinology : RB&E

Article Title: Spam1-associated transmission ratio distortion in mice: Elucidating the mechanism

doi: 10.1186/1477-7827-3-32

Figure Lengend Snippet: Flow cytometric analyses of sperm from hemizygotes for an overexpressed or a Spam1 null allele show bimodal distributions. a) Caudal sperm from the congenic wild-type, Tg11/+ and Tg8/+ show a bimodal distribution only for Tg8/+. The second peak (on the right) with the greater intensity in this bimodal distribution indicates the presence of a subpopulation of sperm with overexpression. The distributions for the wild-type and Tg11/+ are unimodal with lower mean intensities, indicating a lack of Spam1 overexpression. b) Caudal sperm from Tg8 carriers showing the analysis and gating of the sperm for sorting. c) Caput sperm from hemizygous null mice show a bimodal distribution in sperm in A and B. The first peak (on the left) in each shows background levels of fluorescence, likely representing sperm with the null allele.

Article Snippet: After washing and blocking in 2% BSA in PBS they were stained using the rabbit antipeptide mouse Spam1 antiserum generated from a C-terminal 15-mer (#381 – 395) oligopeptide (custom made by Zymed, San Francisco, CA) (diluted 1:400) specific for Spam1 [ , ].

Techniques: Over Expression, Fluorescence

Journal: Reproductive biology and endocrinology : RB&E

Article Title: Spam1-associated transmission ratio distortion in mice: Elucidating the mechanism

doi: 10.1186/1477-7827-3-32

Figure Lengend Snippet: Immunocytochemistry demonstrates that Tg8/+ males have significantly increased numbers of sperm retaining enlarged cytoplasmic droplets (CDs) with overexpressed Spam1 or Hyal5, and a concomitant absence of the proteins on the heads. a) Histograms showing the proportion of sperm, from populations of 200, with CDs in wild-type and Tg8/+ males. The greater than 10-fold increase in caudal sperm compared to wild-type is highly significant (χ 2 = 10.8; P < 0.01), as is the >30-fold increase in caput sperm (χ 2 = 28.5; P < 0.001). b) Retention of CDs with overexpressed Spam1 (green staining) in sperm taken from an aliquot used for flow cytometry in Fig. 2a. A sperm with the normal amount and normal location of Spam1 is shown for comparison with the overexpressed protein in the CD. Note that there is non-specific background staining on the tails. c) Sperm with overexpressed Hyal5 (green staining) in enlarged CDs near the neck and the absence of the protein on the heads are seen in B and C, while A shows a normal CD without Hyal5. FISH signals on flow sorted sperm showing double signals in transgenic cells d) and a single signal in wild-type cells e) .

Article Snippet: After washing and blocking in 2% BSA in PBS they were stained using the rabbit antipeptide mouse Spam1 antiserum generated from a C-terminal 15-mer (#381 – 395) oligopeptide (custom made by Zymed, San Francisco, CA) (diluted 1:400) specific for Spam1 [ , ].

Techniques: Immunocytochemistry, Staining, Flow Cytometry, Transgenic Assay

Journal: Reproductive biology and endocrinology : RB&E

Article Title: Spam1-associated transmission ratio distortion in mice: Elucidating the mechanism

doi: 10.1186/1477-7827-3-32

Figure Lengend Snippet: Spam1 transcripts which are compartmentalized are absent from the bridges and are associated with the cytoskeleton. a) EM autoradiography of seminiferous tubules after in situ hybridization with a tritiated ( 3 H-labeled) Spam-1 antisense RNA probe. Note that silver grains are associated with the ER (arrowheads) but not with any other major spermatid structures such as the chromatoid bodies (A), the radial bodies (B) or the microtubules of the manchette (C). D is an intercellular bridge where the curvatures at the top and bottom represent the outer limits of the bridge. While some grains are seen in association with the ER in the vicinity, they are absent from the bridge. The circles centered over the silver grains include profiles of ER (arrowheads). (E) Late spermatids (S) and Sertoli cells (Se) are unreactive. (F) Shows the cytoplasm of round spermatid (RS) step 8 of a control section hybridized to a sense probe. Co, chromatoid body; Rb, radial body; M, manchette. X19,000 b . Northern hybridization of Spam1 and β- actin mRNAs in free cytosolic-, cytoskeletal-, and membrane-bound testicular RNA fractions. The fractions were separated by subcellular fractionation techniques. A) shows Northern blotting, while B) shows total RNA as a loading control with ethidium bromide staining. The presence of cytoskeletal-bound β-actin RNA in the free cytosolic fraction suggests that Spam1 in the latter could be present as a contaminant due to the preparation procedure.

Article Snippet: After washing and blocking in 2% BSA in PBS they were stained using the rabbit antipeptide mouse Spam1 antiserum generated from a C-terminal 15-mer (#381 – 395) oligopeptide (custom made by Zymed, San Francisco, CA) (diluted 1:400) specific for Spam1 [ , ].

Techniques: Autoradiography, In Situ Hybridization, Labeling, Northern Blot, Hybridization, Fractionation, Staining

Journal: Reproductive biology and endocrinology : RB&E

Article Title: Spam1-associated transmission ratio distortion in mice: Elucidating the mechanism

doi: 10.1186/1477-7827-3-32

Figure Lengend Snippet: Spam1 RNA contains four AREs in the 3' UTR and UV-cross-linking reveals that one or two specifically bind testicular proteins. A) Four AU-rich motifs, 5' AUUUG...AUUUUA...AUUUUUA...AUUUUUG...3', are found in a 77 nucleotide sequence (nt 1909–1985) in Spam1 3' UTR. B) In the upper panel the thin line represents the 77 nt riboprobe sequence containing the AU motifs while the thick lines show the antisense DNA oligomers used in competition assays. C 1 contains antisense oligos for all four AREs, C 2 for the two at the 5' end of the sequence, C 3 for the two at the 3' end, C 5 for the third from the 5' end, and C 6 for the 3' end motif. The lower panel shows the results of in vitro label transfer by UV cross-linking and the RNA-protein complexes formed with the riboprobe and testicular protein extract, without competition, in Lanes T 1 and T 3 . Lanes C 1 and C 2 show the disappearance of the complexes after competition with antisense oligos for all four motifs and the two at the 5' end, respectively. Lane C 3 shows that the complexes are not diminished with competition with antisense oligos for the two 3' end AREs.

Article Snippet: After washing and blocking in 2% BSA in PBS they were stained using the rabbit antipeptide mouse Spam1 antiserum generated from a C-terminal 15-mer (#381 – 395) oligopeptide (custom made by Zymed, San Francisco, CA) (diluted 1:400) specific for Spam1 [ , ].

Techniques: Sequencing, In Vitro

Journal: PLoS ONE

Article Title: Nanoparticle Incorporation of Melittin Reduces Sperm and Vaginal Epithelium Cytotoxicity

doi: 10.1371/journal.pone.0095411

Figure Lengend Snippet: ( A and B ) Size and zeta potential of anti-SPAM1 NPs and anti-SPAM1-mel-NPs immediately following preparation. ( C ) Fluorescence of sperm-bound blank NPs and anti-SPAM1 NPs following incubation with sperm for 30 minutes and removal of unbound NPs by density gradient centrifugation. Control samples did not contain sperm and were used to determine background fluorescence due to remaining unbound NPs. ( D ) Standard curve of blank NP and anti-SPAM1 NP fluorescence used for quantification of sperm binding. Error bars represent S.D. of n = 3 replicates, NS p>0.05, *** p<0.001. ( E , F , G and H ) Brightfield and fluorescence images of semen samples following addition of 3×10 10 blank NPs or anti-SPAM1 NPs. Scale bar = 50 µm.

Article Snippet: To biotinylate the monoclonal anti-SPAM1 antibody, 0.2 mg/ml antibody was mixed with 8.24 µL 1 mM Biotin-NHS (Thermo Scientific, Rockford, IL) and incubated at room temperature for 30 minutes.

Techniques: Fluorescence, Incubation, Gradient Centrifugation, Binding Assay