Dissecting the abilities of murine Siglecs to interact with gangliosides.

Siglecs are cell surface receptors whose functions are tied to the binding of their sialoglycan ligands. Recently, we developed an optimized liposome formulation and used it to investigate the binding of human Siglecs (hSiglec) against a panel of gangliosides. Animal models, more specifically murine models, are used to understand human biology, however, species-specific differences can complicate the interpretation of the results. Herein, we used our optimized liposome formulation to dissect the interactions between murine Siglecs (mSiglecs) and gangliosides to assess the appropriateness of mSiglecs as a proxy to better understand the biological roles of hSiglec-ganglioside interactions. Using our optimized liposome formulation, we found that ganglioside binding is generally conserved between mice and humans with mSiglec-1, -E, -F, and -15 binding multiple gangliosides like their human counterparts. However, in contrast to the hSiglecs, we observed little to no binding between the mSiglecs and ganglioside GM1a. Detailed analysis of mSiglec-1 interacting with GM1a and its structural isomer, GM1b, suggests that mSiglec-1 preferentially binds α2-3-linked sialic acids presented from the terminal galactose residue. The ability of mSiglecs to interact or not interact with gangliosides, particularly GM1a, has implications for using mice to study neurodegenerative diseases, infections, and cancer, where interactions between Siglecs and glycolipids have been proposed to modulate these human diseases.

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket.

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec-glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.

Transcriptome profile of rat genes in bone marrow-derived macrophages at different activation statuses by RNA-sequencing.

The underlying mechanisms of macrophage polarization have been detected by genome-wide transcriptome analysis in a variety of mammals. However, the transcriptome profile of rat genes in bone marrow-derived macrophages (BMM) at different activation statuses has not been reported. Therefore, we performed RNA-Sequencing to identify gene expression signatures of rat BMM polarized in vitro with different stimuli. The differentially expressed genes (DEGs) among unactivated (M0), classically activated pro-inflammatory (M1), and alternatively activated anti-inflammatory macrophages (M2) were analyzed by using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. In this study, not only we have identified the changes of global gene expression in rat M0, M1 and M2, but we have also made clear systematically the key genes and signaling pathways in the differentiation process of M0 to M1 and M2. These will provide a foundation for future researches of macrophage polarization.

Correction to: Antidepressant-Like Action of Single Facial Injection of Botulinum Neurotoxin A is Associated with Augmented 5-HT Levels and BDNF/ERK/CREB Pathways in Mouse Brain.

In the original publication, Figure 4G was incorrectly published. The correct version of Figure 4G is presented in this correction. This correction does not affect the conclusions of the paper.

Antidepressant-Like Action of Single Facial Injection of Botulinum Neurotoxin A is Associated with Augmented 5-HT Levels and BDNF/ERK/CREB Pathways in Mouse Brain.

The present study was designed to examine the therapeutic effects of Botulinum neurotoxin A (BoNT/A) on depression-like behaviors in mice and to explore the potential mechanisms. These results revealed that a single facial injection of BoNT/A induced a rapid and prolonged improvement of depression-like behaviors in naïve and space-restriction-stressed (SRS) mice, reflected by a decreased duration of immobility in behavioral despair tests. BoNT/A significantly increased the 5-hydroxytryptamine (5-HT) levels in several brain regions, including the hippocampus and hypothalamus, in SRS mice. BoNT/A increased the expression of the N-methyl-D-aspartate receptor subunits NR1 and NR2B in the hippocampus, which were significantly decreased in SRS mice. Furthermore, BoNT/A significantly increased the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus, hypothalamus, prefrontal cortex, and amygdala, which were decreased in SRS mice. Finally, BoNT/A transiently increased the levels of phosphorylated extracellular signal-regulated kinase (p-ERK) and cAMP-response element binding protein (p-CREB), which were suppressed in the hippocampus of SRS mice. Collectively, these results demonstrated that BoNT/A treatment has anti-depressant-like activity in mice, and this is associated with increased 5-HT levels and the activation of BDNF/ERK/CREB pathways in the hippocampus, supporting further investigation of BoNT/A therapy in depression.

Subcutaneous Administration of PDGF-AA Improves the Functional Recovery After Spinal Cord Injury.

Previous studies by our group have demonstrated that the transplantation of exogenous platelet-derived growth factor (PDGF)-AA-overexpressing oligodendrocyte progenitor cells (OPCs) promotes tissue repair and recovery of neurological function in a rat model of spinal cord injury (SCI). However, it remains unclear whether treatment with PDGF-AA also affects endogenous oligodendrocytes (OLs) or even neurons, thus promoting further functional recovery after SCI. In the present study, we evaluated the therapeutic potential of PDGF-AA treatment by direct subcutaneous injection of PDGF-AA immediately after SCI. We demonstrated that PDGF-AA injection resulted in increased tissue sparing, myelination and functional recovery in rats following SCI. Further experimentation confirmed that PDGF-AA increased the survival of endogenous OPCs and OLs, and promoted the proliferation of OPCs and their differentiation into OLs. Moreover, PDGF-AA also protected motor neurons from death in the injured spinal cord. These results indicated that PDGF-AA administration may be an effective treatment for SCI.

Expression and localization of absent in melanoma 2 in the injured spinal cord.

In traumatic brain injury, absent in melanoma 2 (AIM2) has been demonstrated to be involved in pyroptotic neuronal cell death. Although the pathophysiological mechanism of spinal cord injury is similar to that of brain injury, the expression and cellular localization of AIM2 after spinal cord injury is still not very clear. In the present study, we used a rat model of T9 spinal cord contusive injury, produced using the weight drop method. The rats were randomly divided into 1-hour, 6-hour, 1-day, 3-day and 6-day (post-injury time points) groups. Sham-operated rats only received laminectomy at T9 without contusive injury. Western blot assay revealed that the expression levels of AIM2 were not significantly different among the 1-hour, 6-hour and 1-day groups. The expression levels of AIM2 were markedly higher in the 1-hour, 6-hour and 1-day groups compared with the sham, 3-day and 7-day groups. Double immunofluorescence staining demonstrated that AIM2 was expressed by NeuN+ (neurons), GFAP+ (astrocytes), CNPase+ (oligodendrocytes) and CD11b+ (microglia) cells in the sham-operated spinal cord. In rats with spinal cord injury, AIM2 was also found in CD45+ (leukocytes) and CD68+ (activated microglia/macrophages) cells in the spinal cord at all time points. These findings indicate that AIM2 is mainly expressed in neurons, astrocytes, microglia and oligodendrocytes in the normal spinal cord, and that after spinal cord injury, its expression increases because of the infiltration of leukocytes and the activation of astrocytes and microglia/macrophages.

Increased ceruloplasmin expression caused by infiltrated leukocytes, activated microglia, and astrocytes in injured female rat spinal cords.

Ceruloplasmin (Cp), an enzyme containing six copper atoms, has important roles in iron homeostasis and antioxidant defense. After spinal cord injury (SCI), the cellular components in the local microenvironment are very complex and include functional changes of resident cells and the infiltration of leukocytes. It has been confirmed that Cp is elevated primarily in astrocytes and to a lesser extent in macrophages following SCI in mice. However, its expression in other cell types is still not very clear. In this manuscript, we provide a sensible extension of these findings by examining this system within a female Sprague-Dawley rat model and expanding the scope of inquiry to include additional cell types. Quantitative reverse transcription polymerase chain reaction and Western blot analysis revealed that the Cp mRNA and protein in SCI tissue homogenates were quite consistent with prior publications. However, we observed that Cp was expressed not only in GFAP+ astrocytes (consistent with prior reports) but also in CD11b+ microglia, CNPase+ oligodendrocytes, NeuN+ neurons, CD45+ leukocytes, and CD68+ activated microglia/macrophages. Quantitative analysis proved that infiltrated leukocytes, activated microglia/macrophages, and astrocytes should be the major sources of increased Cp.

[Toxicity thresholds and predicted model of Pb added to soils with various properties and its leaching factors as determined by barley root-elongation test].

Ten field soils with various properties were added with Pb at 8 levels, and treated with or without leaching using simulated rain to test the Pb toxicity threshold (EC10, EC50) according to the ISO 11269-1 root elongation toxicity testing method. The leaching factors of Pb toxicity to barley root elongation were determined and the predicted models of Pb toxicity thresholds in soils with various properties were developed. The results indicated that the determined toxicity threshold (EC10, EC50) of Pb varied significantly (P<0.01) among the soils, and the EC10 and EC50 of Pb to barley in the soils ranged from 300-4130 mg . kg-1 and 55-633 mg . kg-1, respectively. The Pb toxicity decreased sharply after leaching treatment. The determined leaching factors (LF) ranged from 0.96-1.96 (LFEC50) and 1.03-1.81 (LFEC10) respectively, and the obvious effect was observed for the Pb toxicity decreasing in the soils with pH<6.81. The predicted models for Pb toxicity thresholds in soils were developed based on main soil properties (soil pH, organic carbon content and cation exchange capacity), and the determined Pb toxicity thresholds (ECx, x = 10,50) in soils fell in the range of mean values with ± 2 standard error (SD) prediction interval for the leached and unleached soils except for the red soil of Jiangxi Province, which indicated that the models based on the main soil properties could well predict the Pb toxicity in soils with various soil properties.

[Toxicity and accumulation of copper and nickel in wheat plants cropped on alkaline and acidic field soils].

Field experiments were conducted to study the toxicity of added copper (Cu) and nickel (Ni) in soils to wheat and metal accumulation in wheat plants. The results showed that the yields of wheat straw and grain were decreased with the increasing concentration of Cu and Ni added to soils. The added Cu concentrations yielding 10% inhibition of wheat yield (EC10) were 499.6 mg x kg(-1) for alkaline soils (Dezhou, pH 8.90), and 55.7 mg x kg(-1) for acidic soils (Qiyang, pH 5.31). The toxicity of Cu or Ni in acidic soils were significantly higher than that in alkaline soils. With increasing addition of Cu or Ni, the contents of Cu in wheat grains initially increased and then keep at constant level, while the accumulation of Ni in grains linearly increased. The contents of Cu and Ni in Qiyang wheat grains were 6.07-9.26 mg x kg(-1) and 0.53-31.78 mg x kg(-1), and those of in Dezhou were 5.24-10. 52 mg x kg(-1) and 0.16-25.33 mg x kg(-1). In both field experimental sites, the contents of Cu in wheat grains meet the national standard for food safety. These findings showed that Cu is more relevant to ecological risk assessments than to food safety assessments for wheat grown in soils that have been contaminated with Cu.

[The potential role of Id1 in COX-2 mediated angiogenesis in gastric cancer].

OBJECTIVE: To investigate the potential role of inhibitor of DNA binding/differentiation 1 (Id1) in cyclooxygenase-2 (COX-2) mediated angiogenesis in gastric cancer. METHODS: Two human gastric cancer sub-cell lines (SGC7901/COX-2 and SGC7901/COX-2RNAi) highly expressing COX-2 and COX-2 RNAi respectively were established. Western blotting and RT-PCR were performed to detect the protein and mRNA expression levels of Id1 in these transfectants. ELISA was performed to detect the levels of VEGF protein in the supernatants of these cells. Humane umbilical vein endothelial cells of the line HU-LT were cultured in condition medium, supernatant of SGC7901/COX2 cells transfected with COX-2 sense strand, COX-2 SiRNA and Id1. MTT assay was used to examine the proliferation ability of these cells. Suspensions of SGC7901/COX-2 and SGC7901/COX-2RNAi cells were injected subcutaneously to nude mice respectively. Eight weeks later the mice were killed and the tumors were taken out to undergo immunohistochemistry and detection of mcrovessel density (MVD). RESULTS: Western blotting and RT-PCR showed that the expression levels of COX-2 protein and mRNA, as well as those of Id1 in the SGC7901/COX-2 cells were high, and the expression level of Id1 was down-regulated in the SGC7901/COX-2RNAi cells transfected with Id1 RNAi. The VEGF level of the SGC7901/COX-2 cells was 2060 +/- 42, significantly higher than that of the control SGC7901/PC cells (1248 +/- 28, P = 0.000) and VEGF level of the supernatant of then SGC7901/COX-2 RNAi cells was 1024 +/- 20, significantly lower than that of the SGC7901/COX-2/PC cells (2033 +/- 27, P = 0.000). The proliferation rate of the HU-LT cells cultured in SGC7901/COX-2 condition medium was higher than that of the HU-LT cells cultured in SGC7901/COX-2RNAi condition medium. The increase of VEGF in the SGC7901/COX-2 and the effect of contribution to the proliferation of HU-LT were both abrogated when Id1 was knocked out in the SGC7901/COX-2. The tumor weight of the COX-2 RNAi group was (353 +/- 12) mg, significantly lower than that of the SGC7901/COX-2 group [(1020 +/- 91) mg, P = 0.038]. The MVD of the tumor of the COX-2 RNAi group was 8.8 +/- 1.6, significantly lower than that of the SGC7901/COX-2 group (20 +/- 1.7, P = 0.001). CONCLUSION: COX-2 can stimulate VEGF and enhance the proliferation of endothelial cells by upregulating Id1, and blocking this pathway may be helpful to the tumor therapeutics, so COX-2 and Id1 can be exploited as therapeutic targets of gastric cancer.

[Prokaryotic expression and purification of RPS13 and preparation of polyclonal antibody against RPS13].

AIM: To construct prokaryotic expression plasmid of RPS13, express and purify the protein for the preparation of polyclonal antibody. METHODS: RPS13 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the highly expressed cell of gastric multidrug cell SGC7901/VCR. After sequenced, the gene was cloned into the expression vector pET-28a(+) to construct RPS13 expression plasmid pET-28a(+)-RPS13. The expression plasmid was transformed into the E.coli BL21 and screened by ampiline, and then the E.coli BL21 containing the expression plasmid was induced by IPTG and the protein was purifed by Histag column. BALB/c mice were immunized by the immunogen His-RPS13. The titer was measured by ELISA and the polyclonal antibody was obtained. RESULTS: The expression plasmid pET-28a(+)-RPS13 was constructed. The 19 kDa recombined protein was successfully expressed in the E.coli BL21 by IPTG for 3 hours, purified by Histag column and confirmed by SDS-PAGE and Western blot. The polyclonal anti-His-RPS13 antibody was obtained by immunizing the mice with RPS13 protein. RPS13 could be specifically recognized by the antibody based on Western blot. CONCLUSION: RPS13 has bene expressed and purified successfully and polyclonal anti-His-RPS13 antibody has been prepared. Our study can be used for the preparation of monoclonal anti-His-RPS13 antibody and for further research of the function of the RPS13 protein in tumor.