Cytotoxicity, cytokine release and ER stress-autophagy gene expression in endothelial cells and alveolar-endothelial co-culture exposed to pristine and carboxylated multi-walled carbon nanotubes.

Recently we found that direct exposure of human umbilical vein endothelial cells (HUVECs) to multi-walled carbon nanotubes (MWCNTs) might induce toxicological responses through the modulation of ER stress gene expression, but whether this signal could be transferred from other cells to endothelial cells (ECs) is unknown. This study investigated the toxicity of pristine and carboxylated MWCNTs to HUVECs and alveolar-endothelial co-culture, the later of which could mimic the possible signaling communications between ECs and MWCNT exposed alveolar cells. The results showed that direct contact with high levels of MWCNTs induced cytotoxicity and modulated expression of genes associated with ER stress (HSPA5, DDIT3 and XBP-1s) and autophagy (BECN1 and ATG12) both in A549-THP-1 macrophages cultured in the upper chambers as well as HUVECs. However, most of these responses were minimal or negligible in HUVECs cultured in the lower chambers. Moreover, significantly increased cytokine release (interleukin-6 and soluble vascular cell adhesion molecule-1) was only observed in MWCNT exposed HUVECs (p < 0.01) but not HUVECs cultured in the lower chambers (p > 0.05). The minimal or even absent response was likely due to relatively low translocation of MWCNTs from upper chambers to lower chambers, whereas A549-macrophages cultured in the upper chambers internalized large amount MWCNTs. The results indicated that ER stress-autophagy signaling might not be able to transfer from alveolar cells to endothelial cells unless sufficient MWCNTs are translocated.

Efficient cryopreservation of mouse embryos by modified droplet vitrification (MDV).

The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-µL vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P>0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P<0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P<0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P>0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P>0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P<0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages.

Technological innovation, militarization, and environmental change: evidence from BRICS economies.

In the complex international society, the economic development and defense construction of BRICS countries have attracted increasing attention. This article incorporates technological innovation, militarization, and environmental change into a unified analytical framework to assess the potential impact of technological progress and defense military expenditure on the environment in BRICS countries. Based on CSD tests, unit root tests, and cointegration tests, this study constructs a CS-ARDL model to examine the long-term and short-term relationships among various variables from 1990 to 2021. The results show that technological innovation, military expenditure, and economic growth can significantly increase ecological footprint in the long run, while in the short term, technological innovation and economic growth significantly increase ecological footprint, and the impact of military expenditure is not significant. It is suggested that BRICS countries should focus on supporting low-carbon technology policies and research and development investment, while also considering the use of cutting-edge technology to improve military intelligence capabilities in order to reduce the negative impact of technological innovation and military activities on the environment.

Albumin pre-opsonized membrane-active iPep nanomedicine potentiates chemo to immunotherapy of cancer.

Chemotherapy-conjugated immunotherapy in clinical oncology conceptually resembles the combined effects of cytoreduction and immunostimulation in membrane targeted cell killings mediated by pore-forming proteins or host defense peptides. Of the similar concept, targeting cancer cell membrane using membrane active peptides is a hopeful therapeutic modality but had long been hindered from in vivo application. Here we report an enabling strategy of pre-opsonizing a membrane penetrating Ir-complexed octa-arginine peptide (iPep) with serum albumin via intrinsic amphipathicity-driven bimodal interactions into nanoparticles (NP). We found that NP triggered stress-mediated 4T1 cell oncosis which induced potent immunological activation, surpassing several well-known immunogenic medicines. Vested with albumin-enhanced in vivo tumor targeting specificity and pharmacokinetic properties, NP showed combined chemo to immunotherapies of s. c. tumors in mice, with decreased percentages of MDSC, Treg, M2-like macrophage and improved infiltration of CTLs in tumor site, caused complete regression of 4T1 and CT26 tumors, outperforming clinical medicines. In a challenging orthotopic breast cancer model, boost i. v. injections of NP acted as in situ tumor vaccine that drastically enhanced 4T1-specific cellular and humoral immunities to reverse disease progression. Thus, with combined effects of direct cytoreduction, immune activation and tumor vaccine, iPep-NP presents the promise and potential of a new modality of cancer medicine.

The toxicity of multi-walled carbon nanotubes (MWCNTs) to human endothelial cells: The influence of diameters of MWCNTs.

The biological applications of multi-walled carbon nanotubes (MWCNTs) may lead to their exposure to human blood vessels, but the influence of their physicochemical properties on toxicity to endothelial cells is incompletely known. Here, human umbilical vein endothelial cells (HUVECs) were exposed to three commercially available MWCNTs, namely XFM4, XFM22, and XFM34 (diameters XFM4 < XFM22 < XFM34), to understand the possible role of their diameter on toxicity. Based on the same mass concentration, XFM4 induced significantly higher level of cytotoxicity than the other two MWCNTs, and HUVECs internalized more XFM4. Cytokine release, monocyte adhesion, and intracellular reactive oxygen species levels were significantly induced only after XFM4 treatment. The exposure to XFM4 significantly reduced the expression of autophagic genes autophagy-related 7 (ATG7), autophagy-related 12 (ATG12), and beclin 1 (BECN1) and increased the expression of endoplasmic reticulum (ER) stress genes DNA damage inducible transcript 3 (DDIT3) and X-box binding protein 1 spliced (XBP-1s). Moreover, the modulation of autophagy-ER stress by chemicals resulted in a significant increase in the cytotoxicity of XFM4 but had minimal impact on the cytotoxicity of XFM34. These data indicate that the diameter of MWCNTs may influence their toxicity to HUVECs, probably through autophagy dysfunction and ER stress.

Synergistic effect of cysteamine, leukemia inhibitory factor, and Y27632 on goat oocyte maturation and embryo development in vitro.

Goat oocyte in vitro maturation is associated with a variable efficiency of embryo development after in vitro fertilization (IVF). Here, we developed a novel maturation procedure to evaluate the cellular effect of cysteamine (Cys), leukemia inhibitory factor (LIF) and Y27632 on oocyte in vitro maturation in native Chinese Yangtze river white goats. Oocytes were collected by slicing ovary tissues and matured for 24 h in vitro prior to IVF. Presumptive fertilized oocytes were cultured in embryo media for 8 days. Maturation rates were similar in gonadotropin basal maturation medium and the same medium supplemented with Cys, LIF, or Y27362 (41.0-48.0%; P > 0.05). However, when two substances were co-supplemented into the medium, the maturation rate was higher in the Cys+LIF group than in the LIF+Y27362 and Cys+Y27362 groups (60.0% vs. 43.1% and 25.8%, respectively; P < 0.05). Co-supplementation of all three substances into the medium achieved the highest maturation rate (67.5%; P < 0.05). Compared with oocytes in gonadotropin basal maturation medium, those in medium supplemented with Cys showed increased fertilization (56.1% vs. 72.1%), cleavage (36.7% vs. 44.8%), and blastocyst development (1.7% vs. 4.2%), respectively (P < 0.05). Cys+LIF supplementation further improved fertilization (81.6%), cleavage (54.9%), and blastocyst development (6%; P < 0.05). Furthermore, combined supplementation of all three substances resulted in the best fertilization (84.9%), cleavage (70.7%), and blastocyst development (10.3%; P < 0.05). Resultant IVF blastocysts possessed an average cell number as high as 276 ± 45 per embryo. This is the first study to report increased efficiency of caprine oocyte maturation by combined Cys, LIF, and Y27632 supplementation into basal maturation medium, leading to improved fertilization and embryo development in vitro post-IVF.

Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs.

We investigated the effects of 5'-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7-2 vs. F7-2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7-1 vs.F7-1, 12.1 vs. 23.6%; Site Three, tF7-3 vs.F7-3, 7.7 vs 10.9%) (P < 0.05). Out of 15 predicated off-target sites, tru-RGNs showed significantly decreased frequencies at 5 sites. By microinjecting tru-RGN RNAs into zygotes, FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P < 0.05), but not at Site three (39.4 vs 27.8%) (P > 0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing.

Autologous endothelial progenitor cells improve allograft survival in porcine lung transplantation with prolonged ischemia.

BACKGROUND: As endothelial progenitor cells (EPCs) attenuated acute lung injury (ALI) in rabbit model, we hypothesized that autologous EPCs preserved lung graft function during the acute reperfusion period of lung transplantation and tested the therapeutic potential of EPCs in a porcine model of lung transplantation with prolonged graft ischemia. METHODS: Day-7 EPCs isolated from the recipient subjects or plain culture media were administered into the left pulmonary artery immediately before restoration of pulmonary blood flow in a porcine lung allotransplantation model, with the transplantation surgeons blinded to the content of injection. Hemodynamics and arterial blood gas were recorded, and the right pulmonary artery was occluded 30 min after reperfusion to evaluate the lung graft function. The lung grafts were sectioned for histological examination at the end of experiments. The total ischemic time for lung graft was approximately 14 h. RESULTS: All animals receiving plain medium died within 40 min after reperfusion, but 3 out of 5 (60%) piglets receiving EPCs survived up to 4 h after diversion of the entire cardiac output into the lung graft (P<0.01). The donor body weight, recipient body weight, cold ischemic time, and time for anastomosis were comparable between the EPC and control group (P=0.989, 0.822, 0.843, and 0.452, respectively). The mean aortic pressure decreased, and the cardiac output and mean pulmonary artery pressure elevated after right pulmonary artery occlusion. All these parameters were gradually compensated in the EPC group but decompensated in the control group. Better preservation of gas exchange function, reduced thrombi formation in the terminal pulmonary arterioles, and attenuated interstitial hemorrhage of the lung graft were observed in the EPC group. CONCLUSIONS: We concluded autologous EPCs significantly enhanced the function of lung allograft and improved survival in a porcine model of lung transplantation with prolonged ischemia.