Ultrasound-propelled nanomotors for efficient cancer cell ferroptosis.

Ferroptosis is a non-apoptotic form of cell death that is dependent on the accumulation of intracellular iron that causes elevation of toxic lipid peroxides. Therefore, it is crucial to improve the levels of intracellular iron and reactive oxygen species (ROS) in a short time. Here, we first propose ultrasound (US)-propelled Janus nanomotors (Au-FeOx/PEI/ICG, AFPI NMs) to accelerate cellular internalization and induce cancer cell ferroptosis. This nanomotor consists of a gold-iron oxide rod-like Janus nanomotor (Au-FeOx, AF NMs) and a photoactive indocyanine green (ICG) dye on the surface. It not only exhibits accelerating cellular internalization (∼4-fold) caused by its attractive US-driven propulsion but also shows good intracellular motion behavior. In addition, this Janus nanomotor shows excellent intracellular ROS generation performance due to the synergistic effect of the "Fenton or Fenton-like reaction" and the "photochemical reaction". As a result, the killing efficiency of actively moving nanomotors on cancer cells is 88% higher than that of stationary nanomotors. Unlike previous passive strategies, this work is a significant step toward accelerating cellular internalization and inducing cancer-cell ferroptosis in an active way. These novel US-propelled Janus nanomotors with strong propulsion, efficient cellular internalization and excellent ROS generation are suitable as a novel cell biology research tool.

Occurrence state of fluoride in barite ore and the complexation leaching process.

Barite ore is typically associated with difficult-to-remove vein minerals, but commercial barite products require a high BaSO4 content. We investigated the occurrence state of fluoride in barite ore using various analytical techniques, which indicated that elemental fluorine in barite predominantly exists as fluorite. Fluoride was then leached from barite ore via complexation. The effects of HCl and AlCl3 concentrations, temperature, time, and liquid-solid ratio on the leaching rate were examined, and the leaching conditions were optimized using an orthogonal array method. The fluorine leaching rate approached 93.11% after stirring for 30 min at 90 °C and 300 rpm with 3 mol/L HCl, 0.4 mol/L AlCl3, a liquid-solid ratio of 10:1 mL/g, and an ore sample size of -75 µm + 48 µm. According to the leaching kinetics, the process conformed to the solid membrane diffusion control model at a high temperature and the joint chemical reaction-diffusion control model at a low temperature. The apparent activation energy was 56.88 kJ/mol. Furthermore, aluminum and fluorine coordination numbers increased with increasing Al3+/F- molar concentration ratios. Competing complexation reactions of Al3+, H+, and F- occurred at three levels. This complexation approach effectively leaches fluoride from barite, improves barite product quality, and reduces environmental pollution.

Determination of carbonate content in barite ore by headspace gas chromatography.

This paper reports a method for determining the carbonate content in barite ore using headspace gas chromatography. Based on the acidification reaction, the carbonate in the barite ore was converted to CO2 in a closed headspace vial. When the carbonate content was significant, the pressure caused changes in the CO2 and O2 signals and affected the measurement accuracy. It was found that carbonate content is proportional to the intensity ratio of the CO2 to O2 signals. Thus, the carbonate content in barite ore can be measured indirectly using a theoretical model. The results showed that the carbonate in 3 g of barite ore sample with a particle size of 74 µm could react completely with a hydrochloric acid solution (2 mol/L) at 65°C for 5 min. The method described herein had good precision (relative standard deviation < 4.14%) and accuracy (relative differences < 6.12%). Further, the limit of quantification was 0.07 mol/L. Owing to its simplicity and speed, this method can be used for the batch determination of carbonate content in barite ore.

Determination of barium sulfate in barite ore by phase conversion-partial pressure-corrected headspace gas chromatography.

Barium sulfate (BaSO4) content is used to evaluate the grade of barite ore. In the present study, we report a method to determine the BaSO4 content in barite ore by phase conversion-headspace gas chromatography with partial pressure correction. In this method, the ore sample is roasted with sodium carbonate and potassium carbonate after pretreatment with hydrochloric acid. The roasted product is subsequently placed in a closed headspace bottle to react with hydrochloric acid. The ratio of CO2 to O2 signals is detected by a thermal conductivity detector for gas chromatography. Finally, the BaSO4 content in barite ore is calculated using this ratio. The method demonstrates good precision (relative standard deviation < 0.84%) and accuracy (relative error < 3.40%), with the uncertainty at 95% confidence interval at approximately +/- 0.57%. Moreover, this approach is expected to be used for the batch testing of BaSO4 content in barite ores in industrial applications.

Pathogenicity of the newly emerged Lagovirus europaeus GI.2 strain in China in experimentally infected rabbits.

In April 2020, rabbit hemorrhagic virus type 2 (Lagovirus europaeus GI.2), which causes highly infectious fatal rabbit hemorrhagic disease, was emerged in China. The phylogenetic analyses of the complete genome sequence of GI.2 showed that it belonged to the non-recombinant GI.3/GI.2 genotype. However, the pathogenicity of this GI.2 strain differed from that of early typical GI.2 strains in Europe. To prevent the spread of the new strain in China, its pathogenicity urgently needs to be studied. Thus, viral shedding and distribution as well as clinical symptoms, histopathological changes, and serum cytokines were studied in experimentally GI.2/SC2020-infected rabbit adults and kits. The kit group showed a shorter survival time after the challenge than the adult group did. The mortality rate was higher in the kits (80 %) than in the adults (30 %). Viral RNA could be detected in both nasal and fecal swabs, and the main dissemination route appeared to be the fecal route. Viral RNA rapidly increased in the blood of the adults and kits at 6 h post-infection, indicating that blood viral load testing can be used for early diagnosis. The most affected organs were the liver and spleen, and the lesions were more severe in the kits than in the adults. The liver contained the highest viral RNA levels. Moreover, serum interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor-alpha levels were increased in the infected rabbits. In conclusion, our findings will help to understand the evolutionary trends and pathogenic characteristics of GI.2 strains in China.

NF-κB and Keap1 Interaction Represses Nrf2-Mediated Antioxidant Response in Rabbit Hemorrhagic Disease Virus Infection.

The rabbit hemorrhagic disease virus (RHDV), which belongs to the family Caliciviridae and the genus Lagovirus, causes lethal fulminant hepatitis in rabbits. RHDV decreases the activity of antioxidant enzymes regulated by Nrf2 in the liver. Antioxidants are important for the maintenance of cellular integrity and cytoprotection. However, the mechanism underlying the regulation of the Nrf2-antioxidant response element (ARE) signaling pathway by RHDV remains unclear. Using isobaric tags for relative and absolute quantification (iTRAQ) technology, the current study demonstrated that RHDV inhibits the induction of ARE-regulated genes and increases the expression of the p50 subunit of the NF-κB transcription factor. We showed that RHDV replication causes a remarkable increase in reactive oxygen species (ROS), which is simultaneously accompanied by a significant decrease in Nrf2. It was found that nuclear translocation of Keap1 plays a key role in the nuclear export of Nrf2, leading to the inhibition of Nrf2 transcriptional activity. The p50 protein partners with Keap1 to form the Keap1-p50/p65 complex, which is involved in the nuclear translocation of Keap1. Moreover, upregulation of Nrf2 protein levels in liver cell nuclei by tert-butylhydroquinone (tBHQ) delayed rabbit deaths due to RHDV infection. Considered together, our findings suggest that RHDV inhibits the Nrf2-dependent antioxidant response via nuclear translocation of Keap1-NF-κB complex and nuclear export of Nrf2 and provide new insight into the importance of oxidative stress during RHDV infection.IMPORTANCE Recent studies have reported that rabbit hemorrhagic disease virus (RHDV) infection reduced Nrf2-related antioxidant function. However, the regulatory mechanisms underlying this process remain unclear. The current study showed that the NF-κB p50 subunit partners with Keap1 to form the Keap1-NF-κB complex, which plays a key role in the inhibition of Nrf2 transcriptional activity. More importantly, upregulated Nrf2 activity delayed the death of RHDV-infected rabbits, strongly indicating the importance of oxidative damage during RHDV infection. These findings may provide novel insights into the pathogenesis of RHDV.

Rabbit Hemorrhagic Disease Virus Non-structural Protein 6 Induces Apoptosis in Rabbit Kidney Cells.

Rabbit hemorrhagic disease (RHD) is a highly contagious disease caused by rabbit hemorrhagic disease virus (RHDV). Previous research has shown that RHDV induces apoptosis in numerous cell types, although the molecular mechanisms underlying the apoptosis induced by RHDV are not well understood. One possible factor is non-structural protein 6 (NSP6), a 3C-like protease that plays an important role in processing viral polyprotein precursors into mature non-structural proteins. To fully establish a role for NSP6, the present study examined the effects of ectopic expression of the protein in rabbit (RK13) and human (HeLa and HepG2) cells. We found that NSP6 suppressed cell viability and promoted apoptosis in all three cell types in a dose-dependent manner. We also identified increased caspase-3, -8, and -9 activities in RK13 cell, and an increased Bax to Bcl2 mRNA ratio. Mechanistically, the ability of NSP6 to induce apoptosis was impaired by mutation of the catalytic His27 residue. Our study has shown that RHDV NSP6 can induce apoptosis in host cells and is likely an important contributor to RHDV-induced apoptosis and pathogenesis.

Recombination between G2 and G6 strains of rabbit hemorrhagic disease virus (RHDV) in China.

Rabbit hemorrhagic disease (RHD) is an acute fatal disease caused by the lagovirus rabbit hemorrhagic disease virus (RHDV), which was first reported in 1984 in China. Genetic characterization of RHDV has demonstrated that two different genogroups (G2 and G6) are present in China. To gain a better understanding of the molecular evolution of RHDV, we searched for recombination events by analyzing all full-length RHDV capsid VP60 sequences of Chinese isolates belonging to the genogroups 2 and 6. Our results revealed a recombinant origin for the NanBu/China/2011 isolate. This recombination event occurred between G2 and G6 strains with two breakpoints located at nucleotide positions 393 and 1079 of the VP60 sequence. Phylogenetically, the NanBu/China/2011 strain clustered with genogroup G6 in the entire capsid gene sequence except in the fragment between nucleotides 394 and 1078, where it clustered with genogroup G2. As the consequences of the presence of a G2/G6 recombinant strain in China are unpredictable, the circulation of RHDV in the populations should be carefully monitored.

Molecular cloning of the rabbit interleukin 6 promoter: Functional characterization of rabbit hemorrhagic disease virus response elements in RK-13 cells.

Infection with rabbit hemorrhagic disease virus (RHDV) can cause acute liver failure (ALF), leading to severe mortality in rabbits. Inflammatory response, especially the expression of inflammatory cytokines such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6, may play major roles in mediating and amplifying the ALF. Among these cytokines, IL-6 is a multifunctional cytokine with a central role in various physiological inflammatory and immunological processes. In this study, we found that RHDV infection significantly upregulated IL-6 gene expression in vivo. Next, the rabbit IL-6 promoter was cloned and analyzed. Transfection of full-length RHDV cDNA in RK-13 cells upregulated the activity of the IL-6 promoter. A series of 5' deletion constructs demonstrated that AP-1 (activator protein 1), NF-IL6 (nuclear factor interleukin-6), and NF-κB (nuclear factor kappa B) elements were critical for RHDV-induced IL-6 transcription. Besides, the CREB (cAMP-response element binding protein) element may also play an accessory effect on RHDV-induced IL-6 transcription. Collectively, the results elucidate the mechanism of IL-6 induction, and enrich the RHDV pathogenesis in rabbit.

A new variant of rabbit hemorrhagic disease virus G2-like strain isolated in China.

To investigate the genetic variability and evolution of rabbit hemorrhagic disease virus (RHDV) strains in China, VP60 gene sequences of eight new isolates collected from farms with RHD occurrences in China between 2009 and 2014 were analyzed, and compared with the reference sequence of the vaccine strain WF/China/2007. We conducted a comprehensive analysis of the Chinese RHDV strains, including hemagglutination tests, western blot and immunosassays of capsid proteins, and phylogenetic analysis, and identified a new distinct antigenic variant. Specifically, strain HB/2014 collected in North China was identified as a non-hemagglutinating strain, and belongs to the original RHDV (G1-G5) group. The other seven isolates were classified in genogroup G6 (RHDVa), which was widely distributed across China before 2014, and was thought to replace the earlier groups. Antigenic characterization of the VP60 genes revealed a large degree of nucleotide sequence divergence between HB/2014 and the other Chinese strains. However, the current vaccine showed complete cross-protection against HB/2014 challenge in inoculated rabbits. Collectively, these data provide new tools and insight for further understanding the molecular evolution of RHDV in China.

Identification of novel rabbit hemorrhagic disease virus B-cell epitopes and their interaction with host histo-blood group antigens.

Rabbit haemorrhagic disease, caused by rabbit hemorrhagic disease virus (RHDV), results in the death of millions of adult rabbits worldwide, with a mortality rate that exceeds 90%. The sole capsid protein, VP60, is divided into shell (S) and protruding (P) domains, and the more exposed P domain likely contains determinants for cell attachment and antigenic diversity. Nine mAbs against VP60 were screened and identified. To map antigenic epitopes, a set of partially overlapping and consecutive truncated proteins spanning VP60 were expressed. The minimal determinants of the linear B-cell epitopes of VP60 in the P domain, N(326)PISQV(331), D(338)MSFV(342) and K(562)STLVFNL(569), were recognized by one (5H3), four (1B8, 3D11, 4C2 and 4G2) and four mAbs (1D4, 3F7, 5G2 and 6B2), respectively. Sequence alignment showed epitope D(338)MSFV(342) was conserved among all RHDV isolates. Epitopes N(326)PISQV(331) and K(562)STLVFNL(569) were highly conserved among RHDV G1-G6 and variable in RHDV2 strains. Previous studies demonstrated that native viral particles and virus-like particles (VLPs) of RHDV specifically bound to synthetic blood group H type 2 oligosaccharides. We established an oligosaccharide-based assay to analyse the binding of VP60 and epitopes to histo-blood group antigens (HBGAs). Results showed VP60 and its epitopes (aa 326-331 and 338-342) in the P2 subdomain could significantly bind to blood group H type 2. Furthermore, mAbs 1B8 and 5H3 could block RHDV VLP binding to synthetic H type 2. Collectively, these two epitopes might play a key role in the antigenic structure of VP60 and interaction of RHDV and HBGA.

[Mechanisms for reversal of pulmonary hypertension by rapamycin in rats].

OBJECTIVE: To investigate the effects of rapamycin (RAP) on pulmonary hypertension (PH) in rats, and to provide new insights into medication selection for the clinical treatment of PH. METHODS: Fifty male Sprague-Dawley rats were randomly divided into blank control, PH model, solvent control, RAP 1, and RAP 2 groups. A rat model of PH was induced by left pneumonectomy (PE) and monocrotaline (MCT). At 5 days after PH model establishment, the solvent control group and the RAP 1 group received an intramuscular injection of solvent and RAP, respectively. At 35 days after PH model establishment, the RAP 2 group received an intramuscular injection of RAP. The mean pulmonary artery pressure (mPAP) and the right ventricle/left ventricle plus septum weight ratio (RV/LV+S) were measured in each group. Histopathological changes in the right lung were evaluated by hematoxylin-eosin (HE) staining. The relative expression of alpha-smooth muscle actin (α-SMA) and smooth muscle protein 22-alpha (SM22α) in each group was determined using real-time PCR. RESULTS: At 35 days after surgery, the PH model and the solvent control groups had significantly higher mPAP and RV/LV+S than the blank control group, while the RAP 1 and the RAP 2 groups had significantly lower mPAP than the solvent control group (P<0.05). The RV/LV+S in the RAP 1 group was significantly lower than that in the solvent control group (P<0.05); however, there was no significant difference in RV/LV+S between the RAP 2 and the solvent control groups (P>0.05). HE staining in the right lung showed the substantially thickened pulmonary artery wall and narrowed arterial lumen in the PH model and the solvent control groups compared with the blank control group. Different degrees of reversal of the pulmonary artery wall thickening were observed after RAP administration. The results of real-time PCR revealed that the relative expression of α-SMA and SM22α in the PH model and the solvent control groups was significantly lower than in the blank control group, while the relative expression of α-SMA and SM22α in the RAP 1 and the RAP 2 groups was significantly higher than in the solvent control group (P<0.05). CONCLUSIONS: RAP can reverse the increase in pulmonary artery pressure and the right ventricular hypertrophy probably by regulation of the phenotypic conversion of vascular smooth muscle cells.

Role of the PI3K/AKT pathway in modulating cytoskeleton rearrangements and phenotype switching in rat pulmonary arterial vascular smooth muscle cells.

Pulmonary arterial smooth muscle cell (PASMC) phenotype switching, which is characterized by changes in smooth muscle (SM)-specific gene expression, contributes to vascular remodeling in pulmonary hypertension. In addition, it has been shown that the transcription of SM-specific genes is modulated by cytoskeleton rearrangement. However, the intracellular mechanisms and signaling pathways that regulate these relationships are largely unknown. In the present study, we aimed to investigate the roles that phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB), also known as AKT, play in modulating the cytoskeleton and phenotype of rat PASMCs. To observe the downstream effects of inhibiting or enhancing PI3K/AKT pathway activity, we used various approaches to manipulate protein function and gene expression. Treatment of PASMCs with platelet-derived growth factor (PDGF)-BB or PIK3CA-adenovirus induced cytoskeleton rearrangements and downregulated SM22α and α-SM actin gene expression. Inhibition of PI3K led to blocking of AKT phosphorylation and attenuated the PDGF-BB-induced downregulation of F-actin and SM-specific genes, the downstream effector of PI3K. The decrease in SM22α and α-SM actin mRNA levels induced by PDGF-BB was markedly and reproducibly blocked by LY294002. PI3K/AKT pathway plays a vital role in the modulation of PASMCs cytoskeleton rearrangement and phenotype switching.

[Effects of paclitaxel on proliferation and transition of phenotype in platelet-derived growth factor-BB-induced pulmonary vascular smooth muscle cells].

OBJECTIVE: To investigate the effects of paclitaxel on the phenotypic modulation induced by platelet-derived growth factor (PDGF-BB) in rat pulmonary vascular smooth muscle cells (PVSMC). METHODS: The proliferation of PVSMC isolated from SD rats cultured in vitro was induced by PDGF-BB and then intervened by different concentration of paclitaxel. MTT and [³H]-thymidine incorporation were used to detect the changes of cell proliferation. The expression level of alpha-smooth muscle-actin (SM-α-actin) and smooth muscle protein 22alpha (SM22α) were tested by Western blot. Confocal laser scanning microscopy was applied to observe the change of fluorescence intensity. RESULTS: Treatment with PDGF-BB for 24 hours results in a significant increase in [³H]-thymidine incorporation and marked change in phenotype and cytoskeleton, Paclitaxel inhibited the proliferation of PVSMC induced by PDGF-BB, the inhibition rate was 45.4%, 35.4%, 21.6% (P < 0.01) tested by[³H]-thymidine incorporation and 40.0%, 30.0%, 18.0% (P < 0.01) tested by MTT. Meanwhile, the paclitaxel promoted the expression level of SM-α-actin and SM22α. Fluorescence intensity of F-actin decreased significantly. CONCLUSION: Paclitaxel may play an important role in vascular remodeling by changing the phenotypes and cytoskeleton of VSMC stimulated by PDGF-BB.