Ononin promotes radiosensitivity in lung cancer by inhibiting HIF-1α/VEGF pathway.

BACKGROUND: In our previous study, we provided evidence that Astragalus mongholicus Bunge(AM) and its extracts possess a protective capability against radiation-induced damage, potentially mediated through the reduction of reactive oxygen species (ROS) and nitric oxide (NO). However, we were pleasantly surprised to discover during our experimentation that AM not only offers protection against radiation damage but also exhibits a radiation sensitization effect. This effect may be attributed to a specific small molecule present in AM known as ononin. Currently, radiation sensitizers are predominantly found in nitrazole drugs and nanomaterials, with no existing reports on the radiation sensitization properties of ononin, nor its underlying mechanism. PURPOSE: This study aims to investigate the sensitization effect of the small molecule ononin derived from AM on lung cancer radiotherapy, elucidating its specific molecular mechanism of action. Additionally, the safety profile of combining astragalus small molecule ononin with radiation therapy will be evaluated. METHODS: The effective concentration of ononin was determined through cell survival experiments, and the impact of ononin combined with varying doses of radiation on lung cancer cells was observed using CCK-8 and cell cloning experiments. The apoptotic effect of ononin combined with radiation on lung cancer cells was assessed using Hochester staining, flow cytometry, and WB assay. Additionally, WB and immunofluorescence analysis were conducted to investigate the influence of ononin on HIF-1α/VEGF pathway. Furthermore, Molecular Dynamics Simulation was employed to validate the targeted binding ability of ononin and HIF-1α. A lung cancer cell line was established to investigate the effects of knockdown and overexpression of HIF-1α. Subsequently, the experiment was repeated using tumor bearing nude mice and C57BL/6 mouse models in an in vivo study. Tumor volume was measured using a vernier caliper, while HE, immunohistochemistry, and immunofluorescence techniques were employed to observe the effects of ononin combined with radiation on tumor morphology, proliferation, and apoptosis. Additionally, Immunofluorescence was employed to examine the impact of ononin on HIF-1α/VEGF pathway in vivo, and its effect on liver function in mice was assessed through biochemistry analysis. RESULTS: At a concentration of 25 µM, ononin did not affect the proliferation of lung epithelial cells but inhibited the survival of lung cancer cells. In vitro experiments demonstrated that the combination of ononin and radiation could effectively inhibit the growth of lung cancer cells, induce apoptosis, and suppress the excessive activation of the Hypoxia inducible factor 1 alpha/Vascular endothelial growth factor pathway. In vivo experiments showed that the combination of ononin and radiation reduced the size and proliferation of lung cancer tumors, promoted cancer cell apoptosis, mitigated abnormal activation of the Hypoxia inducible factor 1 alpha pathway, and protected against liver function damage. CONCLUSION: This study provides evidence that the combination of AM and its small molecule ononin can enhance the sensitivity of lung cancer to radiation. Additionally, it has been observed that this combination can specifically target HIF-1α and exert its effects. Notably, ononin exhibits the unique ability to protect liver function from damage while simultaneously enhancing the tumor-killing effects of radiation, thereby demonstrating a synergistic and detoxifying role in tumor radiotherapy. These findings contribute to the establishment of a solid basis for the development of novel radiation sensitizers derived from traditional Chinese medicine.

Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation.

Objective: To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation (IR). Methods: Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237 (MLN) and/or p21 depletion by small interfering RNA (siRNA). Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator (FUCCI) system combined with histone H3 phosphorylation at Ser10 (pS10 H3) detection. Senescence was assessed using senescence-associated-ß-galactosidase (SA-ß-Gal), Ki67, and γH2AX staining. Protein expression levels were determined using western blotting. Results: Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment. The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells, ultimately leading to senescence in G1. During this process, the p53/p21 pathway is hyperactivated. Accompanying p21 accumulation, Aurora A kinase levels declined sharply. MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction. Conclusion: Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation, leading to senescence via mitotic skipping.

Simulated microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts can be prevented by protection of primary cilia.

Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts.

BAG3: Nature's Quintessential Multi-Functional Protein Functions as a Ubiquitous Intra-Cellular Glue.

BAG3 is a 575 amino acid protein that is found throughout the animal kingdom and homologs have been identified in plants. The protein is expressed ubiquitously but is most prominent in cardiac muscle, skeletal muscle, the brain and in many cancers. We describe BAG3 as a quintessential multi-functional protein. It supports autophagy of both misfolded proteins and damaged organelles, inhibits apoptosis, maintains the homeostasis of the mitochondria, and facilitates excitation contraction coupling through the L-type calcium channel and the beta-adrenergic receptor. High levels of BAG3 are associated with insensitivity to chemotherapy in malignant cells whereas both loss of function and gain of function variants are associated with cardiomyopathy.

Protection of primary cilia is an effective countermeasure against the impairment of osteoblast function induced by simulated microgravity.

The molecular mechanism for the microgravity-induced decrease in bone formation remains unclear and there is a lack of effective specific preventative therapies. We recently reported that primary cilia of osteoblasts became shorter and even disappeared when the cells were exposed to random positioning machine (RPM)-simulated microgravity and that the microgravity-induced loss of osteogenic potential of osteoblasts could be attenuated when the resorption of primary cilia was prevented by treatment with 0.1 µM cytochalasin D. In the current study, it was further found that the loss of the osteogenic capacity of rat calvarial osteoblasts (ROBs) was associated with the inhibition of the BMP-2/Smad1/5/8 signalling pathway, of which most of the signalling proteins including BMP-2, BMPRII, Smad1/5/8 and p-Smad1/5/8 were found localized to primary cilia. Accompanying the resorption of primary cilia following the cells being exposed to simulated microgravity, the expression levels of these signalling proteins were reduced significantly. Furthermore, the expression of miRNA-129-3p, a microRNA previously reported to control cilium biogenesis, was found to be reduced quickly and changed in a similar tendency with the length of primary cilia. Moreover, overexpression of miRNA-129-3p in ROBs significantly attenuated microgravity-induced inhibition of BMP-2 signalling and loss of osteogenic differentiation and mineralization. These results indicated the important role of miRNA-129-3p in microgravity-induced resorption of primary cilia of osteoblasts and the potential of replenishing the miRNA-129-3p as an effective countermeasure against microgravity-induced loss of primary cilia and impairment of osteoblast function.

Radiation-Induced Bystander Effect on the Genome of Bone Marrow Mesenchymal Stem Cells in Lung Cancer.

Aims: Radiation by-radiation effect (RIBE) can induce the genomic instability of bone marrow mesenchymal stem cells (BMSCs) adjacent to lung cancer, and this effect not only exists in the short-term, but also accompanies it in the long-term, but its specific mechanism is not clear. Our goal is to explore the similarities and differences in the mechanism of genomic damage in tumor-associated BMSCs induced by short-term and long-term RIBE, and to provide a theoretical basis for adjuvant drugs for protection against RIBE at different clinical time periods. Results: We found that both short- and long-term RIBE induced genomic instability. We could show a high expression of TGF-ß1, TNF-α, and HIF-1α in tumor-associated BMSCs after short-term RIBE whereas only TNF-α and HIF-1α expression was increased in long-term RIBE. We further confirmed that genomic instability is associated with the activation of the HIF-1α pathway and that this is mediated by TNF-α and TGF-ß1. In addition, we found differences in the mechanisms of genomic instability in the considered RIBE windows of analysis. In short-term RIBE, both TNF-α and TGF-ß1 play a role, whereas only TNF-α plays a decisive role in long-term RIBE. In addition, there were differences in BMSC recruitment and genomic instability of different tissues with a more pronounced expression in tumor and bone marrow than compared to lung. Innovation and Conclusion: We could show dynamic changes in the expression of the cytokines TGF-ß1 and TNF-α during short- and long-term RIBE. The differential expression of the two is the key to causing the genomic damage of tumor-associated BMSCs in the considered windows of analysis. Therefore, these results may serve as a guideline for the administration of radiation protection adjuvant drugs at different clinical stages. Antioxid. Redox Signal. 38, 747-767.

MiR-663a Inhibits Radiation-Induced Epithelium-to-Mesenchymal Transition by Targeting TGF-ß1.

Objective: miR-663a has been reported to be downregulated by X-ray irradiation and participates in radiation-induced bystander effect via TGF-ß1. The goal of this study was to explore the role of miR-663a during radiation-induced Epithelium-to-mesenchymal transition (EMT). Methods: TGF-ß1 or IR was used to induce EMT. After miR-663a transfection, cell migration and cell morphological changes were detected and the expression levels of miR-663a, TGF-ß1, and EMT-related factors were quantified. Results: Enhancement of cell migration and promotion of mesenchymal changes induced by either TGF-ß1 or radiation were suppressed by miR-663a. Furthermore, both X-ray and carbon ion irradiation resulted in the upregulation of TGF-ß1 and downregulation of miR-663a, while the silencing of TGF-ß1 by miR-663a reversed the EMT process after radiation. Conclusion: Our findings demonstrate an EMT-suppressing effect by miR-663a via TGF-ß1 in radiation-induced EMT.

Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells.

Objective: To investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM). Methods: GBM cells were treated with TMZ or X-ray/carbon ion. The primary cilia were examined by immunostaining with Arl13b and γ-tubulin, and the cellular resistance ability was measured by cell viability assay or survival fraction assay. Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride, the autophagy was measured by acridine orange staining assay. The DNA damage repair ability was estimated by the kinetic curve of γH2AX foci, and the DNA-dependent protein kinase (DNA-PK) activation was detected by immunostaining assay. Results: Primary cilia were frequently preserved in GBM, and the induction of ciliogenesis decreased cell proliferation. TMZ and IR promoted ciliogenesis in dose- and time-dependent manners, and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR. The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR via the impairment of autophagy and DNA damage repair. The interference of ciliogenesis reduced DNA-PK activation, and the knockdown of DNA-PK led to cilium formation and elongation. Conclusion: Primary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair.

Astragalus polysaccharide inhibits radiation-induced bystander effects by regulating apoptosis in Bone Mesenchymal Stem Cells (BMSCs).

The purpose of this study was to investigate the effects of astragalus polysaccharides (APS) on the proliferation and apoptosis of bone marrow mesenchymal stem cells (BMSCs) induced by X-ray radiation-induced A549 cells bystander effect (RIBE), and to explore their mechanisms. In this study, APS increased the reduced cell proliferation rate induced by RIBE and inhibiting the apoptosis of bystander cells. In terms of mechanism, APS up-regulates the proteins Bcl-2, Bcl-xl, and down-regulates the proteins Bax and Bak, which induces a decrease in mitochondrial membrane potential, which induces the release of Cyt-c and AIF, which leads to caspase-dependent and caspase-independent pathway to cause apoptosis. In addition, we believe that ROS may be the main cause of these protein changes. APS can inhibit the generation of ROS in bystander cells and thus inhibit the activation of the mitochondrial pathway, further preventing cellular damage caused by RIBE.

Flavonoid genistein protects bone marrow sinusoidal blood vessels from damage by methotrexate therapy in rats.

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy-derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated ( p < 0.001) in the MTX-alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 ( p < 0.001) in bone. In MTX-treated rats, genistein suppressed MTX-induced apoptosis of BM SECs ( p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF ( p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs.

Icariin prevents bone loss by inhibiting bone resorption and stabilizing bone biological apatite in a hindlimb suspension rodent model.

Bone loss induced by microgravity is a substantial barrier to humans in long-term spaceflight. Recent studies have revealed that icariin (ICA) can attenuate osteoporosis in postmenopausal women and ovariectomized rats. However, whether ICA can protect against microgravity-induced bone loss remains unknown. In this study, the effects of ICA on a hindlimb suspension rodent model were investigated. Two-month-old female Wistar rats were hindlimb suspended and treated with ICA (25 mg·kg-1·d-1, i.g.) or a vehicle for 4 weeks (n = 6). The bone mass density of the hindlimbs was analyzed using dual-energy X-ray absorptiometry and micro-CT. mRNA expression of osteogenic genes in the tibia and the content of bone metabolism markers in serum were measured using qRT-PCR and ELISA, respectively. The bone mineral phase was analyzed using X-ray diffraction and atomic spectrometry. The results showed that ICA treatment significantly rescued the hindlimb suspension-induced reduction in bone mineral density, trabecular number and thickness, as well as the increases in trabecular separation and the structure model index. In addition, ICA treatment recovered the decreased bone-related gene expression, including alkaline phosphatase (ALP), bone glaprotein (BGP), and osteoprotegerin/receptor activator of the NF-κB ligand ratio (OPG/RANKL), in the tibia and the decreased bone resorption marker TRACP-5b levels in serum caused by simulated microgravity. Notably, ICA treatment restored the instability of bone biological apatite and the metabolic disorder of bone mineral elicited by simulated microgravity. These results demonstrate that ICA treatment plays osteoprotective roles in bone loss induced by simulated microgravity by inhibiting bone resorption and stabilizing bone biological apatite.

Values of aortic dissection detection risk score combined with ascending aorta diameter >40 mm for the early identification of type A acute aortic dissection.

BACKGROUND: Type A acute aortic dissection (A-AAD), involving the ascending aorta, is a life-threatening disease. To detect A-AAD early and rapidly in patients with acute chest pain, especially in patients with acute myocardial infarction (AMI) secondary to A-AAD, we investigated values of combined use of the risk score and the ascending aorta diameter >40 mm for the early identification of A-AAD. METHODS: Our study retrospectively encompassed 239 patients with acute chest pain on admission to our hospital between July 2010 and December 2016. The risk score was calculated according to the aortic dissection detection (ADD) risk score system, and the ascending aorta diameter was accurately obtained from the transthoracic echocardiography (TTE). RESULTS: A risk score ≥1 had an excellent sensitivity of 94.9% and a fair negative predictive value (NPV) of 77.8%, with a poor specificity of 8.7% and a positive predictive value (PPV) of 33.5% for the diagnosis of A-AAD. A risk score ≥2 had an excellent specificity of 91.3% and a fair NPV of 73.1%, whereas it had a lower sensitivity of 30.8% and a PPV of 63.2%. A risk score ≥1, combined with an ascending aorta diameter >40 mm, had a sensitivity, a specificity, a PPV, and an NPV of 84.6%, 87.6%, 76.7%, and 92.2% for the diagnosis of A-AAD, respectively. The combined use of a risk score ≥2 and an ascending aorta diameter >40 mm had an excellent specificity of 98.1% and a PPV of 86.4%, a fair NPV of 72.8%, and a poor sensitivity of 24.4% for the detection of A-AAD. Moreover, the omission diagnostic rate for A-AAD was significantly decreased from 33.3% to 7.4% using a risk score ≥1 combined with an ascending aorta diameter >40 mm in patients with AMI secondary to A-AAD. CONCLUSIONS: The combined use of an ADD risk score ≥1 and an ascending aorta diameter >40 mm was highly indicative of A-AAD in patients presenting with acute chest pain, especially in patients with AMI secondary to A-AAD, which urgently needed computed tomography angiography (CTA) or magnetic resonance imaging (MRI) to confirm the diagnosis of A-AAD.

Bone Loss Induced by Simulated Microgravity, Ionizing Radiation and/or Ultradian Rhythms in the Hindlimbs of Rats.

OBJECTIVE: To better understand the pathological causes of bone loss in a space environment, including microgravity, ionizing radiation, and ultradian rhythms. METHODS: Sprague Dawley (SD) rats were randomly divided into a baseline group, a control group, a hindlimb suspension group, a radiation group, a ultradian rhythms group and a combined-three-factor group. After four weeks of hindlimb suspension followed by X-ray exposure and/or ultradian rhythms, biomechanical properties, bone mineral density, histological analysis, microstructure parameters, and bone turnover markers were detected to evaluate bone loss in hindlimbs of rats. RESULTS: Simulated microgravity or combined-three factors treatment led to a significant decrease in the biomechanical properties of bones, reduction in bone mineral density, and deterioration of trabecular parameters. Ionizing radiation exposure also showed adverse impact while ultradian rhythms had no significant effect on these outcomes. Decrease in the concentration of the turnover markers bone alkaline phosphatase (bALP), osteocalcin (OCN), and tartrate-resistant acid phosphatase-5b (TRAP-5b) in serum was in line with the changes in trabecular parameters. CONCLUSION: Simulated microgravity is the main contributor of bone loss. Radiation also results in deleterious effects but ultradian rhythms has no significant effect. Combined-three factors treatment do not exacerbate bone loss when compared to simulated microgravity treatment alone.

Evaluation of left atrial volume and function using single-beat real-time three-dimensional echocardiography in atrial fibrillation patients.

BACKGROUND: This study was aimed to evaluate the feasibility and accuracy of real-time three-dimensional echocardiography (RT-3DE) measurement of left atrial (LA) volume and function in comparison with two-dimensional echocardiography (2DE) measurements in atrial fibrillation (AF) patients. METHODS: A total of 50 pairs of AF patients and healthy controls were enrolled in this study. Indexed LA end-diastole volume (ILAEDV) and indexed LA end-systolic volume (ILAESV), as well as LA function indices such as segmental LA ejection fraction (LAEF), were assessed using 2DE Simpson's method and the RT-3DE method. RESULTS: The images showed that regional LA volume-time curves and LAEF were disordered in AF patients. ILAEDV and ILAESV were markedly increased and global LAEF was significantly decreased in AF patients compared with those in healthy controls (P < 0.01). No significant differences were found in ILAEDV, ILAESV, and LAEF levels as determined by the RT-3DE method or 2DE Simpson's method. Bland-Altman analysis showed that the two methods agreed well for measuring ILAEDV, ILAESV, and segmental LAEF. CONCLUSION: The RT-3DE method may be a feasible and accurate method for evaluating LA volume and function of AF patients in clinical practice.

Pulsed electromagnetic fields stimulate osteogenic differentiation and maturation of osteoblasts by upregulating the expression of BMPRII localized at the base of primary cilium.

Pulsed electromagnetic fields (PEMFs) have been considered as a potential candidate for the prevention and treatment of osteoporosis, however, the mechanism of its action is still elusive. We have previously reported that 50Hz 0.6mT PEMFs stimulate osteoblastic differentiation and mineralization in a primary cilium- dependent manner, but did not know the reason. In the current study, we found that the PEMFs promoted osteogenic differentiation and maturation of rat calvarial osteoblasts (ROBs) by activating bone morphogenetic protein BMP-Smad1/5/8 signaling on the condition that primary cilia were normal. Further studies revealed that BMPRII, the primary binding receptor of BMP ligand, was readily and strongly upregulated by PEMF treatment and localized at the bases of primary cilia. Abrogation of primary cilia with small interfering RNA sequence targeting IFT88 abolished the PEMF-induced upregulation of BMPRII and its ciliary localization. Knockdown of BMPRII expression level with RNA interference had no effects on primary cilia but significantly decreased the promoting effect of PEMFs on osteoblastic differentiation and maturation. These results indicated that PEMFs stimulate osteogenic differentiation and maturation of osteoblast by primary cilium-mediated upregulation of BMPRII expression and subsequently activation of BMP-Smad1/5/8 signaling, and that BMPRII is the key component linking primary cilium and BMP-Smad1/5/8 pathway. This study has thus revealed the molecular mechanism for the osteogenic effect of PEMFs.

High Mobility Group Box1 Protein Is Involved in Endoplasmic Reticulum Stress Induced by Clostridium difficile Toxin A.

High Mobility Group Box1 (HMGB1), a damage-associated inflammatory factor, plays an important role in the pathogenesis of numerous chronic inflammatory and autoimmune diseases. In this study, the role of the HMGB1 in TcdA-induced ER stress was identified. Clostridium difficile toxin A is one of the major virulence factors of C. difficile infection (CDI) and has been proved to induce apoptotic cell death through ER stress. Our results showed that HMGB1 might play an important role in the TcdA-induced ER stress and unfolded protein response. HMGB1 activated molecular markers and induced the C/EBP homologous protein upregulation (CHOP). This study may provide the essential information for better understanding of the molecular mechanisms involved in CDI.

p21 is Responsible for Ionizing Radiation-induced Bypass of Mitosis.

OBJECTIVE: To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest. METHODS: Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence- associated-ß-galactosidase (SA-ß-gal) staining combined with Ki67 staining, a cell proliferation marker. RESULTS: Accompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells. CONCLUSION: Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis.

High mobility group box1 protein is involved in acute inflammation induced by Clostridium difficile toxin A.

High mobility group box1 (HMGB1), as a damage-associated inflammatory factor, contributes to the pathogenesis of numerous chronic inflammatory and autoimmune diseases. In this study, we explored the role of HMGB1 in CDI (Clostridium difficile infection) by in vivo and in vitro experiments. Our results showed that HMGB1 might play an important role in the acute inflammatory responses to C. difficile toxin A (TcdA), affect early inflammatory factors, and induce inflammation via the HMGB1-TLR4 pathway. Our study provides the essential information for better understanding the molecular mechanisms of CDI and the potential new therapeutic strategies for the treatment of this infection.

Enhancing resolution of free-flow zone electrophoresis via a simple sheath-flow sample injection.

In this work, a simple and novel sheath-flow sample injection method (SFSIM) is introduced to reduce the band broadening of free-flow zone electrophoresis separation in newly developed self-balance free-flow electrophoresis instrument. A needle injector was placed in the center of the separation inlet, into which the BGE and sample solution were pumped simultaneously. BGE formed sheath flow outside the sample stream, resulting in less band broadening related to hydrodynamics and electrodynamics. Hemoglobin and C-phycocyanin were successfully separated by the proposed method in contrast to the poor separation of free-flow electrophoresis with the traditional injection method without sheath flow. About 3.75 times resolution enhancement could be achieved by sheath-flow sample injection method.

An enhanced sensitive electrochemical immunosensor based on efficient encapsulation of enzyme in silica matrix for the detection of human immunodeficiency virus p24.

We report a new electrochemical immunosensor for enhanced sensitive detection of human immunodeficiency virus p24 (HIV-p24) based on graphene oxide (GO) as a nanocarrier and enzyme encapsulated in carbon nanotubes-silica as a matrix in a multienzyme amplification strategy. Greatly enhanced sensitivity was achieved by using the bioconjugates featuring horseradish peroxidase-HIV-p24 signal antibody (HRP-HIV-p24) linked to functionalized GO and thionine (TH) as well as efficient encapsulation of enzyme (HRP) in the silica matrix with retained bioactivity. After a sandwich immunoreactions, the HRP in carbon nanotubes-silica matrix and the HRP-HIV-p24-TH/GO captured onto the electrode surface produced an amplified electrocatalytic response by the reduction of enzymatically oxidized thionine in the presence of hydrogen peroxide. The increase of response current was proportional to the HIV-p24 concentration in the range of 0.5 pg/mL-8.5 ng/mL with the detection limit of 0.15 pg/mL, which was lower than that of the traditional sandwich electrochemical measurement for HIV-p24. The amplified immunoassay developed in this work shows acceptable stability and reproducibility, and the assay results for HIV-p24 spiked in human plasma also show good accuracy. This simple and low-cost immunosensor shows great promise for detection of other proteins and clinical applications.