Tirapazamine combined with photodynamic therapy improves the efficacy of ABZSO nanoparticles on Echinococcosis granulosus via further enhancing "breaking-then-curing".

BACKGROUND: Photodynamic therapy (PDT) has a promising application prospect in Echinococcus granulosus (Egs), however, the hypoxic environment of Egs and the hypoxia associated with PDT will greatly limit its effects. As a hypoxic-activated pre-chemotherapeutic drug, tirapazamine (TPZ) can be only activated and produce cytotoxicity under hypoxia environment. Albendazole sulfoxide (ABZSO) is the first choice for the treatment of Egs. This study aimed to explore the effects of ABZSO nanoparticles (ABZSO NPs), TPZ combined with PDT on the activity of Egs in vitro and in vivo. METHODS: The Egs were divided into control, ABZSO NPs, ABZSO NPs + PDT, and ABZSO NPs + TPZ + PDT groups, and the viability of Egs was determined using methylene blue staining. Then, the ROS, LDH and ATP levels were measured using their corresponding assay kit, and H2AX and TopoI protein expression was detected by western blot. The morphology of Egs with different treatments was observed using hematoxylin eosin (HE) staining and scanning electron microscopy (SEM). After that, the in vivo efficacy of ABZSO NPs, TPZ and PDT on Egs was determined in a Egs infected mouse model. RESULTS: In vitro experiments showed that the combined treatment of TPZ, ABZSO NPs and PDT significantly inhibited Egs viability; and significantly increased ROS levels and LDH contents, while decreased ATP contents in Egs; as well as up-regulated H2AX and down-regulated TopoI protein expression. HE staining and SEM results showed that breaking-then-curing treatment seriously damaged the Egs wall. Additionally, in vivo experiments found that the combination of ABZSO NPs, PDT and TPZ had more serious calcification and damage of the wall structure of cysts. CONCLUSIONS: ABZSO NPs combined with TPZ and PDT has a better inhibitory effect on the growth of Egs in vitro and in vivo based on the strategy of "breaking-then-curing".

Efficacy and Safety of Citric Acid and Heparin Anticoagulation in Patients with Septic Acute Kidney Injury Undergoing Continuous Renal Replacement Therapy: A Meta-Analysis.

Objective: This meta-analysis compares the clinical efficacy and safety of citrate anticoagulation with heparin anticoagulation in continuous renal replacement therapy for acute kidney injury in sepsis. Methods: The experimental group underwent local anticoagulation with citrate, whereas the control group received systemic anticoagulation with heparin. Relevant data from randomized controlled trials (RCTs) meeting the inclusion criteria were independently extracted through computer searches of the China Journal Full Text Database (CNKI), Wanfang, and Vipul databases. Additionally, references to included literature were searched to expand the dataset. Extracted RCTs that met inclusion criteria underwent independent quality evaluation and cross-checking using the Cochrane systematic review method. Subsequently, a meta-analysis was conducted using Stata 12.0 software. Results: The analysis included seven studies involving a total of 652 patients. After treatment, renal function improvement was significantly more significant in the citrate group, while creatinine and urea nitrogen levels showed a more significant decrease in the heparin group, with statistically significant differences (WMD = -51.30, 95% CI = -68.54 ~ -34.06, P = .000 and WMD = 3.68, 95% CI = -4.52 ~ -2.85, P = .000). The filter lifespan in the citrate group was significantly longer than in the heparin group, with a statistically significant difference (WMD = 6.93, 95% CI = 6.30 ~ 7.55, P = .000). Adverse bleeding reactions were significantly less common in the citrate group compared to the heparin group, with a statistically significant difference (RR = 0.14, 95% CI = 0.06 ~ 0.32, P = .000). Conclusions: The results of this meta-analysis indicate that citrate anticoagulation is more effective than heparin anticoagulation in continuous renal replacement therapy for patients with acute kidney injury in sepsis. Citrate anticoagulation contributes to improved renal function and extended filter usage and reduces the incidence of adverse bleeding reactions.

A multi-bioresponsive self-assembled nano drug delivery system based on hyaluronic acid and geraniol against liver cancer.

Herein, a multi-bioresponsive self-assembled nano-drug delivery system (HSSG) was constructed by conjugating the anticancer drug Geraniol (GER) to hyaluronic acid (HA) via a disulfide bond. The HSSG NPs displayed a uniform spherical shape with an average diameter of ∼110 nm, maintained high stability, and realized controlled drug release in the tumor microenvironment (pH/glutathione/hyaluronidase). Results of fluorescence microscopy and flow cytometry verified that HSSG NPs were selectively uptaken by human hepatocellular carcinoma cell lines HepG2 and Huh7 via CD44 receptor-mediated internalization. Studies on H22 tumor-bearing mice demonstrate that HSSG NPs could effectively accumulate at the tumor site for a long period. In vitro and in vivo studies show that HSSG NPs significantly promoted the death of cancer cells while reducing the toxicity as compared to GER. Therefore, the HSSG NPs have great potential in the treatment of tumors.

A genome-wide CRISPR screen identifies HuR as a regulator of apoptosis induced by dsRNA and virus.

We performed an unbiased whole-genome CRISPR/Cas9 screen in A549 cells to identify potential regulators involved in cell death triggered by double-stranded RNA (dsRNA). Of several top candidate genes, we identified the RNA-binding gene ELAV like protein 1 (256529), which encodes the protein Hu antigen R (HuR). Depletion of HuR led to less cell death induced by dsRNA. HuR is mainly involved in apoptosis, and all of its RNA recognition motifs are essential for its pro-apoptotic function. We further showed that the HuR depletion had no influence on the mRNA level of the anti-apoptotic gene BCL2, but instead that HuR downregulates BCL2 translation in a cap-independent way. Polysome fractionation studies showed that HuR retarded the BCL2 mRNA in the non-translating pool of polysomes. Moreover, protection from dsRNA-induced apoptosis by HuR depletion required the presence of BCL2, indicating that the pro-apoptotic function of HuR is executed by suppressing BCL2. Consistent with this, HuR regulated apoptosis induced by infection of encephalomyocarditis or Semliki Forest virus. Collectively, our work identified a suite of proteins that regulate dsRNA-induced cell death, and elucidated the mechanism by which HuR acts as a pro-apoptotic factor.

AMBRA1 promotes dsRNA- and virus-induced apoptosis through interacting with and stabilizing MAVS.

Apoptosis is an important cellular response to viral infection. In this study, we identified activating molecule in Beclin1-regulated autophagy protein 1 (AMBRA1) as a positive regulator of apoptosis triggered by double-stranded (ds)RNA. Depletion of AMBRA1 by gene editing significantly reduced dsRNA-induced apoptosis, which was largely restored by trans-complementation of AMBRA1. Mechanistically, AMBRA1 interacts with mitochondrial antiviral-signaling protein (MAVS), a key mitochondrial adaptor in the apoptosis pathway induced by dsRNA and viral infection. Further co-immunoprecipitation analysis demonstrated that the mitochondrial localization of MAVS was essential for their interaction. The impact of AMBRA1 on dsRNA-induced apoptosis relied on the presence of MAVS and caspase-8. AMBRA1 was involved in the stabilization of MAVS through preventing its dsRNA-induced proteasomal degradation. Consistently, AMBRA1 upregulated the apoptosis induced by Semliki Forest virus infection. Taken together, our work illustrated a role for AMBRA1 in virus-induced apoptosis through interacting with and stabilizing MAVS.

Positive Feedback Loop of Long Noncoding RNA OASL-IT1 and Innate Immune Response Restricts the Replication of Zika Virus in Epithelial A549 Cells.

Expression of host noncoding RNAs and coding mRNAs is significantly altered by viral infection. In the current study, we screened the transcriptional profile of human lung epithelial A549 cells infected with Zika virus (ZIKV) by microarray assay. Seventy-nine long noncoding RNAs (lncRNAs) and 140 mRNAs were differentially expressed (DE). The bioinformatics analysis revealed that the mRNAs adjacent to the DE lncRNAs were closely related to the host responses to viral infection. We selected 7 lncRNAs from the top 50 hits for validation. The quantitative real-time PCR data confirmed that expression of selected lncRNAs was induced by ZIKV infection. Moreover, the expression of 7 lncRNAs was induced by infection of dengue virus, Japanese encephalitis virus, or vesicular stomatitis virus, or by treatment of poly(I:C) and IFN-ß. Furthermore, loss of innate immune adaptor IPS-1 or receptor IFNAR1 resulted in lower induction levels of several lncRNAs by ZIKV. Overexpression of 3 lncRNAs (RPL27-OT1, OASL-IT1, and REC8-OT3) reduced the virus yields of ZIKV. Knockout of OASL-IT1 significantly enhanced ZIKV replication. In OASL-IT1 knockout cells, the levels of interferons (IFNs) and the activation of 3 innate immune signaling pathways triggered by ZIKV were dramatically reduced. Collectively, our work found a positive feedback loop in the IFN system, in which IFNs and OASL-IT1 regulate each other, thereby promoting establishment of antiviral defense.

In situ deposition of MOF-74(Cu) nanosheet arrays onto carbon cloth to fabricate a sensitive and selective electrocatalytic biosensor and its application for the determination of glucose in human serum.

A new electrocatalytic biosensor (MOF-74(Cu) NS-CC) based on the in situ deposition of MOF-74(Cu) nanosheet on carbon cloth via a bottom-up synthetic approach in a glass tube was developed. The electrocatalytic activity of the deposited MOF-74(Cu) NS was demonstrated in the oxidation of glucose to gluconate under alkaline conditions. The results revealed that the proposed method of in situ formation of MOF-74(Cu) NS onto a carbon cloth surface in a multi-layer solution is capable to generate a stable MOF-74(Cu) NS-CC electrode with excellent sensing performance. When the as-synthesized MOF-74(Cu) NS-CC was applied directly as the working electrode for glucose sensing, it showed much higher conductivity and redox activity than MOF-74(Cu) NS-GCE. With the potential applied at 0.55 V (vs. Ag/AgCl), this new electrocatalytic biosensor exhibits an excellent linear relationship between current density and concentration of glucose. Moreover, a wide linear range of detection (1.0 to 1000 µM) was observed. The limit of detection was found to be 0.41 µM (S/N = 3). The response sensitivity is 3.35 mA mM-1 cm-2 when the concentration of glucose is in the range 1-100 µM and 3.81 mA mM-1 cm-2 for the 100-1000 µM concentration range. This study provides a low-cost, easy to prepare, and reproducible methodology for the synthesis of highly redox-active nanomaterials based on the in situ formation of two-dimensional MOF-74(Cu) NS for the development of new electrocatalytic biosensors. Graphical abstract.

Inositol-Requiring Enzyme 1α Promotes Zika Virus Infection through Regulation of Stearoyl Coenzyme A Desaturase 1-Mediated Lipid Metabolism.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which has become a global epidemic threat due to its rapid spread and association with serious consequences of infection, including neonatal microcephaly. Inositol-requiring enzyme 1α (IRE1α) is an endoplasmic reticulum (ER)-related transmembrane protein that mediates unfolded protein response (UPR) pathway and has been indicated to play an important role in flavivirus replication. However, the mechanism of how IRE1α affects ZIKV replication remains unknown. In this study, we explored the role of IRE1α in ZIKV infection in vitro and in vivo by using CRISPR/Cas9-based gene knockout and RNA interference-based gene knockdown techniques. Both knockout and knockdown of IRE1α dramatically reduced ZIKV replication levels, including viral RNA levels, protein expression, and titers in different human cell lines. Trans-complementation with IRE1α restored viral replication levels decreased by IRE1α depletion. Furthermore, the proviral effect of IRE1α was dependent on its kinase and RNase activities. Importantly, we found that IRE1α promoted the replication of ZIKV through upregulating the accumulation of monounsaturated fatty acid (MUFA) rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1), which further affected the production of oleic acid (OA) and lipid droplet. Finally, our data demonstrated that in the brain tissues of ZIKV-infected mice, the replication levels of ZIKV and virus-related lesions were significantly suppressed by both the kinase and RNase inhibitors of IRE1α. Taken together, our results identified IRE1α as a ZIKV dependency factor which promotes viral replication through affecting SCD1-mediated lipid metabolism, potentially providing a novel molecular target for the development of anti-ZIKV agents.IMPORTANCE Zika virus (ZIKV) has been linked to serious neurologic disorders and causes widespread concern in the field of global public health. Inositol requiring enzyme 1α (IRE1α) is an ER-related transmembrane protein that mediates unfolded protein response (UPR) pathway. Here, we revealed that IRE1α is a proviral factor for ZIKV replication both in culture cells and mice model, which relies on its kinase and RNase activities. Importantly, we further provided evidence that upon ZIKV infection, IRE1α is activated and splices XBP1 mRNA which enhances the expression of monounsaturated fatty acids rate-limiting enzyme stearoyl coenzyme A (stearoyl-CoA) desaturase 1 (SCD1) and subsequent lipid droplet production. Our data uncover a novel mechanism of IRE1α proviral effect by modulating lipid metabolism, providing the first evidence of a close relationship between IRE1α-mediated UPR, lipid metabolism, and ZIKV replication and indicating IRE1α inhibitors as potentially effective anti-ZIKV agents.

Double-stranded RNA deaminase ADAR1 promotes the Zika virus replication by inhibiting the activation of protein kinase PKR.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as a threat to global health. The family of adenosine deaminases acting on dsRNA (ADARs) are human host factors important for the genetic diversity and evolution of ZIKV. Here, we further investigated the role of ADAR1 in ZIKV replication by utilizing CRISPR/Cas9-based gene editing and RNAi-based gene knockdown techniques. Both ADAR1 knockout and knockdown significantly reduced ZIKV RNA synthesis, protein levels, and viral titers in several human cell lines. Trans-complementation with the full-length ADAR1 form p150 or the shorter form p110 lacking the Zα domain restored viral replication levels suppressed by the ADAR1 knockout. Moreover, we observed that the nuclear p110 form was redistributed to the cytoplasm in response to ZIKV infection. ADAR1 was not involved in viral entry but promoted viral protein translation by impairing ZIKV-induced activation of protein kinase regulated by dsRNA (PKR). Of note, the RNA-editing activity of ADAR1 was not required to promote ZIKV replication. We also found that the proviral role of ADAR1 was partially mediated through its ability to suppress IFN production and PKR activation. Our work identifies ADAR1 as a proviral factor involved in ZIKV replication, suggesting that ADAR1 could be a potential antiviral target.

Role of heparan sulfate in the Zika virus entry, replication, and cell death.

Zika virus (ZIKV) is an emerging arbovirus and its infection associates with neurologic diseases. Whether heparan sulfate (HS), an attachment factor for many viruses, plays a role in the ZIKV infection remains controversial. Our study generated several HS biosynthesis-deficient cell clones by disrupting SLC35B2, B3GAT3, or B4GALT7 gene using the CRISPR/Cas9 system. The HS deficiency did not affect the viral attachment and internalization of ZIKV, but reduced the attachment of Dengue virus (DENV) 2. The early RNA and protein levels of ZIKV and DENV2 were impaired in the HS deficient cells, while the viral yields were not accordingly reduced. Our data further showed that HS promoted the cell death induced by virus infection, and inhibition of cell death significantly increased the viral replication of ZIKV and DENV2. Collectively, our study described an unexpected role of HS in the viral attachment, replication and cell death induced by ZIKV.

The biological effect and mechanism of the Wnt/ß-catenin signaling pathway on malignant melanoma A375 cells.

The present study aimed to determine the influence of the Wnt/ß-catenin signaling pathway on the proliferation, invasion, migration and apoptosis of malignant melanoma (MM) A375 cells. ß-catenin interfering lentivirus liquid (ß-catenin-RNAi-LV) and empty vector lentivirus liquid (ß-catenin-negative-LV) were used to infect A375 cells. Infected cells were obtained and marked as A375-RNA interference (A375-RNAi) or A375-negative, respectively. Western blotting was used to measure the expression of ß-catenin in infected cells and uninfected cells were utilized as a control. An MTT assay was adopted to measure cell proliferation and the clone formation of cells was assessed. In addition, the Transwell method was used to detect cell invasion and migration in vitro and flow cytometry was utilized to determine cell apoptosis. Western blot analysis demonstrated that ß-catenin was highly expressed in uninfected A375 cells but exhibited reduced expression in A375-RNAi cells. These results indicate that ß-catenin expression is effectively silenced by ß-catenin-RNAi-LV. The proliferative and clone forming abilities of A375-RNAi cells were impaired compared with A375-negative and A375 cells. Additionally, the apoptosis rate was increased and the invasion and migration of A375-RNAi cells was decreased. However, no significant differences were identified in the proliferation, clone formation, apoptosis rate, invasion and migration of A375-negative cells compared with A375 cells. Therefore, the current study demonstrated that the inhibition of ß-catenin expression or activity inhibits cell proliferation and invasion and migration, further downregulating the expression of anti-apoptotic genes and accelerating cellular apoptosis.

Sphingosine kinase 1 enhances the invasion and migration of non-small cell lung cancer cells via the AKT pathway.

Sphingosine kinase 1 (SphK1) has been shown to play an important role in the progression of a number of human cancers. It has been reported that the expression of SphK1 is greatly elevated in non-small cell lung cancer (NSCLC) tissues. However, its role and underlying mechanisms in NSCLC have not been fully elucidated. In the present study, we found that SphK1 was highly expressed in NSCLC cells. Overexpression of SphK1 promoted the invasion and migration of NSCLC cells, while knockdown of SphK1 suppressed the invasion and migration. Furthermore, we demonstrated that SphK1 decreased the protein level of E-cadherin, yet increased the protein level of Snail. In addition, SphK1 was able to stimulate the activation of AKT. Inhibition of the AKT pathway attenuated the biological functions of NSCLC cells induced by overexpression of SphK1. Taken together, our findings suggest that SphK1 can enhance the invasion and migration of NSCLC cells via activation of the AKT pathway and regulation of E-cadherin and Snail expression. Thus, SphK1 could be a potential target for the detection and treatment of NSCLC.