Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury.

BACKGROUND: The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes. METHODS: A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms. RESULTS: A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

Silk Fibroin Formed Bioadhesive Ophthalmic Gel for Dry Eye Syndrome Treatment.

PURPOSE: Dry eye syndrome (DES), arising from various etiologic factors, leads to tear film instability and ocular surface damage. Given its anti-inflammatory effects, cyclosporine A (CsA) has been widely used as a short-term treatment option for DES. However, poor bioavailability and solubility of CsA in aqueous phase make the development of a cyclosporine A-based eye drop for ocular topical application a huge challenge. METHODS: In this study, a novel strategy for preparing cyclosporine A-loaded silk fibroin nanoemulsion gel (CsA NBGs) was proposed to address these barriers. Additionally, the rheological properties, ocular irritation potential, tear elimination kinetics, and pharmacodynamics based on a rabbit dry eye model were investigated for the prepared CsA NBGs. Furthermore, the transcorneal mechanism across the ocular barrier was also investigated. RESULTS: The pharmacodynamics and pharmacokinetics of CsA NBGs exhibited superior performance compared to cyclosporine eye drops, leading to a significant enhancement in the bioavailability of CsA NBGs. Furthermore, our investigation into the transcorneal mechanism of CsA NBGs revealed their ability to be absorbed by corneal epithelial cells via the paracellular pathway. CONCLUSION: The CsA NBG formulation exhibits promising potential for intraocular drug delivery, enabling safe, effective, and controlled administration of hydrophobic drugs into the eye. Moreover, it enhances drug retention within the ocular tissues and improves systemic bioavailability, thereby demonstrating significant clinical translational prospects.

RhoA promotes osteoclastogenesis and regulates bone remodeling through mTOR-NFATc1 signaling.

BACKGROUND: The cytoskeletal architecture of osteoclasts (OCs) and bone resorption activity must be appropriately controlled for proper bone remodeling, which is associated with osteoporosis. The RhoA protein of GTPase plays a regulatory role in cytoskeletal components and contributes to osteoclast adhesion, podosome positioning, and differentiation. Although osteoclast investigations have traditionally been performed by in vitro analysis, however, the results have been inconsistent, and the significance of RhoA in bone physiology and pathology is still unknown. METHODS: We generated RhoA knockout mice by specifically deleting RhoA in the osteoclast lineage to understand more about RhoA's involvement in bone remodeling. The function of RhoA in osteoclast differentiation and bone resorption and the mechanisms were assessed using bone marrow macrophages (BMMs) in vitro. The ovariectomized (OVX) mouse model was adopted to examine the pathological effect of RhoA in bone loss. RESULTS: Conditional deletion of RhoA in the osteoclast lineage causes a severe osteopetrosis phenotype, which is attributable to a bone resorption suppression. Further mechanistic studies suggest that RhoA deficiency suppresses Akt-mTOR-NFATc1 signaling during osteoclast differentiation. Additionally, RhoA activation is consistently related to the significant enhancement the osteoclast activity, which culminates in the development of an osteoporotic bone phenotype. Furthermore, in mice, the absence of RhoA in osteoclast precursors prevented occurring OVX-induced bone loss. CONCLUSION: RhoA promoted osteoclast development via the Akt-mTOR-NFATc1 signaling pathway, resulting a osteoporosis phenotype, and that manipulating RhoA activity might be a therapeutic strategy for osteoporotic bone loss.

A Variable Photo-Model Method for Object Pose and Size Estimation with Stereo Vision in a Complex Home Scene.

Model-based stereo vision methods can estimate the 6D poses of rigid objects. They can help robots to achieve a target grip in complex home environments. This study presents a novel approach, called the variable photo-model method, to estimate the pose and size of an unknown object using a single photo of the same category. By employing a pre-trained You Only Look Once (YOLO) v4 weight for object detection and 2D model generation in the photo, the method converts the segmented 2D photo-model into 3D flat photo-models assuming different sizes and poses. Through perspective projection and model matching, the method finds the best match between the model and the actual object in the captured stereo images. The matching fitness function is optimized using a genetic algorithm (GA). Unlike data-driven approaches, this approach does not require multiple photos or pre-training time for single object pose recognition, making it more versatile. Indoor experiments demonstrate the effectiveness of the variable photo-model method in estimating the pose and size of the target objects within the same class. The findings of this study have practical implications for object detection prior to robotic grasping, particularly due to its ease of application and the limited data required.

One-Step In Situ Polymerization: A Facile Design Strategy for Block Copolymer Electrolytes.

To optimize the rapid transport of lithium ions (Li+ ) inside lithium metal batteries (LMBs), block copolymer electrolytes (BCPEs) have been fabricated in situ in LMBs via a one-step method combining reversible addition-fragmentation chain transfer (RAFT) polymerization and carboxylic acid-catalyzed ring-opening polymerization (ROP). The BCPEs balanced the Li+ coordination characteristics of the polyether- and polyester-based electrolytes to achieve a rapid Li+ migration in the SPEs. The carboxylic acid played a dual role since it both catalyzed the ROP and stabilized the interface. Furthermore, the in situ assembly of LMBs did effectively enable an efficient intercalation/de-intercalation of Li+ at the electrode/electrolyte interface. The in situ assembled Li/BCPE4/LFP exhibited high-capacity retention of 92 % after 400 cycles at 1 C. The one-step in situ fabrication of BCPEs provides a new direction for the design of polymer electrolytes.

Lithium Salt-Induced In Situ Polymerizations Enable Double Network Polymer Electrolytes.

Structural design is an intriguing strategy to improve the physical and electrochemical performance of polymer electrolytes (PEs) for lithium-ion batteries. However, the complex synthetic process and introduction of nonelectrolyte composition severely limit the development and practical application of PEs. Here, a facile method is reported for the fabrication of a double network polymer electrolyte (DN-PE) through combining the lithium salt-accelerated thiol-Michael addition and lithium salt-catalyzed radical polymerization. By adjusting the reaction temperature, the double network with the cross-linking structure can be in situ formed step by step at room temperature and 80 °C. Notably, using lithium salt as the accelerator and catalyst avoids the addition of extra species and the related side reactions in the electrolyte system. Compared with single network polymer electrolyte (SN-PE), DN-PE has a distinctly improved mechanical strength and a better interfacial compatibility with the electrode, which leads to a stable cycling of the symmetric Li|DN-PE|Li cell over 1000 h at a current density of 0.05 mA cm-2 . In addition, the Li|DN-PE|LiFePO4 cell shows a high discharge specific capacity of 150.3 mAh g-1 at 0.1 C and coulomb efficiency of 99%.

Mechanically Strong and Electrochemically Stable Single-Ion Conducting Polymer Electrolytes Constructed from Hydrogen Bonding.

Herein, composite membranes based on a single-ion conducting polymer electrolyte (SIPE) and poly(vinylidene fluoride-hexafluoropropylene) (PVDF-HFP) were prepared by an electrospinning technology. The SIPE with hydrogen bonding was obtained via reversible addition-fragmentation chain transfer (RAFT) copolymerization of 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido)ethyl methacrylate (UPyMA), poly(ethylene glycol) methyl ether methacrylate (PEGMA), and lithium 4-styrenesulfonyl (phenylsulfonyl) imide (SSPSILi). The obtained composite membrane exhibited a highly porous network structure, superior thermal stability (>300 °C), and high mechanical strength (17.3 MPa). The fabricated SIPE/PVDF-HFP composite membrane without lithium salts possessed a high ionic conductivity of 2.78 × 10-5 S cm-1 at 30 °C, excellent compatibility with the lithium metal electrode, and high lithium-ion transference number (0.89). The symmetric Li//Li cell exhibited a superior cycle performance without short circuit, indicating the generation of a stable interface between SIPE and the lithium metal electrode during the process of lithium plating/stripping, which could inhibit lithium dendrite growth in lithium metal batteries (LMBs). The Li//LiFePO4 cell also exhibited superior cycle life and excellent rate capability at 60 or 25 °C. In consequence, the composite membrane exhibits a considerable future prospect for advanced LMBs.

Protein tyrosine phosphatase 11 acts through RhoA/ROCK to regulate eosinophil accumulation in the allergic airway.

Src homology domain 2-containing protein tyrosine phosphatase 2 (SHP2) participates in multiple cell functions including cell shape, movement, and differentiation. Therefore, we investigated the potential role of SHP2 in eosinophil recruitment into lungs in allergic airway inflammation and explored the underlying mechanism. Both SHP2 and Ras homolog family member A (RhoA) kinase were robustly activated in the airway eosinophils of children with allergic asthma and of a mouse model with allergic airway inflammation. Moreover, inhibition of SHP2 activity by its specific inhibitors reverses the dephosphorylation of p190-A Rho GTPase-activating protein and in turn attenuates RhoA/Rho-associated protein kinase (ROCK) signaling, resulting in the attenuation of eosinophil migration in response to platelet-activating factor stimulation. Specifically, SHP2 deletion in myeloid cells did not affect the number and classification of circulating leukocytes but significantly attenuated the allergen-induced inflammatory cell, especially eosinophil, infiltration into lungs, and airway hyperreactivity. Notably, genetic interaction between RhoA and SHP2 indicated that RhoA inactivation and SHP2 deletion synergistically attenuated the allergen-induced eosinophil infiltration into lungs and airway hyperreactivity, whereas overexpression of active RhoA robustly restored the SHP2 deletion-resultant attenuation of allergen-induced eosinophil recruitment into lungs and airway hyperreactivity as well. Thus, this study demonstrates that SHP2 via RhoA/ROCK signaling regulates eosinophil recruitment in allergic airway inflammation and possibly in allergic asthma.-Xu, C., Wu, X., Lu, M., Tang, L., Yao, H., Wang, J., Ji, X., Hussain, M., Wu, J., Wu, X. Protein tyrosine phosphatase 11 acts through RhoA/ROCK to regulate eosinophil accumulation in the allergic airway.

Stabilizing Liquid Electrolytes in a Porous PVDF Matrix Incorporated with Star Polymers with Linear PEG Arms and CycloPEG Cores.

Porous membranes fabricated from poly(vinylidene fluoride) (PVDF) and a star polymer with linear poly(ethylene glycol) (PEG) arms and cycloPEG cores were fabricated via the phase-separation method. The porous gel polymer electrolytes (PGPEs) were obtained by immersing the porous membranes in the electrolyte solution. When the additive amount of star polymer was up to 20 wt %, the prepared membrane had the largest porosity and the pores were uniformly distributed in the membrane. The star polymer can not only decrease the crystallization of PVDF and enhance the absorption of liquid electrolyte but also offer ion conduction channels (cycloPEG cores). Therefore, the PGPE with 20 wt % star polymers exhibited competitive ionic conductivities of 1.27 mS cm-1 at 30 °C and 2.89 mS cm-1 at 80 °C. To stabilize the liquid electrolyte in the holes of porous membranes, a gelator was introduced in the liquid electrolyte to form gelled porous gel polymer electrolytes (GPGPEs), and the leakage of liquid electrolytes was thus remarkably reduced. The ionic conductivity of GPGPEs with 20 wt % star polymer and 1.5 wt % gelator was importantly improved at high temperatures (6.02 mS cm-1 at 80 °C). We systematically investigated the electrochemical performances of PGPEs without star polymer, PGPEs with star polymer, and GPGPEs with star polymer. The incorporation of star polymers with linear PEG arms and cycloPEG cores into the PGPEs and GPGPEs significantly improved the electrochemical performances of the lithium metal/LiFePO4 cell assembled with the PGPEs or GPGPEs.

Long noncoding RNA MAPKAPK5-AS1 promotes colorectal cancer proliferation by partly silencing p21 expression.

Colorectal cancer (CRC) is the third most common malignancy in the world, and long noncoding RNA (lncRNA) plays a critical role in carcinogenesis. Here, we report a novel lncRNA, MAPKAPK5-AS1, that acts as a critical oncogene in CRC. In addition, we attempted to explore the functions of MAPKAPK5-AS1 on tumor progression in vitro and in vivo. Quantitative RT-PCR was used to examine the expression of MAPKAPK5-AS1 in CRC tissues and cells. Expression of MAPKAPK5-AS1 was significantly upregulated in 50 CRC tissues, and increased expression of MAPKAPK5-AS1 was found to be associated with greater tumor size and advanced pathological stage in CRC patients. Knockdown of MAPKAPK5-AS1 significantly inhibited proliferation and caused apoptosis in CRC cells. We also found that p21 is a target of MAPKAPK5-AS1. In addition, we are the first to report that MAPKAPK5-AS1 plays a carcinogenic role in CRC. MAPKAPK5-AS1 is a novel prognostic biomarker and a potential therapeutic candidate for CRC cancer.

Ultrasonic-Assisted Dispersion of ZnO Nanoparticles and Its Inhibition Activity to Trichoderma viride.

The activity inhibition of fungi by ZnO nanoparticles (NPs) has shown huge potential applications in the area of hygienic coatings. However, the inhibition efficiency was limited due to the agglomeration of NPs. To obtain well-dispersed and highly stabilized ZnO nanofluids, ZnO NPs were capped with four kinds of surfactants under ultrasonication. The capping procedure was optimized by varying the dosage of surfactants, the ultrasonic duration, ultrasonic power and temperature. Capped ZnO nanofluids were then used for the inhibition of Trichoderma viride. The influence on the activity of the capping conditions, illumination, ZnO NPs content, humidity and temperature were investigated in details. Results suggest that well-dispersed ZnO NPs were obtained through ultrasonic-assisted functionalization using sodium polyacrylate as a dispersant. Moreover, capped ZnO nanofluids revealed long-term stability at pH above 6. The optimal capping procedure was obtained for a sonication power of 250 W, treatment duration of 40 min, dosage of 0.4% and temperature of 60 °C. Antifungal tests indicated that capped ZnO NPs showed an inhibition ability versus T. viride even in the dark. The antifungal ability of ZnO NPs increased with the increasing ZnO content, and humidity and temperature only affected the growth of fungi. Capped ZnO NPs showed an excellent antifungal performance even in the circumstance that was beneficial for the fungi growth (temperature of 30 °C, humidity of 95%), demonstrating the antimicrobial capability in practical applications.

Smoothened-independent activation of hedgehog signaling by rearranged during transfection promotes neuroblastoma cell proliferation and tumor growth.

BACKGROUND: Rearranged during transfection (RET) proto-oncogene encodes a receptor tyrosine kinase for glial cell line-derived neurotrophic factor (GDNF) signaling, and high RET expression is closely related to the tumorigenesis and malignancy of neuroblastoma(NB). METHODS: We have investigated whether RET signals through hedgehog (HH) pathway in NB cell proliferation and tumor growth by in vitro cell culture and in vivo xenograft approaches. RESULTS: The key members of both GDNF/RET and HH/GLI pathways are expressed in NB cell lines to different extents. Knockdown of RET in NB cells significantly attenuates the activity of HH signaling, whereas overexpression of RET robustly enhances the output of transcriptional activation by HH. Likewise, activation of RET by GDNF induces HH signaling, whereas knockdown of RET attenuates both basal and GDNF-induced activities of HH signaling. Moreover, protein kinase B lies on the downstream of GDNF/RET signaling module to inhibit the GSK3ß, resulting in activation of HH signaling. Furthermore, either knockdown of RET by shRNA or inhibition of HH pathway by cyclopamine attenuates not only basal but also GDNF-induced proliferation of SH-SY5Y cells, and knockdown of either RET or smoothened in SH-SY5Y cell xenografts significantly attenuated the tumor growth. Finally, inhibition of HH signaling by GLI1 and GLI2 inhibitor, Gant61, reduces not only basal but also RET-induced proliferation of SH-SY5Y cells and outgrowth of xenografts. CONCLUSION: GDNF/RET/AKT/GSK3ß signaling module activates HH pathway to stimulate NB cells proliferation and tumor outgrowth. GENERAL SIGNIFICANCE: Targeting HH pathway is a rational approach for therapeutic intervention of NB with high RET expression.

The long noncoding RNA HOXA transcript at the distal tip promotes colorectal cancer growth partially via silencing of p21 expression.

Accumulating evidence strongly suggests that dysregulation of long noncoding RNAs (lncRNAs) is associated with human carcinogenesis. The lncRNA HOXA transcript at the distal tip (HOTTIP) is involved in the development of several cancers. However, the biological role of HOTTIP in colorectal cancer (CRC) has not yet been discussed. Here, we report that HOTTIP acts as a functional oncogene in the pathogenesis of CRC. In this study, quantitative polymerase chain reaction (qPCR) was performed to detect the expression of HOTTIP in 48 pairs of colorectal cancer samples. We found that overexpression of HOTTIP is correlated with an advanced pathological stage and a larger tumor size. Moreover, functional analyses revealed that the knockdown of HOTTIP expression by small interfering RNA (siRNA) or small hairpin RNA (shRNA) could inhibit cell proliferation and induce cell apoptosis. More importantly, we observed that HOTTIP knockdown induced a marked increase in the number of cells in the G0/G1 phase and a reduction in the number of cells in the S phase in both DLD-1 cells and SW480 cells. An in vivo experiment also revealed that the knockdown of HOTTIP inhibited tumor growth. Western blot and immunohistochemistry analyses indicated that HOTTIP potentially contributed to CRC cell growth partially through the silencing of p21 expression. Collectively, our results suggest that HOTTIP is involved in the progression of CRC and may provide evidence for HOTTIP being a target for therapy of this disease.

The association between serum lipids and colorectal neoplasm: a systemic review and meta-analysis.

OBJECTIVE: There have been inconsistent results published regarding the relationship between dyslipidaemia and an increased risk of colorectal neoplasia (CRN), including colorectal adenoma (CRA) and colorectal cancer (CRC). We conducted a meta-analysis to explore the relationship between dyslipidaemia and CRN. DESIGN: We identified studies by performing a literature search using PubMed, EMBASE and the Science Citation Index through October 2013. SETTING: We analysed thirty-three independent studies reporting the association between CRN and at least one of the selected lipid components, including total cholesterol (TC), TAG, HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C). SUBJECTS: CRN cases (n 21 809) were identified. RESULTS: Overall, people with high levels of serum TAG (risk ratio (RR)=1.08; 95% CI 1.05, 1.12, P<0.00001) and LDL-C (RR=1.07; 95% CI 1.00, 1.14, P=0.04) presented an increased prevalence of CRN. Subgroup analyses revealed that high levels of serum TC (RR=1.04; 95% CI 1.01, 1.09, P=0.02), TAG (RR=1.06; 95% CI 1.03, 1.10, P=0.0009) and LDL-C (RR=1.11; 95% CI 1.04, 1.19, P=0.003) increased the risk of CRA but not of CRC. No association between serum HDL-C and risk for CRN (including CRA and CRC) was observed. CONCLUSIONS: Both TAG and LDL-C were significantly associated with an increasing prevalence of CRN. High levels of serum TC, TAG and LDL-C were positively associated with CRA but not with CRC. No significant association was observed between levels of serum HDL-C and CRN.

High-mobility group nucleosome-binding domain 5 increases drug resistance in osteosarcoma through upregulating autophagy.

Although tumor therapy has been improved in the past decades, the survival outcomes for osteosarcoma remain unsatisfactory, and one of the primary reasons for the failure of current treatment is that patients with late-stage cancer often develop resistance to anticancer drugs. High-mobility group nucleosome-binding domain 5 (HMGN5) is a newly identified gene associated with cancer and autophagy, which could inhibit apoptosis induced by anticancer agents. However, it is still unclear whether HMGN5 regulated autophagy in osteosarcoma, and the mechanism and significance of HMGN5-mediated autophagy in tumor therapy is never investigated. In this study, we first detected HMGN5 in vivo and in vitro. HMGN5 was highly expressed in osteosarcoma tumor, especially in posttreatment tumor. Next, we employed adenovirus-mediated overexpression of HMGN5 in U-2OS and MG63 to investigate the role of HMGN5 in osteosarcoma cell lines. Adenovirus-mediated overexpression of HMGN5 could efficiently upregulate the expression level of HMGN5 in osteosarcoma cell lines at both messenger RNA (mRNA) and protein levels. Anticancer agents namely doxorubicin, cisplatin, and methotrexate each induced HMGN5 upregulation in human U-2OS and MG63 osteosarcoma cell lines. In addition, overexpression of HMGN5 reduced the chemosensitivity of osteosarcoma cells in vitro, and the mechanistic investigation revealed that HMGN5 increased drug resistance by upregulating autophagy. Therefore, HMGN5 is a critical factor in the development of chemoresistance through regulating autophagy, and it offers a novel target for improving osteosarcoma therapy.

Physiological and biochemical characterization of egg extract of black widow spiders to uncover molecular basis of egg toxicity.

BACKGROUND: Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity. RESULTS: In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na+, K+ and Ca2+ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed. CONCLUSIONS: The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs' toxic actions.

[Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene].

OBJECTIVE: To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene. METHODS: Based on the human ILK gene sequences, RNAi target sequences were designed and cloned into the lentiviral vector pSicoR-eGFP by restriction endonuclease HpaI and XhoI double digestion and T4 DNA ligase ligation. Based on the human mda7 gene sequences, PCR primers were designed to clone the full-length mda7, and were cloned into the lentiviral vector pLVX-Puro. After the candidate clones were identified by DNA sequencing, the recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral particles. Human prostate cancer PC-3 cells were infected with the constructed lentiviral vector. The ILK and mda7 expression levels in PC-3 cells were quantified by qPCR and Western blot, respectively. The effect of ILK and mda7 on proliferation and migration of PC-3 cells were assessed by MTT method and Transwell assay, respectively. RESULTS: ILK-pSicoR-eGFP and mda7-pLVX-Puro lentiviral vectors were successfully constructed. Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of 293T cells with 4 plasmids of lentiviral vector. The transfection efficiency of the collected virus exceeded 90% in the 293T cells and the PC-3 cells were infected with the lentiviral particles with high efficiency. The A and B lentiviral vector inhibited the expression of ILK at both the mRNA and protein levels in PC-3 cells significantly. The mda7-pLVX-Puro lentiviral vector increased the expression of mda7 in PC-3 cells, and the ability was maintained for one month. Within 96 h, ILK and mad7 significantly inhibited the proliferation and migration of PC-3 cells (Ps<0.05). CONCLUSION: The lentiviral vectors of ILK knockdown and mda7 over-expression have been successfully constructed and identified. The recombinant lentivirus can efficiently infect human prostate cancer PC-3 cells, in which ILK expression is inhibited and mda7 is over-expressed.

COL10A1 as a Prognostic Biomarker in Association with Immune Infiltration in Prostate Cancer.

BACKGROUND: The collagen type X alpha 1 (COL10A1) has recently been found to play an important role in the development and progression of cancer. However, the link between COL10A1 and the tumor immune microenvironment remains understood scantily. METHODS: In the current study, the pan-cancer data of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were used to investigate the expression mode, the clinical prognostic and diagnostic value of COL10A1 in different tumors. We used TCGA data to assess the correlations between COL10A1 and clinical symptoms of prostate cancer. The R packages "edgR" and "clusterProfiler" were used for differential expression gene and enrichment analysis of COL10A1. Immunohistochemistry was further employed to corroborate the expression of COL10A1 gene in prostate cancer. After that, we used TIMER to evaluate the pertinence of COL10A1 expression to immune infiltration level in prostate cancer. RESULTS: On the whole, COL10A1 was expressed at significantly higher levels in a variety of tumor tissues than in the corresponding normal tissues. Besides, significant correlations with tumor prognosis and relative exactitude in predicting tumors show that COL10A1 may be a probable prognostic and diagnostic biomarker of prostate cancer. In addition, the evidence indicates a significant correlation between COL10A1 and clinical symptoms of prostate cancer. Furthermore, the main molecular functions of COL10A1 included humoral immune response, complement activation, immunoglobulin, regulation of complement activation, and regulation of humoral immune response. Finally, we found that COL10A1 expression is positively correlated with enhanced macrophage and M2 macrophage infiltration in prostate cancer. CONCLUSION: The study indicates that COL10A1 might participate in M2 macrophage polarization in prostate cancer. COL10A1 might be an innovative biomarker to evaluate tumor microenvironment immune cell infiltration and prognosis in prostate cancer.

Facile Fabrication of Polymer Electrolytes with Branched Structure via Deep Eutectic Electrolyte-Enabled In Situ Polymerizations.

The demand for higher energy density in energy storage devices drives further research on lithium metal batteries (LMBs) because of the high theoretical capacity and low voltage of lithium metal anode. Polymer electrolytes (PEs) exhibit obvious advantages in combating volatilization and leakage compared with liquid electrolytes, which improves the safety of LMBs. However, it is still difficult to construct PEs with a stable electrolyte-electrode interface for high-performance and long-term life LMBs. Herein, the gel polymer electrolyte (GPE-SL) containing deep eutectic electrolyte (DEE) and branchlike polymer skeleton are designed and prepared by the DEE-induced in situ cationic and radical polymerizations. The DEE provides a smooth Li+ migration pathway to ensure the electrochemical properties, and the multibrominated polymer matrix formed in situ enables a LiBr-rich solid electrolyte interphase (SEI) layer on lithium metal anode and prolongs the life span of LMBs. Hence, the Li|GPE-SL|LiFePO4 battery displays an excellent cycling stability with 84% capacity retention after 1200 cycles at 1C. This simple deep eutectic electrolyte-induced polymerization method provides a promising direction for high-performance LMBs with improved anode-electrolyte compatibility through the construction of a stable SEI layer in situ.