The siEGFR nanoplexes for the enhanced brain glioma treatment: Endoplasmic reticulum biomimetic strategy to induce homing effect and non-degradable intracellular transport.

The epidermal growth factor receptor (EGFR) plays a pivotal role in tumor progression and is an essential therapeutic target for treating malignant gliomas. Small interfering RNA (siRNA) has the potential to selectively degrade EGFR mRNA, yet its clinical utilization is impeded by various challenges, such as inefficient targeting and limited escape from lysosomes. Our research introduces polyethylene glycol (PEG) and endoplasmic reticulum membrane-coated siEGFR nanoplexes (PEhCv/siEGFR NPs) as an innovative approach to brain glioma therapy by overcoming several obstacles: 1) Tumor-derived endoplasmic reticulum membrane modifications provide a homing effect, facilitating targeted accumulation and cellular uptake; 2) Endoplasmic reticulum membrane proteins mediate a non-degradable "endosome-Golgi-endoplasmic reticulum" transport pathway, circumventing lysosomal degradation. These nanoplexes demonstrated significantly enhanced siEGFR gene silencing in both in vitro and in vivo U87 glioma models. The findings of this study pave the way for the advanced design and effective application of nucleic acid-based therapeutic nanocarriers.

An efficient and universal In silico screening strategy for acquisition of high-affinity Aptamer and its application in analytical utility.

Numerous aptamers against various targets have been identified through the technology of systematic evolution of ligands by exponential enrichment (SELEX), but the affinity of these aptamers are often insufficient due to the limitations of SELEX. Therefore, a more rational in silico screening strategy (ISS) was developed for efficient screening of high affinity aptamers, which took shape complementarity and thermodynamic stability into consideration. Neuron specific enolase (NSE), a tumor marker, was selected as the target molecule. In the screening process, three aptamer candidates with good shape complementarity, lower ΔG values, and higher ZDOCK scores were produced. The dissociation constant (Kd) of these candidates to NSE was determined to be 10.13 nM, 14.82 nM, and 2.76 nM, respectively. Each of them exhibited higher affinity to NSE than the parent aptamer (Kd = 23.83 nM). Finally, an antibody-free fluorescence aptasensor assay, based on the aptamer with the highest affinity, P-5C8G, was conducted, resulting in a limit of detection (LOD) value of 1.8 nM, which was much lower than the parental aptamer (P, LOD = 12.6 nM). The proposed ISS approach provided an efficient and universal strategy to improve the aptamer to have a high affinity and good analytical utility.

A novel loop-mediated isothermal amplification-lateral flow dipstick method for Helicobacter pylori detection.

Introduction: To eradicate Helicobacter pylori (H. pylori) and reduce the risk of gastric cancer, a sensitive, specific, convenient, and simple detection method is needed. This study aimed to establish a novel loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for H. pylori detection. Methods: LAMP primer design software was used to design primers for the conserved sites of the H. pylori ureB gene. UreB-FIP-labeled biotin was used for LAMP amplification, and FAM-labeled probes were specifically hybridized with LAMP amplification products, which were then detected by LFD. In addition, a clinical study was conducted to assess LAMP-LFD in 20 fecal samples. Results: The results of the optimization indicated that H. pylori could be specifically detected by LFD without cross-reaction with other non-H. pylori bacteria when the LAMP was performed at 65°C for 60 min. The lower limit of the detection method was 102 copies/µL, which was 100 times the sensitivity of polymerase chain reaction (PCR). H. pylori-positive fecal samples were detected by LAMP-LFD in 13/20 patients. Discussion: In conclusion, a new LAMP-LFD assay has been fully established and confirmed for H. pylori detection. The entire process can be completed in approximately 1.5 h, with the advantages of strong specificity, high sensitivity, and simple operation. This study provides a novel potential method for the detection of H. pylori in the clinical settings of primary hospitals and low-resource countries.

Development of local injectable, bone-targeting nanocarriers of triptolide for treatment of bone-only metastasis.

Triptolide (TP) is known for its diverse pharmacological activities but also its delivery and toxicity issues. This study aimed at exploiting TP's anticancer effects at lower risk of systemic toxicity by developing local-injectable "bone-targeting TP nanoparticle" (TPN) for bone-only metastasis treatment. The lipid/oil-based TPNs decorated with alendronate (ALE) achieved size of 70.4-111.2 nm with good dispersion stability. The drug encapsulation efficiency reached 97 % and drug release profiles were in biphasic, controlled manner lasting for 5 days in medium with serum proteins and calcium. TPNs were more cytotoxic than free TP against MDA-MB-231 breast cancer cells (IC50: 16.40 ± 0.80 nM vs 25.45 ± 1.83 nM, P < 0.05) but less cytotoxic against MC3T3-E1 osteoblasts (P < 0.05). When combined with paclitaxel or docetaxel, low dose TPN (containing 10 nM) significantly increased the effectiveness of the two chemotherapy drugs against MDA-MB-231 (IC50 values decreased from 7.3 nM to 2.5 nM for docetaxel; from 4.6 nM to 1.1 nM), indicating potent chemosensitization effects. Retardation of in vitro cancer cell migration by TPN was also observed in the standard scratch assay. ALE decoration significantly enhanced the TPN affinity for both calcium hydroxyapatite and porcine bone chip models, which led to enhancement in TP retention in the bones up to 8.1-fold versus free drug. Overall, TPN demonstrated good potential as a local-injectable, bone-targeted nanotherapy tailored for eradication of bone-only metastasis at reduced risk of systemic toxicity.

Estrogen Suppresses Cytokines Release in cc4821 Neisseria meningitidis Infection via TLR4 and ERß-p38-MAPK Pathway.

Estrogen has long been known to possess immune-modulatory effects in diseases, and multiple pathological conditions show great sex disparities. However, the impact of estrogen in Neisseria meningitidis infection has not been determined. The present study aimed to investigate the role of estrogen in N. meningitidis infection and the molecular mechanism. We selected 35 N. meningitidis isolates representing different clonal complexes (cc), serogroups, and isolation sources to infect the HBMEC cell line. Results showed that the expression of estrogen receptor (ER) ß in N. meningitidis-infected cells was downregulated compared with that in normal cells. The expression of ERß induced by invasive isolates was lower than that in carriers. Serogroup C isolates induced the lowest expression of ERß compared with serogroup A and B isolates. We used four cc4821 N. meningitidis isolates to infect two kinds of host cells (human brain microvascular endothelial cells and meningeal epithelial cells). The results showed that 17 ß-estradiol (E2) could inhibit the release of inflammatory factors interleukin (IL)-6, IL-8, and tumor necrosis factor-α after N. meningitidis infection via TLR4. E2 could inhibit the activation of the p38-MAPK signal pathway induced by N. meningitidis infection through binding to ERß, and significantly inhibit the release of inflammatory factors in N. meningitidis-infected host cells. This study demonstrated that estrogen plays a protective role in N. meningitidis infection. ERß is potentially associated with the release of inflammatory cytokines in N. meningitidis infection, which sheds light on a possible therapeutic strategy for the treatment of invasive diseases caused by N. meningitidis.

[Clinical characteristics and genetic analysis of a Chinese pedigree affected with mitochondrial DNA depletion syndrome due to compound heterozygous variants of RRM2B gene].

OBJECTIVE: To analyze the clinical characteristics and pathogenic gene in a Chinese pedigree affected with mitochondrial DNA depletion syndrome 8A (MTDPS8A). METHODS: Whole exome sequencing was carried out for the patient. Sanger sequencing was used to verify the results, and PolyPhen-2 and PROVEAN software were used to predict the impact of amino acid changes on the function of the protein. RESULTS: The patient, a two-month-old female, was admitted to the hospital for poor milk intake and poor mental response. Her clinical manifestations included feeding difficulty, shortness of breath and low muscle tone. Auxiliary laboratory test indicated that the infant was underdeveloped with abnormal liver, kidney, and heart functions accompanied by hyperlacticacidemia. She responded poorly to treatment and eventually died. Sequencing revealed that the child has carried compound heterozygous missense variants of the RRM2B gene, namely c.16delA (p.R6Gfs*22) and c.175G>C (p.A59P), which were respectively inherited from her father and mother, and both were newly discovered pathologic variants. CONCLUSION: The c.16delA and c.175G>C compound heterozygous variants of the RRM2B gene probably underlay the pathogenesis of MTDPS8A. Above finding has strengthened the understanding of the clinical feature and genetic etiology of this disease and expanded the mutation spectrum of the RRM2B gene.

Comparison of Pathogenicity of Invasive and Carried Meningococcal Isolates of ST-4821 Complex in China.

Serotype 4821 (ST-4821) clonal complex (cc4821) Neisseria meningitidis strains are divided into two groups (groups I and II) according to the core genome-based phylogenetic analysis. Group I contains the greater number of invasive disease isolates. However, the differences in pathogenicity between the two groups are unclear. In this study, the pathogenicity of cc4821 isolates (n = 28) belonging to group I and group II (each containing eight invasive isolates and six isolates from healthy carriers) was investigated, including adhesion, invasion, and induction of interleukin-6 (IL-6) and interleukin-8 (IL-8) release from host cells (Hep2 and A549). The invasive isolates had higher adhesion and invasion capabilities than the carried isolates in both groups. The carried cc4821 isolates in group I had stronger invasion capability than those in group II. Invasive isolates induced more IL-6 and IL-8 secretion than carried isolates in both groups. The carried cc4821 isolates stimulated higher levels of IL-8 in group I than in group II. The isolates were defined as hyperadherent and hypoadherent groups according to their adhesion ability and as hyperinvasive and hypoinvasive groups based on their invasion ability. The hyperadherent and hyperinvasive isolates mediated more IL-6 and IL-8 release than the hypoadherent and hypoinvasive isolates. There was no difference in the level of cytokine release when cc4821 isolates lost their adhesion and invasion capability after lysis. The results revealed that differences in pathogenicity existed between the two groups and that the differences were mainly determined by differences in adhesion and invasion capabilities.

Tripartite motif 31 promotes resistance to anoikis of hepatocarcinoma cells through regulation of p53-AMPK axis.

Anoikis-resistance is an essential feature of cancer cells to obtain successful metastasis, whereas the molecular mechanism involved in this process of hepatocellular carcinoma (HCC) cells is not fully understood. Here we demonstrated that tripartite motif-containing (TRIM) 31, a new member of the TRIM family, was significantly upregulated in the anchorage-deprived HCC cells compared with their attached counterpart. When we blocked TRIM31 expression by its specific interference RNAs, the anoikis-resistance of HCC cells was significantly reversed. We further verified that overactivation of AMPK pathway was responsible for TRIM31-mediated resistance to anoikis of HCC cells; and TRIM31 could directly target p53, the upstream suppressor of AMPK pathway, and mediate K48-linked ubiquitous degradation of p53 in a RING-domain-dependent way. Therefore we demonstrated that TRIM31 promoted anoikis-resistance by targeting p53 for degradation and subsequently overactivating AMPK pathway. Thus our study defined for the first time the role of TRIM31 in the anoikis-resistant process of HCC cells， and it may pave a new avenue for manipulation of metastatic cancer by targeting TRIM31.

Overexpression of NIMA-related kinase 2 is associated with poor prognoses in malignant glioma.

Eleated expression of NIMA-related kinase 2 (NEK2) was frequently observed in a variety of malignant cancers, and it appears to be involved in the initiation, maintenance, progression, metastasis of cancer and is positively associated with poor prognosis. We sought to investigate NEK2 expression and its predictive roles in malignant gliomas, and study the correlation of NEK2 protein expression with proliferation, clinical parameters, overall survival and some other parameters. We investigate NEK2 protein expression in 99 samples of malignant gliomas, including 35 WHO grade II, 22 grade III, and 42 grade IV gliomas, by immunohistochemistry and western blot (n = 50). We then made correlative analysis of protein overexpression using the Kaplan-Meier method, Log rank test, and Cox proportional-hazards model analysis. NEK2 protein was overexpressed in malignant gliomas, but not in normal brain tissues. Overexpression of NEK2 correlated with malignancy, proliferation and adverse overall survival in gliomas. Moreover, chemotherapy, resection extent and WHO grade also correlate with overall survival in gliomas. However, within WHO grade II glioma subgroup, NEK2 overexpression showed no impact on overall survival. The present study firstly reveals that NEK2 protein is widely overexpressed in gliomas. NEK2 overexpression correlates significantly with malignancy (WHO grades), proliferation (Ki-67) and prognosis in malignant gliomas. NEK2 is a potential gene therapy target and prognostic indicator.

Serum microRNA panel for early diagnosis of the onset of hepatocellular carcinoma.

Unique change of circulating microRNAs (miRNAs) was recognized to occur in early oncogenesis, which conferred it the potential as biomarkers for early detection of cancer. However, its diagnostic capability for hepatocellular carcinoma (HCC) has not been fully understood. In this study, microarray analysis was applied to screen the initial candidate miRNA from both the supernatants of anoikis-resistant cellular models and the sera samples of HCC patients. The selected differentially expressed miRNAs were further verified in 115 HCC patients and 40 health controls by qRT-PCR. Among these, 4 miRNAs (miR-16-2-3p, 92a-3p, 107, and 3126-5p) were significantly changed in HCC patients compared with controls. Logistic regression analysis identified a 3-miRNA panel (miR-92-3p, miR-107, and miR-3126-5p) as valuable diagnostic marker for HCC, especially for early stage patients (AUC = 0.975) and for low-level AFP HCC patients (AUC = 0.971). In addition, the combination of 3-miRNA panel and AFP was even more effective for discriminating the early stage HCC patients (AUC = 0.988) and low-level AFP HCC patients (AUC = 0.989) from control. In conclusion, diagnostic efficacy of the combination of 3-miRNA panel and AFP was powerful for HCC diagnosis, especially in early tumor screening and low-level AFP patients.

Loss of AIM2 expression promotes hepatocarcinoma progression through activation of mTOR-S6K1 pathway.

Absent in melanoma (AIM2) is a member of the interferon-inducible HIN-200 protein family and is recently recognized to play an important dual role in both innate immunity and tumor pathology. However, the role of AIM2 in the development of hepatocellular carcinoma (HCC) remains to be clarified. Here we showed that AIM2 expression was significantly decreased in liver cancer tissues, and loss of its expression was significantly correlated with more advanced tumor progression. Exogenous overexpression of AIM2 in HCC cells suppressed mammalian target of rapamycin (mTOR)-S6K1 pathway and further inhibited proliferation, colony formation and invasion of HCC cells. On the contrary, block of AIM2 in HCC cells induced (mTOR)-S6K1 pathway activation and thus promoted HCC progression. Treatment with mTOR pathway inhibitor rapamycin further verified its contribution to HCC progression in AIM2 absent HCC cells. Thus, these data suggested that AIM2 played a critical role as a tumor suppressor and might serve as a potential therapeutic target for future development of AIM2-based gene therapy for human liver cancer. This study also paves a new avenue to treat AIM2-deficient cancer by suppression of mTOR.

Lipid-Based Nanocarriers for RNA Delivery.

RNA-interference (RNAi) agents such as small-interfering RNA (siRNA) and micro-RNA (miRNA) have strong potential as therapeutic agents for the treatment of a broad range of diseases such as malignancies, infections, autoimmune diseases and neurological diseases that are associated with undesirable gene expression. In recent years, several clinical trials of RNAi therapeutics especially siRNAs have been conducted with limited success so far. For systemic administration of these poorly permeable and easily degradable macromolecules, it is obvious that a safe and efficient delivery platform is highly desirable. Because of high biocompatibility, biodegradability and solid track record for clinical use, nanocarriers made of lipids and/or phospholipids have been commonly employed to facilitate RNA delivery. In this article, the key features of the major sub-classes of lipid-based nanocarriers, e.g. liposomes, lipid nanoparticles and lipid nanoemulsions, will be reviewed. Focus of the discussion is on the various challenges researchers face when developing lipid-based RNA nanocarriers, such as the toxicity of cationic lipids and issues related to PEGylated lipids, as well as the strategies employed in tackling these challenges. It is hoped that by understanding more about the pros and cons of these most frequently used RNA delivery systems, the pharmaceutical scientists, biomedical researchers and clinicians will be more successful in overcoming some of the obstacles that currently limit the clinical translation of RNAi therapy.

Estrogen suppresses hepatocellular carcinoma cells through ERß-mediated upregulation of the NLRP3 inflammasome.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. The incidence of HCC is strikingly higher in males than in females. The remarkable gender disparity suggests an important role for sex hormones in HCC pathogenesis. Recently, estrogen has emerged as a protective factor in the development and progression of HCC, but whether it prevents and attenuates HCC, and the mechanism of protection, have not been elucidated. The present study shows that expression of estrogen receptor (ER) ß was significantly downregulated in HCC tissue compared with normal liver tissue; moreover, its expression level showed a significant negative correlation with disease progression and a positive correlation with the expression level of NLRP3 inflammasome components. In a previous study, we showed that loss of NLRP3 inflammasome in HCC tissue contributed to tumor progression, whereas the mechanism of its deregulation was not elucidated. In this study, we investigated the potential link between NLRP3 inflammasome and estrogen. Our data reveal that treatment with 17ß-estradiol (E2) significantly inhibited the malignant behavior of HCC cells through E2/ERß/MAPK pathway-mediated upregulation of the NLRP3 inflammasome. This study shows a novel link between ERß and the NLRP3 inflammasome in HCC progression, which provides a potentially valuable therapeutic strategy for treatment of HCC patients.

MiR-424-5p reversed epithelial-mesenchymal transition of anchorage-independent HCC cells by directly targeting ICAT and suppressed HCC progression.

Resistance to anoikis and Epithelial-mesenchymal transition (EMT) are two processes critically involved in cancer metastasis. In this study, we demonstrated that after anchorage deprival, hepatocellular carcinoma (HCC) cells not only resisted anoikis, but also exhibited EMT process. Microarray expression profiling revealed that expression of miR-424-5p was significantly decreased in anoikis-resistant HCC cells. Ectopic overexpression of miR-424-5p was sufficient to reverse resistance to anoikis, block EMT process and inhibit malignant behaviors of HCC cells. Target analysis showed that a potent ß-catenin inhibitor, ICAT/CTNNBIP1 was a direct target of miR-424-5p. Further study demonstrated that miR-424-5p reversed resistance to anoikis and EMT of HCCs by directly targeting ICAT and further maintaining the E-cadherin/ß-catanin complex on the cellular membrance. In vivo study further demonstrated that miR-424-5p significantly inhibited the tumorigenicity of HCC cells in nude mice. Clinical investigation demonstrated that miR-424-5p was significantly downregulated in HCC tissues compared with that of the non-cancerous liver tissues, and this decreased expression of miR-424-5p was significantly correlated with higher pathological grades and more advanced TNM stages. Therefore, aberrant expression of miR-424-5p is critically involved in resistance to anoikis and EMT during the metastatic process of HCC, and its downregulation significantly contributes to liver cancer progression.

Deregulation of the NLRP3 inflammasome in hepatic parenchymal cells during liver cancer progression.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors worldwide, and it is always the consequence of chronic hepatitis and liver cirrhosis. The nucleotide-binding domain, leucine-rich family (NLR), pyrin-containing 3 (NLRP3) inflammasome has been shown to orchestrate multiple innate and adaptive immune responses. However, little is known about its role in cancer. This study was performed to investigate the role of the NLRP3 inflammasome in the development and progression of HCC. The expression of NLRP3 inflammasome components was analyzed in HCC tissues and corresponding non-cancerous liver tissues at both the mRNA and protein levels. Our data demonstrate that the expression of all of the NLRP3 inflammasome components was either completely lost or significantly downregulated in human HCC, and that the deficiency correlated significantly with advanced stages and poor pathological differentiation. In addition, our data provide an overview of the expression of NLRP3 inflammasome components in the multi-stage development of HCC and indicate a surprising link between deregulation of the NLRP3 inflammasome molecular platform and HCC progression. In conclusion, this study presents a dynamic expression pattern of NLRP3 inflammasome components in multi-stage hepatocarcinogenesis and demonstrates that deregulated expression of the inflammasome is involved in HCC progression.