Fluorinated Lipid Nanoparticles for Enhancing mRNA Delivery Efficiency.

Lipid nanoparticles (LNPs), a nonviral nucleic acid delivery system, have shown vast potential for vaccine development and disease treatment. LNPs assist mRNA to cross physiological barriers such as cell membranes and endosomes/lysosomes, promoting the intracellular presentation of mRNA. However, the endosome escape efficiency and biosafety of currently commercialized LNPs are still unsatisfactory, resulting in underutilization of mRNA. Herein, we report that fluorinated modification of the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 (PEG-DSPE), termed as FPD, in the LNPs can improve the delivery efficiency of mRNA. FPD accounts for only 1.5% of lipids in LNPs but could mediate a 5-fold and nearly 2-fold enhancement of mRNA expression efficiency in B16F10 tumor cells and primary dendritic cells, respectively. Mechanism studies reveal that FPD promotes the cellular internalization of LNPs as well as endosome escape. In vivo studies substantiate that FPD can augment overall mRNA expression at least 3-fold, either by intravenous or intraperitoneal injection, compared to LNPs prepared with nonfluorinated PEG-lipids at a relatively low mRNA dose. Besides, with the introduction of FPD, mRNA expression in the spleen augmented compared to that of the DMG-PEG commercial formulations. Benefiting from a prudent dosage of fluorine, the fluorinated LNPs display favorable biosafety profiles at cellular and zoological levels.

Mechanism of miR-98-5p in gastric cancer cell proliferation, migration, and invasion through the USP44/CTCFL axis.

Objectives: Gastric cancer (GC) is the leading digestive malignancy with high incidence and mortality rate. microRNAs (miRs) play an important role in GC progresssion. This study aimed to investigate the effect of miR-98-5p on proliferation, migration, and invasion of GC cells. Methods: The expression levels of miR-98-5p, ubiquitin specific peptidase 44 (USP44), and CCCTCbinding factor-like (CTCFL) in GC tissues and cells were identified using reversetranscription quantitative polymerase chain reaction and Western blot assay. The relationship between miR-98-5p expression/USP44 and the clinicopathological features in GC patients was analyzed. GC cell proliferation, invasion, and migration were evaluated by cell counting kit-8 and clone formation assays and Transwell assays. The bindings of miR-98-5p to USP44 and USP44 to CTCFL were examined using dualluciferase assay and co-immunoprecipitation. GC cells were treated with MG132 and the ubiquitination level of CTCFL was examined using ubiquitination assay. Rescue experiments were performed to verify the roles of USP44 and CTCFL in GC cells. Results: miR-98-5p was downregulated in GC. miR-98-5p overexpression inhibited the proliferation, migration, and invasion of GC cells. miR-98-5p inhibited USP44 expression. USP44 bound to CTCFL and limited ubiquitination degradation of CTCFL. Overexpression of USP44 and CTCFL attenuated the inhibitory effects of miR-98-5p overexpression on GC cell progression. Conclusion: miR-98-5p overexpression limited USP44-mediated CTCFL deubiquitination, and suppressed CTCFL expression, mitigating GC cell proliferation, migration, and invasion.

Blood-Brain Barrier Integrity Damage in Bacterial Meningitis: The Underlying Link, Mechanisms, and Therapeutic Targets.

Despite advances in supportive care and antimicrobial treatment, bacterial meningitis remains the most serious infection of the central nervous system (CNS) that poses a serious risk to life. This clinical dilemma is largely due to our insufficient knowledge of the pathology behind this disease. By controlling the entry of molecules into the CNS microenvironment, the blood-brain barrier (BBB), a highly selective cellular monolayer that is specific to the CNS's microvasculature, regulates communication between the CNS and the rest of the body. A defining feature of the pathogenesis of bacterial meningitis is the increase in BBB permeability. So far, several contributing factors for BBB disruption have been reported, including direct cellular damage brought on by bacterial virulence factors, as well as host-specific proteins or inflammatory pathways being activated. Recent studies have demonstrated that targeting pathological factors contributing to enhanced BBB permeability is an effective therapeutic complement to antimicrobial therapy for treating bacterial meningitis. Hence, understanding how these meningitis-causing pathogens affect the BBB permeability will provide novel perspectives for investigating bacterial meningitis's pathogenesis, prevention, and therapies. Here, we summarized the recent research progress on meningitis-causing pathogens disrupting the barrier function of BBB. This review provides handy information on BBB disruption by meningitis-causing pathogens, and helps design future research as well as develop potential combination therapies.

The effect of thalidomide on the invasive ability of gastric cancer cells by regulating miR-524-5p/FSTL1.

This study aimed to investigate the effect of thalidomide (Thal) regulating microRNA (miR)-524-5p/follistatin-like protein 1 (FSTL1) on the invasion ability of gastric cancer cells. For this purpose, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the level of miR-524-5p in GES1, SUN-16, MGC-803, SGC-7901, MKN-28, and MKN-45 cells. Then the MGC-803 and MKN-45 cells would proceed to the next research. The MGC-803 and MKN-45 cells were cultured in vitro and added Thal to the final concentration of (6.25, 12.5, 25, 50, 100) µg/mL. The blank control group only added 0.1% dimethyl sulfoxide (DMSO) culture medium, and cultured for 48 hours. CCK8 was used to detect cell proliferation, and Transwell was used to detect cell invasion. The experiment was divided into a blank control group, Thal group (25 µg/mL Thal-treated cells), Thal+inhibitor NC group, and Thal+miR-524-5p inhibitor group (transfected with inhibitor NC and miR-524-5p inhibitor respectively on the basis of Thal group), cultivated for 48 h. The level of miR-524-5p in the cells was detected by RT-qPCR; the cell invasion was detected by Transwell; the expression of matrix metalloproteinase (MMP)-2, MMP-9, FSTL1 protein in the cells was detected by Western blot. The targeting relationship between miR-524-5p and FSTL1 was verified by dual luciferase. Results showed that compared with GES1 cells, the level of miR-524-5p in SUN-16, MGC-803, SGC-7901, MKN-28, and MKN-45 cells decreased (P<0.05). In MGC-803 and MKN-45 cells, compared with the blank control group, the cell proliferation rate and the number of invasions in the (50, 100) µg/mL Thal treatment groups, and the number of invasions in (6.25, 12.5, 25) µg/mL Thal treatment groups decreased (P<0.05). Compared with the blank control group, the level of miR-524-5p in the cells of the Thal group, Thal+inhibitor NC group, and Thal+miR-524-5p inhibitor group increased (P<0.05), the number of invasions, the levels of MMP-2, MMP-9 and FSTL1 proteins in cells decreased (P<0.05); compared with the Thal group and the Thal+inhibitor NC group, the level of miR-524-5p in the cells of the Thal+miR-524-5p inhibitor group decreased (P<0.05), the number of invasions, the levels of MMP-2, MMP-9, and FSTL1 proteins in the cells increased (P<0.05). Dual luciferase verification revealed that there was a targeting relationship between miR-524-5p and FSTL1. In conclusion, that can up-regulate the expression of miR-524-5p to reduce the expression of FSTL1 protein, inhibit the invasion of gastric cancer cells, and achieve alleviation of the disease.

Transcriptional landscape of human neuroblastoma cells in response to SARS-CoV-2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and the neurological symptoms of SARS-CoV-2 infection have already been reported. However, the mechanisms underlying the effect of SARS-CoV-2 infection on patients with central nervous system injuries remain unclear. METHODS: The high-throughput RNA sequencing was applied to analyze the transcriptomic changes in SK-N-SH cells after SARS-CoV-2 infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to identify the functions of differentially expressed genes and related pathways. RESULTS: A total of 820 mRNAs were significantly altered, including 671 upregulated and 149 downregulated mRNAs (showing an increase of ≥ 2-fold or decrease to ≤ 0.5-fold, respectively; p ≤ 0.05). Moreover, we verified the significant induction of cytokines, chemokines, and their receptors, as well as the activation of NF-κB, p38, and Akt signaling pathways, in SK-N-SH by SARS-CoV-2. CONCLUSIONS: To our knowledge, this is the first time the transcriptional profiles of the host mRNAs involved in SARS-CoV-2 infection of SK-N-SH cells have been reported. These findings provide novel insight into the pathogenic mechanism of SARS-CoV-2 and might constitute a new approach for future prevention and treatment of SARS-CoV-2-induced central nervous system infection.

SARS-CoV-2 productively infects human brain microvascular endothelial cells.

BACKGROUND: The emergence of the novel, pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global health emergency. SARS-CoV-2 is highly contagious and has a high mortality rate in severe patients. However, there is very limited information on the effect of SARS-CoV-2 infection on the integrity of the blood-brain barrier (BBB). METHODS: RNA-sequencing profiling was performed to analyze the transcriptomic changes in human brain microvascular endothelial cells (hBMECs) after SARS-CoV-2 infection. Bioinformatic tools were used for differential analysis. Immunofluorescence, real-time quantitative PCR, and Western blotting analysis were used to explore biological phenotypes. RESULTS: A total of 927 differentially expressed genes were identified, 610 of which were significantly upregulated while the remaining 317 were downregulated. We verified the significant induction of cytokines, chemokines, and adhesion molecules in hBMECs by SARS-CoV-2, suggesting an activation of the vascular endothelium in brain. Moreover, we demonstrated that SARS-CoV-2 infection could increase the BBB permeability, by downregulating as well as remodeling the intercellular tight junction proteins. CONCLUSIONS: Our findings demonstrated that SARS-CoV-2 infection can cause BBB dysfunction, providing novel insights into the understanding of SARS-CoV-2 neuropathogenesis. Moreover, this finding shall constitute a new approach for future prevention and treatment of SARS-CoV-2-induced CNS infection.

Resveratrol Attenuates Meningitic Escherichia coli-Mediated Blood-Brain Barrier Disruption.

Meningitic Escherichia coli can infiltrate the central nervous system (CNS), consequently increasing the levels of proinflammatory cytokines and chemokines and deteriorating the integrity of the blood-brain barrier (BBB). Resveratrol has emerged in recent years as a compound with antioxidant and anti-inflammatory properties. However, it is still unknown how resveratrol affects meningitic E. coli-induced CNS dysfunction. Here, by using in vivo and in vitro BBB models, we demonstrated that resveratrol treatment significantly inhibited meningitic E. coli invasion of the BBB, protected the integrity of the BBB, and reduced neuroinflammation and lethality. In mechanism, resveratrol inhibited bacterial penetration of the BBB by attenuating the upregulation of caveolin-1 (CAV-1), a class of lipid rafts maintaining endothelial cell function. Resveratrol treatment also maintained BBB permeability by suppressing the ERK1/2-VEGFA signaling cascade. In vivo treatment of resveratrol decreased the production of inflammatory cytokines and improved the survival rate in mice challenged with meningitic E. coli. These findings collectively indicated that resveratrol could attenuate meningitic E. coli-induced CNS injury, which might constitute a new approach for future prevention and treatment of E. coli meningitis.

Exosomal miR-155-5p promotes proliferation and migration of gastric cancer cells by inhibiting TP53INP1 expression.

Exosomal microRNA (miRNA) secreted by tumor cells plays an important biological role in tumorigenesis and development. We aimed to explore the effects of exosomal miR-155-5p in gastric cancer (GC) and understand its mechanism of action in GC progression. We isolated exosomes from the human gastric mucosal epithelial cell line GES-1 and gastric cancer cell line AGS, and then identified them according to their surface markers by flow cytometry. Later, we detected the miR-155-5p expression levels in tissues and isolated exosomes using RT-qPCR. Bioinformatics analysis showed that miR-155-5p directly binds to the 3' untranslated region (3'-UTR) of tumor protein p53-induced nuclear protein 1 (TP53INP1) mRNA. We also investigated whether the miR-155-5p-rich exosomes caused changes in cell cycle, proliferation, and migration in AGS cells. In this study, we found that the levels of miR-155-5p were significantly increased in GC tissues and AGS cells, and that the TP53INP1 protein level was downregulated in GC tissues using IHC and IFC. TP53INP1 was found to be directly regulated by miR-155-5p following a dual luciferase-based reporter assay. After co-culturing with the isolated miR-155-5p-rich exosomes, the proliferation and migration capabilities of AGS cells were enhanced. Thus, our results reveal that exosomal miR-155-5p acts as an oncogene by targeting TP53INP1 mRNA in human gastric cancer.

CRISPR/Cas9-mediated knockout of the PDEF gene inhibits migration and invasion of human gastric cancer AGS cells.

Gastric cancer is one of the most common malignant tumors worldwide and has the second highest incidence and mortality rate among malignant tumors in China. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors. Although PDEF plays an important role in tumorigenesis, its biological function in gastric cancer is still unclear. Here, we evaluated PDEF expression in 30 cases of human gastric carcinoma and the corresponding peritumoral tissues, using immunohistochemistry and immunofluorescence. Significantly higher levels of PDEF were detected in tumors compared to peritumoral tissues. We then investigated PDEF expression in the gastric cancer cell lines SGC and AGS and the normal gastric epithelial cell line GES; The CRISPR/Cas9 genome-editing system was used to knockout PDEF in AGS cells as a model for gastric cancer. Cell proliferation, apoptosis, migration, and invasion of PDEF-knockout AGS cells were evaluated using CCK-8, flow cytometry, scratch wound, and transwell assays, respectively. The results illustrated that PDEF-knockout inhibited AGS cell proliferation, migration, and invasion. Taken together, the results imply that PDEF plays important roles in the proliferation, migration, and invasion of AGS cells and may serve as a new treatment target in gastric cancer.

[Heavy Metal Pollution and Potential Ecological Risk of Soil from Reclaimed Industrial Sites and Surrounding River Sediments].

Eight heavy metals (As, Cd, Cr, Cu, Ni, Pb, Zn, and Hg) in the soils from 53 reclaimed industrial sites and 23 river sediments in the Shanghai suburbs were determined using the Nemero pollution index and Hakanson potential ecological risk index (RI). The results show that the eight heavy metals exceed the secondary standard limits to different extents. The rate of standard excess is the most prominent for Cu and Zn; it accounts for 47.17% and 43.40%, respectively. The contents of heavy metals in the sediments from surrounding rivers are significantly lower than those in soil from reclaimed industrial sites. The comprehensive Nemero pollution index of heavy metals in reclaimed soil is 11.41, which represents a serious pollution level; Cu and Zn are the most prominent heavy metals. The Nemero pollution index of heavy metals in surrounding river sediments is 1.26, indicating slight pollution. Based on the single-potential ecological risk, the heavy metals at the reclaimed industrial sites all have a slightly latent ecological risk level, except for Cd (strong), Hg (strong), and Cu (moderate). The potential ecological RI is high (385.79). Heavy metals from the surrounding river sediments have a mild potential ecological risk level, except for Hg. Overall, the potential ecological level is mild (RI=83.91). In short, Cu and Zn are more prominent in reclaimed industrial soil. With respect to the toxic response factor, Cd and Hg should be focused on. The heavy metal pollution of surrounding river sediments is light, leading to a mild potential ecological risk level.

The ß2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells.

In this study, ß2-AR level was found to be up-regulated in MCF-7 cells overexpressing Her2 (MCF-7/Her2). Correlation of ß2-AR level with Her2 status was demonstrated in breast cancer tissue samples. Constitutive phosphorylation of ERK, mRNA expression up-regulation of catecholamine-synthesis enzymes, and increased epinephrine release were detected in MCF-7/Her2 cells. ß2-AR expression induced by epinephrine and involvement of ERK signaling were validated. The data indicate that Her2 overexpression and excessive phosphorylation of ERK cause epinephrine autocrine release from breast cancer cells, resulting in up-regulation of ß2-AR expression. The data also showed that catecholamine prominently stimulated Her2 mRNA expression and promoter activity. The activation and nuclear translocation of STAT3 triggered by isoproterenol were observed. Enhanced binding activities of STAT3 to the Her2 promoter after isoproterenol stimulation were verified. Using STAT3 shRNA and dominant negative STAT3 mutant, the role of STAT3 in isoproterenol-induced Her2 expression was further confirmed. The data support a model where ß2-AR and Her2 comprise a positive feedback loop in human breast cancer cells.