Mucus adhesion vs. mucus penetration? Screening nanomaterials for nasal inhalation by MD simulation.

Nanocarrier-aided drug delivery techniques have improved the absorption and permeability of drugs in nose-to-brain delivery. However, the molecular properties of nanocarriers during the delivery process are of great interest; in particular, the characteristics when penetrating barriers in vivo are crucial for the screening and optimization of materials for nasal inhalation. In this study, we have focused on two types of delivery systems: mucoadhesive nanoparticles (MAPs) and mucopenetrating nanoparticles (MPPs); both have been widely used for mucosal delivery, although a method for selecting the more effective type of drug carriers for mucosal delivery has not been established. Molecular dynamics (MD) simulations were used to reveal the all-atom dynamic characteristics of the interaction between different delivery systems and the nasal mucus protein MUC5AC. Among the systems tested, hydroxypropyltrimethyl ammonium chloride chitosan (HTCC) had the strongest interaction with mucin, suggesting it had better mucoadhesive performance, and that it interacted with MUC5AC more strongly than unmodified chitosan. In contrast, the mucus-penetrating material polyethylene glycol-poly lactic acid-co-glycolic acid (PEG-PLGA), had almost no interaction with MUC5AC. The results of the MD simulations were verified by in vitro experiments on nanoparticles (NPs) and mucin binding. The drug delivery performance of the four types of NPs, analyzed by in vitro and ex vivo mucosal penetration, were all generally consistent with the properties of the material predicted from the MD simulation. These clues to the molecular mechanism of MAPs and MPPs may provide useful insight into the screening and optimization of nanomaterials suitable for nasal inhalation.

Versatile modification of the CRISPR/Cas9 ribonucleoprotein system to facilitate in vivo application.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems has created a tremendous wave that is sweeping the world of genome editing. The ribonucleoprotein (RNP) method has evolved to be the most advantageous form for in vivo application. Modification of the CRISPR/Cas9 RNP method to adapt delivery through a variety of carriers can either directly improve the stability and specificity of the gene-editing tool in vivo or indirectly endow the system with high gene-editing efficiency that induces few off-target mutations through different delivery methods. The exploration of in vivo applications mediated by various delivery methods lays the foundation for genome research and variety improvements, which is especially promising for better in vivo research in the field of translational biomedicine. In this review, we illustrate the modifiable structures of the Cas9 nuclease and single guide RNA (sgRNA), summarize the latest research progress and discuss the feasibility and advantages of various methods. The highlighted results will enhance our knowledge, stimulate extensive research and application of Cas9 and provide alternatives for the development of rational delivery carriers in multiple fields.

Knockout of CD147 inhibits the proliferation, invasion, and drug resistance of human oral cancer CAL27 cells in Vitro and in Vivo.

With the development of modern biomedicine, research on the molecular mechanism of tumors has developed gradually. The CD147 gene has been applied to tumor molecular targeted therapy, and significant differences were found in the expression of the CD147 gene in different tumor tissues and normal tissues. Many previous studies have also shown that the expression of the CD147 gene plays a crucial role in the development of tumors. To understand whether CD147 can be used as a therapeutic target for oral cancer, CRISPR/Cas9 gene-editing technology was used to knock out the CD147 gene in cal27 cells to obtain knockout cell lines. Using CCK-8, Transwell, RT-PCR, and Western blotting, the proliferation and invasion abilities of the knockout cell lines were decreased significantly, and the expression of matrix metalloproteinase was also inhibited. Next, a subcutaneously transplanted tumor model in nude mice was constructed to detect the effect of the CD147 gene on tumors. Subcutaneous tumor growth and immunohistochemistry results showed that the proliferation and doxorubicin resistance of knockout cell line were significantly inhibited compared with those in the wild-type group. These results indicated that knocking out CD147 significantly reduced the proliferation and invasion of cal27 cells, and CD147 may be a potential therapeutic target for oral cancer.

Chitosan Hydrogel Doped with PEG-PLA Nanoparticles for the Local Delivery of miRNA-146a to Treat Allergic Rhinitis.

To prepare a binary formulation delivering miRNA-146 and evaluate a nucleic acid nasal delivery system by investigating its pharmacodynamic effects in allergic rhinitis. The gel/NPs/miR-146a thermosensitive in situ chitosan hydrogel carrying a nucleic acid was prepared and evaluated for its characteristics, including temperature sensitivity, gel strength, mucosal adhesion and drug release profile. After nasal administration of the formulation to ovalbumin-sensitized rats, the treatment of allergic rhinitis was verified by assessing nasal symptoms, hematology, hematoxylin-eosin (HE) staining and immunohistochemistry. Western Blot(WB) was used to analyze nasal inflammatory factors as well as miRNA-146-related factors, and the miR146 expression level was measured by PCR. Subsequently, the effects of the gel/NPs/miR-146a binary formulation were evaluated for the nasal delivery of nucleic acids in rhinitis therapy. The prepared binary formulation quickly formed a gel in the nasal cavity at a temperature of 34 °C with good mucosal adhesion, which delivered nucleic acids into the nasal mucosa stably and continuously. Gel/NPs/miR-146a was able to sustain the delivery of miRNA into the mucosa after nasal administration. When compared with the monolithic formulations, the gel/NPs/miR-146a binary formulation performed better regarding its nucleic acid delivery ability and pharmacodynamic effects. The gel/NPs/miR-146a binary preparation has a suitable nasal mucosal drug delivery ability and has a positive pharmacodynamic effect for the treatment of ovalbumin-induced rhinitis in rats. It can serve as a potential nucleic acid delivery platform for the treatment of allergic rhinitis.

Intranasal Delivery of Targeted Nanoparticles Loaded With miR-132 to Brain for the Treatment of Neurodegenerative Diseases.

Effective treatments for neurodegenerative diseases need to be developed. MiR132 is abundantly expressed in the brain, and it modulates neuron morphology and plays a key role in maintaining neuron survival. Regulating miR132 can effectively improve the symptoms of Alzheimer's disease. It can also reduce cell death after cerebral hemorrhage, improve the microenvironment of hematoma lesions and provide a certain protective effect from brain damage after cerebral ischemia. MiR132 has great potential in the treatment of cerebral ischemia and Alzheimer's disease. To prevent the decline of miR132 of miR132 levels in the blood, we used mouse and rat models of Alzheimer's disease with ischemic brain injury, and then delivered Wheat germ agglutinin (WGA)-NPs-miR132 intranasally to treat neurological damage after cerebral ischemia. Synaptic protein expression levels in Alzheimer's mouse models increased significantly after administration. We propose that, nasal delivery of WGA-NPs-miR132 is an interesting novel therapeutic approach for the treatment of neurodegenerative diseases.

Luteolin Protects Against CIRI, Potentially via Regulation of the SIRT3/AMPK/mTOR Signaling Pathway.

Mitochondrial abnormalities accelerate the progression of ischemic brain damage. Sirtuin 3 (SIRT3) is mainly found in mitochondria and affects almost all major aspects of mitochondrial function. Luteolin, a flavonoid with diverse biological properties, including antioxidant activity, inhibition of cell apoptosis and regulation of autophagy. It also modulates the activity of AMP activated kinase and/or sirtuin 1 (SIRT 1) by regulating the expression of sirtuins. We investigated the protective effects of luteolin on cerebral ischemia-reperfusion. It was found through experiments that luteolin reduced the infarcted area of MCAO rat model, and based on the experimental results, it was inferred that luteolin affected the AMPK, mTOR and SIRT3 pathways, thereby protecting brain cells. As expected, we found that luteolin can reduce the neurological function score, the degree of cerebral edema, the cerebral infarction volume, alleviate morphological changes in the cortex and hippocampus, increase neuron survival and decrease the number of apoptotic neurons. At the same time, luteolin significantly reduced the number of GFAP and Iba-1 positive glial cells in the hippocampus while enhanced the scavenging of oxygen free radicals and the activity of SOD in mitochondria. Addtionally, it can also enhance antioxidant capacity via the reversal of mitochondrial swelling and the mitochondrial transmembrane potential. Moreover, luteolin can increase SIRT3-targeted expression in mitochondria, decrease the phosphorylation of AMPK, and increase phosphor-mTOR (p-mTOR) levels, which may have occurred specifically through activation of the SIRT3/AMPK/mTOR pathway. We speculate that luteolin reduces the pathological progression of CIRI by increasing SIRT3 expression and enhancing mitochondrial function. Therefore, the results indicate that luteolin can increase the transduction of SIRT3, providing a potential mechanism for neuroprotective effects in patients with cerebral ischemia.

Co-delivery of brinzolamide and miRNA-124 by biodegradable nanoparticles as a strategy for glaucoma therapy.

Co-delivery nanoparticles with characteristics of intracellular precision release drug have been generally accepted as an effective therapeutic strategy for eye diseases. In this study, we designed a new co-delivery system (miRNA/NP-BRZ) as a lasting therapeutic approach to prevent the neuro-destructive after the long-term treatment of glaucoma. Neuroprotective and intraocular pressure (IOP) response were assessed in in vivo and in vitro models of glaucoma. At the meaning time, we describe the preparation of miRNA/NP-BRZ, drug release characteristics, intraocular tracing, pharmacokinetic and pharmacodynamics study and toxicity test. We found that miRNA/NP-BRZ could remarkably decrease IOP and significantly prevent retinal ganglion cell (RGC) damages. The new formula of miRNA-124 encapsulated in PEG-PSA-BRZ nanoparticles exhibits high encapsulation efficiency (EE), drug-loading capacity (DC), and stable controlled-release efficacy (EC). Moreover, we also verified that the miRNA/NP-BRZ system is significantly neuroprotective and nontoxic as well as lowering IOP. This study shows our co-delivery drug system would have a wide potential on social and economic benefits for glaucoma.

Overexpression of the NDR1/HIN1-Like Gene NHL6 Modifies Seed Germination in Response to Abscisic Acid and Abiotic Stresses in Arabidopsis.

NHL (NDR1/HIN1-like) genes play crucial roles in pathogen induced plant responses to biotic stress. Here, we report the possible function of NHL6 in plant response to abscisic acid (ABA) and abiotic stress. NHL6 was highly expressed in non-germinated seeds, and its expression was strongly induced by ABA and multiple abiotic stress signals. Loss-of-function of NHL6 decreased sensitivity to ABA in the early developmental stages including seed germination and post-germination seedling growth of the nhl6 mutants. However, overexpression of NHL6 increased sensitivity to ABA, salt and osmotic stress of the transgenic plants. Further studies indicated that the increased sensitivity in the 35S::NHL6 overexpressing plants could be a result of both ABA hypersensitivity and increased endogenous ABA accumulation under the stress conditions. It was also seen that the ABA-responsive element binding factors AREB1, AREB2 and ABF3 could regulate NHL6 expression at transcriptional level. Our results indicate that NHL6 plays an important role in the abiotic stresses-induced ABA signaling and biosynthesis, particularly during seed germination and early seedling development in Arabidopsis.

Poplar calcineurin B-like proteins PtCBL10A and PtCBL10B regulate shoot salt tolerance through interaction with PtSOS2 in the vacuolar membrane.

The calcineurin B-like protein (CBL) family represents a unique group of calcium sensors in plants. In Arabidopsis, CBL10 functions as a shoot-specific regulator in salt tolerance. We have identified two CBL10 homologs, PtCBL10A and PtCBL10B, from the poplar (Populus trichocarpa) genome. While PtCBL10A was ubiquitously expressed at low levels, PtCBL10B was preferentially expressed in the green-aerial tissues of poplar. Both PtCBL10A and PtCBL10B were targeted to the tonoplast and expression of either one in the Arabidopsis cbl10 mutant could rescue its shoot salt-sensitive phenotype. Like PtSOS3, both PtCBL10s physically interacted with the salt-tolerance component PtSOS2. But in contrast to the SOS3-SOS2 complex at the plasma membrane, the PtCBL10-SOS2 interaction was primarily associated with vacuolar compartments. Furthermore, overexpression of either PtCBL10A or PtCBL10B conferred salt tolerance on transgenic poplar plants by maintaining ion homeostasis in shoot tissues under salinity stress. These results not only suggest a crucial role of PtCBL10s in shoot responses to salt toxicity in poplar, but also provide a molecular basis for genetic engineering of salt-tolerant tree species.

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis.

Plant responses to developmental and environmental cues are often mediated by calcium (Ca(2+)) signals that are transmitted by diverse calcium sensors. The calcineurin B-like (CBL) protein family represents calcium sensors that decode calcium signals through specific interactions with a group of CBL-interacting protein kinases. We report functional analysis of Arabidopsis CBL2 and CBL3, two closely related CBL members that are localized to the vacuolar membrane through the N-terminal tonoplast-targeting sequence. While cbl2 or cbl3 single mutant did not show any phenotypic difference from the wild type, the cbl2 cbl3 double mutant was stunted with leaf tip necrosis, underdeveloped roots, shorter siliques and fewer seeds. These defects were reminiscent of those in the vha-a2 vha-a3 double mutant deficient in vacuolar H(+)-ATPase (V-ATPase). Indeed, the V-ATPase activity was reduced in the cbl2 cbl3 double mutant, connecting tonoplast CBL-type calcium sensors to the regulation of V-ATPase. Furthermore, cbl2 cbl3 double mutant was compromised in ionic tolerance and micronutrient accumulation, consistent with the defect in V-ATPase activity that has been shown to function in ion compartmentalization. Our results suggest that calcium sensors CBL2 and CBL3 serve as molecular links between calcium signaling and V-ATPase, a central regulator of intracellular ion homeostasis.

AtNHX3 is a vacuolar K+/H+ antiporter required for low-potassium tolerance in Arabidopsis thaliana.

Three vacuolar cation/H+ antiporters, AtNHX1 (At5g27150), 2 (At3g05030) and 5 (At1g54370), have been characterized as functional Na+/H+ antiporters in Arabidopsis. However, the physiological functions of AtNHX3 (At5g55470) still remain unclear. In this study, the physiological functions of AtNHX3 were studied using T-DNA insertion mutant and 35S-driven AtNHX3 over-expression Arabidopsis plants. RT-PCR analyses revealed that AtNHX3 is highly expressed in germinating seeds, flowers and siliques. Experiments with AtNHX3::YFP fusion protein in tobacco protoplasts indicated that AtNHX3 is mainly localized to vacuolar membrane, with a minor localization to pre-vacuolar compartments (PVCs) and endoplasmic reticulum (ER). Seedlings of null nhx3 mutants were hypersensitive to K+-deficient conditions. Expression of AtNHX3 complemented the sensitivity to K+ deficiency in nhx3 seedlings. Tonoplast vesicles isolated from transgenic plants over-expressing AtNHX3 displayed significantly higher K+/H+ exchange rates than those isolated from wild-type plants. Furthermore, nhx3 seeds accumulated less K+ and more Na+ when both wild-type and nhx3 were grown under normal growth condition. The overall results indicate that AtNHX3 encodes a K+/H+ antiporter required for low-potassium tolerance during germination and early seedling development, and may function in K+ utilization and ion homeostasis in Arabidopsis.

Overexpression of G10H and ORCA3 in the hairy roots of Catharanthus roseus improves catharanthine production.

A number of genes that function in the terpenoid indole alkaloids (TIAs) biosynthesis pathway have been identified in Catharanthus roseus. Except for the geraniol 10-hydroxylase (G10H) gene, which encodes a cytochrome P450 monooxygenase, several of these genes are up-regulated by ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcript factor. In this study, the G10H gene was transformed independently, or co-transformed with ORCA3 into C. roseus, using Agrobacterium rhizogenes MSU440. Hairy root clones expressing the G10H gene alone, or both the G10H and ORCA3 genes, were obtained. Alkaloid accumulation level analyses showed that all transgenic clones accumulated more catharanthine, with the highest accumulation level in the transgenic clone OG12 (6.5-fold higher than that of the non-expression clone). Following treatment with ABA, accumulation of catharanthine reached 1.96 mg/g DW in the transgenic clone OG12. The expression levels of TIAs biosynthesis genes in transgenic and non-transgenic clones were also investigated.

The Arabidopsis AP2/ERF transcription factor RAP2.6 participates in ABA, salt and osmotic stress responses.

AP2/ERF proteins play crucial roles in various biological processes. RAP2.6, an Arabidopsis AP2/ERF family member, has been reported to function in plant response to biotic stress, but whether it also functions in plant response to abiotic stress is not known. In this work, we demonstrate that in wild-type Arabidopsis, the expression of RAP2.6 is responsive to abscisic acid (ABA) and different stress conditions such as high salt, osmotic stress, and cold. Trans-activating ability tests in yeast demonstrate that RAP2.6 could act as a transactivator. RAP2.6 is able to bind to the GCC and CE1 cis-elements, as confirmed by both electrophoretic mobility shift assay (EMSA) and yeast one-hybrid assay. Experiments with RAP2.6-YFP fusion protein indicated that RAP2.6 is nuclear localized. Overexpression of RAP2.6 conferred hypersensitivity to exogenous ABA and abiotic stresses during seed germination and early seedling growth in Arabidopsis. The ABA content in RAP2.6 overexpressor lines decreased after being treated with salt. Furthermore, transcripts of AtABI4 and some stress inducible genes increased, and loss of ABI4 function rescues the hypersensitive phenotype of RAP2.6 overexpression lines under ABA and stress treatment. These results suggest that RAP2.6 participates in abiotic stress, possibly through the ABA-dependent pathway.

Fatty acid desaturase-6 (Fad6) is required for salt tolerance in Arabidopsis thaliana.

Fatty acid desaturases play important role in plant responses to abiotic stresses including cold, high temperature, drought, and osmotic stress. In this work, we provide the evidence that Fad6, a chloroplast desaturase, is required for salt tolerance during the early seedling development of Arabidopsis. Expression of Fad6 was responsive to salt and osmotic stress. Compared with the wild-type plants, the fad6 mutant showed reduced tolerance to salt stress, and accumulated more Na(+) and less K(+) under high NaCl stress condition. Furthermore, cellular oxidative damage was more severe in fad6 when treated with high concentrations of NaCl, as indicated by increased electrolyte leakage rate and malondialdehyde production, as well as by decreased activities of anti-oxidative enzymes. All these results suggest that Fad6 is required for salt resistance in Arabidopsis.

Knock-out of Arabidopsis AtNHX4 gene enhances tolerance to salt stress.

AtNHX4 belongs to the monovalent cation:proton antiporter-1 (CPA1) family in Arabidopsis. Several members of this family have been shown to be critical for plant responses to abiotic stress, but little is known on the biological functions of AtNHX4. Here, we provide the evidence that AtNHX4 plays important roles in Arabidopsis responses to salt stress. Expression of AtNHX4 was responsive to salt stress and abscisic acid. Experiments with CFP-AtNHX4 fusion protein indicated that AtNHX4 is vacuolar localized. The nhx4 mutant showed enhanced tolerance to salt stress, and lower Na(+) content under high NaCl stress compared with wild-type plants. Furthermore, heterologous expression of AtNHX4 in Escherichia coli BL21 rendered the transformants hypersensitive to NaCl. Deletion of the hydrophilic C-terminus of AtNHX4 dramatically increased the hypersensitivity of transformants, indicating that AtNHX4 may function in Na(+) homeostasis in plant cell, and its C-terminus plays a role in regulating the AtNHX4 activity.

Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene.

Nitrate reductase (NR) catalyzes NAD (P) H dependent reduction of nitrate to nitrite. Transformation systems have been established in several species of green algae by nitrate reductase gene functional complementation. In this report, an endogenous NR cDNA (3.4 kb) and a genomic fragment (14.6 kb) containing the NR gene (DvNIA1) were isolated from the D. viridis cDNA and genomic libraries respectively. Southern blot and Northern blot analyses showed that this gene exists as a single copy in D. viridis and is induced by nitrate. To obtain a NR defective mutant as a recipient strain, D. viridis cells were treated with a chemical mutagen and then cultured on a chlorate-containing plate to enrich chlorate tolerant mutants. Southern analysis showed that one isolate, B14, had a deletion in the DvNIA1 gene region. Using electroporation conditions determined in this laboratory, plasmid pDVNR containing the intact DvNIA1 gene has been electroporated into the defective mutant B14. Strains retaining a nitrate assimilation phenotype were obtained from nitrate plates after spreading the electroporated cells. In some individual strains, transcription of the introduced gene was detected. NR activity in these strains was slightly higher than that in the defective B14 cell, but excretion of nitrite into culture media was almost as high as that of the wild-type cell. Possible episomal presence of the introduced DNA in D. viridis is discussed.

Isolation and characterization of a sodium-dependent phosphate transporter gene in Dunaliella viridis.

A sodium-dependent phosphate transporter gene, DvSPT1, was isolated from a cDNA library using a probe derived from a subtracted cDNA library of Dunaliella viridis. Sequencing analyses revealed a cDNA sequence of 2649 bp long and encoded an open-reading frame consisting of 672 amino acids. The deduced amino acid sequence of DvSPT1 exhibited 31.2% identity to that of TcPHO from Tetraselmis chui. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa. Northern blot analysis indicated that the DvSPT1 expression was induced upon NaCl hyperosmotic stress or phosphate depletion. Functional characterization in yeast Na+ export pump mutant G19 suggested that DvSPT1 encoded a Na+ transporter protein. The gene sequence of GDvSPT1 (7922 bp) was isolated from a genomic library of D. viridis. Southern blot analysis indicated that there exist at least two homologous genes in D. viridis.

Expression of foreign genes in Dunaliella by electroporation.

An electroporation procedure has been described for introducing plasmid DNA into Dunaliella salina cells. By this procedure, a bulk of plasmid DNA was delivered into the cells and retained for at least 3 d. Reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing analyses indicated that the transcription and pre-mRNA splicing of ble gene (contributing the Zeocin resistance) were detected in the cells as early as 1 h after the electroporation. Individual colonies could retain the resistance to 10 mg/L Zeocin for at least 6 mo. Subsequent Southern blot analysis showed the existence of introduced plasmid DNA inside these colonies. However, most of the cells (approx 90%) lost the resistance in the presence of 5 mg/L Zeocin during subculturing, which was consistent with the observations of both rearranged and episomal plasmid DNA existed in the cells. Nevertheless, the electroporation procedure allows introducing a gene of interest and studying its expression and function in D. salina cells.