Improving ocular bioavailability of hydrophilic drugs through dynamic covalent complexation.

The clinical benefits of diquafosol tetrasodium (DQS), a hydrophilic P2Y2 receptor agonist for dry eye, have been hindered by a demanding dosing regimen. Nevertheless, it is challenging to achieve sustained release of DQS with conventional drug delivery vehicles which are mainly designed for hydrophobic small molecule drugs. To address this, we developed an affinity hydrogel for DQS by taking advantage of borate-mediated dynamic covalent complexation between DQS and hydroxypropyl guar. The resultant formulation (3% DQS Gel) was characterized by sustained release, low corneal permeation, and extended ocular retention, which were desirable attributes for ocular surface drug delivery. Both in vitro and in vivo studies had been carried out to verify the biocompatibility of 3% DQS Gel. Using corneal fluorescein staining, the Schirmer's test, PAS staining, quantitative PCR and immunohistological analyses as outcome measures, the superior therapeutic effects of 3% DQS Gel over PBS, the hydrogel vehicle and free DQS were demonstrated in a mouse dry eye model. Our DQS delivery strategy reported herein is readily applicable to other hydrophilic small molecule drugs with cis-diol moieties, thus providing a general solution to improve clinical outcomes of numerous diseases.

A topical fluorometholone nanoformulation fabricated under aqueous condition for the treatment of dry eye.

Fluorometholone (FMT) is a frequently prescribed drug for the alleviation of dry eye. However, due to low aqueous solubility, it has been routinely used as an ophthalmic suspension, which is characterized by low bioavailability, inconvenience of administration, and difficulty in delivering accurate dose. Furthermore, the opaque appearance of the ophthalmic suspension is not desirable for optical purpose. In the present study, a transparent FMT nanoformulation (FMT-CD NPs) was fabricated by the cyclodextrin (CD) nanoparticle technology without organic solvents. It was demonstrated that FMT was encapsulated in an amorphous form, which was associated with increased release rate and enhanced corneal penetration efficiency. The biocompatibility of FMT-CD NPs was confirmed by the Live/Dead assay, CCK-8 assay and the wound healing assay. Most importantly, FMT-CD NPs alleviated dry eye signs more efficiently than the commercial eye drop, with one-fifth the dosage of FMT in the latter. Collectively, our study provides a promising FMT formulation for improved management of dry eye while reducing drug related side effects.

Engineering Advanced Drug Delivery Systems for Dry Eye: A Review.

Dry eye disease (DED) is a widespread and frequently reported multifactorial ocular disease that not only causes ocular discomfort but also damages the cornea and conjunctiva. At present, topical administration is the most common treatment modality for DED. Due to the existence of multiple biological barriers, instilled drugs generally exhibit short action times and poor penetration on the ocular surface. To resolve these issues, several advanced drug delivery systems have been proposed. This review discusses new dosage forms of drugs for the treatment of DED in terms of their characteristics and advantages. Innovative formulations that are currently available in the market and under clinical investigation are elaborated. Meanwhile, their deficiencies are discussed. It is envisioned that the flourishing of advanced drug delivery systems will lead to improved management of DED in the near future.

Artemether Attenuates Aß25-35-Induced Cognitive Impairments by Downregulating Aß, BACE1, mTOR and Tau Proteins.

BACKGROUND: Alzheimer's disease (AD) is clinically characterized as a progressive cognitive impairment and behavioral disorder. Pathological hallmarks of AD include extracellular senile plaques (SPs), intracellular neurofibrillary tangles (NFTs) and massive neuronal loss. Although the exact cause of AD is not well understood, a mounting body of evidence has demonstrated that the pathogenesis of AD is associated with oxidative stress, neu-roinflammation, and amyloid beta (Aß) induced neural apoptosis. Moreover, overexpression of ß-secretase 1 (BACE1), Aß, mammalian target of rapamycin (mTOR), and Tau proteins are closely related to cognitive symptoms in AD. Studies have demonstrated that artemether, an antimalarial drug with acceptable side effects, possesses protective effects against neuroinflammation and oxidative stress. Importantly, artemether can easily penetrate the blood brain barrier, thereby representing an ideal drug candidate for AD treatment. METHODS: The effect of artemether on memory protection and the associated molecular mechanisms were investigated in an Aß25-35 induced cognitive impairments rat model. RESULTS: Results of the in vivo study showed that oral administration of artemether significantly attenuated Aß25-35-induced cognitive impairment in rats. Results of the in vitro study revealed that artemether significantly downregulated the endogenous expression of Aß, BACE1, mTOR, and Tau proteins in N2a cells. CONCLUSIONS: The beneficial effect of artemether against Aß 25-35-induced cognitive impairments was attributable to the downregulation of the expression of Aß, BACE1, mTOR, and Tau proteins, suggesting the potential of artemether as an effective, neuronal protective, and multi-targeted drug candidate for AD treatment.

Effects and mechanism of microRNA­218 against lung cancer.

Lung cancer is the most prevalent and observed type of cancer in Xuanwei County, Yunnan, South China. Lung cancer in this area is called Xuanwei lung cancer. However, its pathogenesis remains largely unknown. To date, a number of studies have shown that microRNA (miR)­218 functions as a tumor suppressor in multiple types of cancer. However, the role of miR­218 and its regulatory gene network in Xuanwei lung cancer have yet to be investigated. The current study identified that the expression levels of miR­218 in XWLC­05 cells were markedly lower compared with those in immortalized lung epithelial BEAS­2B cells. The present study also demonstrated that overexpression of miR­218 could decrease cell proliferation, invasion, viability and migration in Xuanwei lung cancer cell line XWLC­05 and NSCLC cell line NCI­H157. Additionally, the results revealed that overexpression of miR­218 could induce XWLC­05 and NCI­H157 cell apoptosis by arresting the cell cycle at G2/M phase. Finally, the present study demonstrated that overexpression of miR­218 could lead to a significant increase in phosphatase and tensin homolog (PTEN) and YY1 transcription factor (YY1), and a decrease in B­cell lymphoma 2 (BCL­2) and BMI1 proto­oncogene, polycomb ring finger (BMI­1) at the mRNA and protein level in XWLC­05 and NCI­H157 cell lines. However, we did not observe any remarkable difference in the roles of miR­218 and miR­218­mediated regulation of BCL­2, BMI­1, PTEN and YY1 expression in the progression of Xuanwei lung cancer. In conclusion, miR­218 could simultaneously suppress cell proliferation and tumor invasiveness and induce cell apoptosis by increasing PTEN and YY1 expression, while decreasing BCL­2 and BMI­1 in Xuanwei lung cancer. The results demonstrated that miR­218 might serve a vital role in tumorigenesis and progression of Xuanwei lung cancer and overexpression of miR­218 may be a novel approach for the treatment of Xuanwei lung cancer.

Assessment of high sensitivity C-reactive protein and coronary plaque characteristics by computed tomography in patients with and without diabetes mellitus.

BACKGROUND: To evaluate the coronary plaque characteristics of coronary arteries using computed tomography angiography (CTA) in order to assess the risk of coronary artery disease and the relevance of high sensitivity C reactive protein (hs-CRP) in patients with Diabetes Mellitus (DM). METHODS: The clinical data of 400 DM patients and 400 non-DM patients from January 2017 to December 2019 were collected, including the results of coronaryCTA. The plasma hs-CRP level of the two groups were divided into three groups: CRP ≤ 1, 1 < CRP ≤ 2, CRP > 2. The correlation of the degree of stenosis, the number of plaques, the nature of plaques and hs-CRP value between the two groups was evaluated. RESULTS: Compared with non-DM patients, the incidence of coronary artery plaques and lumen stenosis in DM patients was more higher than that in non-DM patients. DM patients were more likely to have more diseased vessels, especially diffuse vascular disease (12.00% vs 1.75%; P < 0.001). Subjects with high hs-CRP levels were more likely to have any plaque compared with individuals showing normal hs-CRP levels (p<0.01). There was no statistical significance in non calcified plaque with high level of hs-CRP, but the occurrence of plaque types in DM group was statistically significant compared with other hs-CRP levels in non DM group. Subjects with high hs-CRP were observed to be at increased risk for the presence of calcified plaque and severe narrowing in the unadjusted values. CONCLUSIONS: Coronary CTA combined with hs-CRP can accurately detect the characteristics of coronary artery stenosis and plaque in DM patients, which has an important clinical value in the risk assessment of coronary heart disease in DM patients.

Identification and characterization of long non-coding RNAs involved in osmotic and salt stress in Medicago truncatula using genome-wide high-throughput sequencing.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been shown to play crucially regulatory roles in diverse biological processes involving complex mechanisms. However, information regarding the number, sequences, characteristics and potential functions of lncRNAs in plants is so far overly limited. RESULTS: Using high-throughput sequencing and bioinformatics analysis, we identified a total of 23,324 putative lncRNAs from control, osmotic stress- and salt stress-treated leaf and root samples of Medicago truncatula, a model legume species. Out of these lncRNAs, 7,863 and 5,561 lncRNAs were identified from osmotic stress-treated leaf and root samples, respectively. While, 7,361 and 7,874 lncRNAs were identified from salt stress-treated leaf and root samples, respectively. To reveal their potential functions, we analyzed Gene Ontology (GO) terms of genes that overlap with or are neighbors of the stress-responsive lncRNAs. Enrichments in GO terms in biological processes such as signal transduction, energy synthesis, molecule metabolism, detoxification, transcription and translation were found. CONCLUSIONS: LncRNAs are likely involved in regulating plant's responses and adaptation to osmotic and salt stresses in complex regulatory networks with protein-coding genes. These findings are of importance for our understanding of the potential roles of lncRNAs in responses of plants in general and M. truncatula in particular to abiotic stresses.

Genome variations account for different response to three mineral elements between Medicago truncatula ecotypes Jemalong A17 and R108.

BACKGROUND: Resequencing can be used to identify genome variations underpinning many morphological and physiological phenotypes. Legume model plant Medicago truncatula ecotypes Jemalong A17 (J. A17) and R108 differ in their responses to mineral toxicity of aluminum and sodium, and mineral deficiency of iron in growth medium. The difference may result from their genome variations, but no experimental evidence supports this hypothesis. RESULTS: A total of 12,750 structure variations, 135,045 short insertions/deletions and 764,154 single nucleotide polymorphisms were identified by resequencing the genome of R108. The suppressed expression of MtAACT that encodes a putative aluminum-induced citrate efflux transporter by deletion of partial sequence of the second intron may account for the less aluminum-induced citrate exudation and greater accumulation of aluminum in roots of R108 than in roots of J. A17, thus rendering R108 more sensitive to aluminum toxicity. The higher expression-level of MtZpt2-1 encoding a TFIIIA-related transcription factor in J. A17 than R108 under conditions of salt stress can be explained by the greater number of stress-responsive elements in its promoter sequence, thus conferring J. A17 more tolerant to salt stress than R108 plants by activating the expression of downstream stress-responsive genes. YSLs (Yellow Stripe-Likes) are involved in long-distance transport of iron in plants. We found that an YSL gene was deleted in the genome of R108 plants, thus rendering R108 less tolerance to iron deficiency than J. A17 plants. CONCLUSIONS: The deletion or change in several genes may account for the different responses of M. truncatula ecotypes J. A17 and R108 to mineral toxicity of aluminum and sodium as well as iron deficiency. Uncovering genome variations by resequencing is an effective method to identify different traits between species/ecotypes that are genetically related. These findings demonstrate that analyses of genome variations by resequencing can shed important light on differences in responses of M. truncatula ecotypes to abiotic stress in general and mineral stress in particular.

Genome-wide identification of microRNAs in Medicago truncatula by high-throughput sequencing.

MicroRNAs (miRNAs) are small, endogenous RNAs that play important regulatory roles in development and stress response in plants by negatively regulating gene expression post-transcriptionally. Medicago truncatula has been used as a model plant to study functional genomics of legume plants. It has also been widely used to functionally study miRNAs. Identification of miRNAs at the whole-genome level is essential for functional characterization of miRNAs in plants. High-throughput sequencing is a powerful technology to identify miRNAs. In this chapter, the methods used for construction of a small RNA library and high-throughput sequencing involving total RNA isolation, small RNA purification, adapter ligation, reverse transcription, PCR amplification, and Solexa sequencing are described. Bioinformatics and analysis of differential expression of miRNAs including primary disposal, miRNA identification, target prediction, and expression analysis are also discussed. These methodologies associated with identification and functional characterization of miRNAs may provide useful tools for readers to study miRNAs in plants in general and Medicago truncatula in particular.

A Medicago truncatula EF-hand family gene, MtCaMP1, is involved in drought and salt stress tolerance.

BACKGROUND: Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize genes of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic Arabidopsis seedlings expressing MtCaMP1 exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na(+) in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H2O2 and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress. CONCLUSIONS/SIGNIFICANCE: The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na(+) accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na(+) under drought and salt stress would protect plants from water default and Na(+) toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.

Flixweed is more competitive than winter wheat under ozone pollution: evidences from membrane lipid peroxidation, antioxidant enzymes and biomass.

To investigate the effects of ozone on winter wheat and flixweed under competition, two species were exposed to ambient, elevated and high [O3] for 30 days, planted singly or in mixculture. Eco-physiological responses were examined at different [O3] and fumigating time. Ozone reduced the contents of chlorophyll, increased the accumulation of H2O2 and malondialdehyde in both wheat and flixweed. The effects of competition on chlorophyll content of wheat emerged at elevated and high [O3], while that of flixweed emerged only at high [O3]. The increase of H2O2 and malondialdehyde of flixweed was less than that of wheat under the same condition. Antioxidant enzyme activities of wheat and flixweed were seriously depressed by perennial and serious treatment using O3. However, short-term and moderate fumigation increased the activities of SOD and POD of wheat, and CAT of flixweed. The expression levels of antioxidant enzymes related genes provided explanation for these results. Furthermore, the increase of CAT expression of flixweed was much higher than that of SOD and POD expression of wheat. Ozone and competition resulted in significant reductions in biomass and grain yield in both winter wheat and flixweed. However, the negative effects on flixweed were less than wheat. Our results demonstrated that winter wheat is more sensitive to O3 and competition than flixweed, providing valuable data for further investigation on responses of winter wheat to ozone pollution, in particular combined with species competition.

Expression of a Medicago falcata small GTPase gene, MfARL1 enhanced tolerance to salt stress in Arabidopsis thaliana.

To understand the role of small GTPases in response to abiotic stress, we isolated a gene encoding a small GTPase, designated MfARL1, from a subtracted cDNA library in Medicago falcata, a native legume species in semi-arid grassland in northern China. The function of MfARL1 in response to salt stress was studied by expressing MfARL1 in Arabidopsis. Wild-type (WT) and transgenic plants constitutively expressing MfARL1 showed comparable phenotype when grown under control conditions. Germination of seeds expressing MfARL1 was less suppressed by salt stress than that of WT seeds. Transgenic seedlings had higher survival rate than WT seedlings under salt stress, suggesting that expression of MfARL1 confers tolerance to salt stress. The physiological and molecular mechanisms underlying these phenomena were elucidated. Salt stress led to a significant decrease in chlorophyll contents in WT plants, but not in transgenic plants. Transgenic plants accumulated less amounts of H(2)O(2) and malondialdehyde than their WT counterparts under salt stress, which can be accounted for by the higher catalase activities, lower activities of superoxide dismutase, and peroxidase in transgenic plants than in WT plants. Transgenic plants displayed lower Na(+)/K(+) ratio due to less accumulation of Na(+) than wild-type under salt stress conditions. The lower Na(+)/K(+) ratio may result from less accumulation of Na(+) due to reduced expression of AtHKT1 that encodes Na(+) transporter in transgenic plants under salt stress. These findings demonstrate that MfARL1 encodes a novel stress-responsive small GTPase that is involved in tolerance to salt stress.