International Journal of Molecular Sciences — Open Access Journal

Rebamipide ophthalmic solution is a mucin secretagogue which is an important therapeutic agent in the treatment of dry eye. It has been noted that dry eye in office workers is associated with a decrease in secretory mucin. This study aimed to evaluate the effects of 2% rebamipide ophthalmic solution in mice subjected to environmental dry eye stress (EDES), which mimics the conditions of office workers. Thirty eyes from thirty BALB/c mice (eight-week-old males) were divided into three treatment groups: artificial tear (vehicle), 2% rebamipide ophthalmic solution, and 0.1% hyaluronic acid (HA) ophthalmic solution. After four days of pretreatment, mice were exposed to EDES for three days. The corneal subbasal nerve and inflammatory cells were then examined using in vivo confocal microscopy. Following EDES exposure, the lissamine green staining score was significantly lower and corneal sensitivity was more preserved in the 2% rebamipide group than in the HA group. In addition, the subbasal nerve fiber density was significantly higher and the DC density was significantly lower in the 2% rebamipide group than in the HA group. Overall, the topical rebamipide ophthalmic solution showed more favorable therapeutic effects when compared to the HA ophthalmic solution in a mouse model of EDES, likely owing to its anti-inflammatory and neuroprotective effects. Full article

Eucommia ulmoides Oliver is widely distributed in China. This species has been used mainly in medicine due to the high concentration of chlorogenic acid (CGA), flavonoids, lignans, and other compounds in the leaves and barks. However, the categories of metabolites, dynamic changes in metabolite accumulation and overall molecular mechanisms involved in metabolite biosynthesis during E. ulmoides leaf growth and development remain unknown. Here, a total of 515 analytes, including 127 flavonoids, 46 organic acids, 44 amino acid derivatives, 9 phenolamides, and 16 vitamins, were identified from four E. ulmoides samples using ultraperformance liquid chromatography–mass spectrometry (UPLC-MS) (for widely targeted metabolites). The accumulation of most flavonoids peaked in growing leaves, followed by old leaves. UPLC-MS analysis indicated that CGA accumulation increased steadily to a high concentration during leaf growth and development, and rutin showed a high accumulation level in leaf buds and growing leaves. Based on single-molecule long-read sequencing technology, 69,020 transcripts and 2880 novel loci were identified in E. ulmoides. Expression analysis indicated that isoforms in the flavonoid biosynthetic pathway and flavonoid metabolic pathway were highly expressed in growing leaves and old leaves. Co-expression network analysis suggested a potential direct link between the flavonoid and phenylpropanoid biosynthetic pathways via the regulation of transcription factors, including MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic/helix-loop-helix). Our study predicts dynamic metabolic models during leaf growth and development and will support further molecular biological studies of metabolite biosynthesis in E. ulmoides. In addition, our results significantly improve the annotation of the E. ulmoides genome. Full article

Primary open-angle glaucoma (POAG) represents the leading cause of irreversible blindness worldwide and is a multifactorial, chronic neurodegenerative disease characterized by retinal ganglion cell and visual field loss. There are many factors that are associated with the risk of developing POAG, with increased intraocular pressure being one of the most prevalent. Due to the asymptomatic nature of the disease, the diagnosis of POAG often occurs too late, which necessitates development of new effective screening strategies for early diagnosis of the disease. However, this task still remains unfulfilled. In order to provide further insights into the pathophysiology of POAG, we applied a targeted metabolomics strategy based on a high-throughput screening method for the determination of tear amino acids, free carnitine, acylcarnitines, succinylacetone, nucleosides, and lysophospholipids in naïve to therapy glaucomatous patients and normal controls. Also, we conducted proteomic analyses of the whole lacrimal fluid and purified extracellular vesicles obtained from POAG patients and healthy subjects. This multi-omics approach allowed us to conclude that POAG patients had lower levels of certain tear amino acids and lysophospholipids compared with controls. These targeted analyses also highlighted the low amount of acetylcarnitine (C2) in POAG patient which correlated well with proteomics data. Moreover, POAG tear proteins seemed to derive from extracellular vesicles, which carried a specific pro-inflammatory protein cargo. Full article

The high invasive phenotype of glioblastoma is one of the main causes of therapy inefficacy and tumor relapse. Cell adhesion molecules of the cadherin family are involved in cell migration and are known as master regulators of epithelial tumor invasiveness, but their role in glioblastoma is less understood. In particular, we recently demonstrated, in the syngeneic murine model, the occurrence of a previously undescribed cadherin switch between Cdh2 and Cdh4 during gliomagenesis, which is necessary for the acquisition of the highly infiltrative and tumorigenic phenotype of these cells. In the present study, we tested the role of Cdh4 in human gliomas. Our results on patient-derived glioma cells demonstrate a positive correlation between Cdh4 expression levels and the loss of cell–cell contact inhibition of proliferation controls that allows cells to proliferate over confluence. Moreover, the silencing of Cdh4 by artificial microRNAs induced a decrease in the infiltrative ability of human glioma cells both in vitro and in vivo. More strikingly, Cdh4 silencing induced an impairment of the tumorigenic potential of these cells after orthotopic transplantation in immunodeficient mice. Overall, we conclude that in human glioblastoma, Cdh4 can also actively contribute in regulating cell invasiveness and malignancy. Full article

Titanium micro-scale topography offers excellent osteoconductivity and bone–implant integration. However, the biological effects of sub-micron topography are unknown. We compared osteoblastic phenotypes and in vivo bone and implant integration abilities between titanium surfaces with micro- (1–5 µm) and sub-micro-scale (0.1–0.5 µm) compartmental structures and machined titanium. The calculated average roughness was 12.5 ± 0.65, 123 ± 6.15, and 24 ± 1.2 nm for machined, micro-rough, and sub-micro-rough surfaces, respectively. In culture studies using bone marrow-derived osteoblasts, the micro-rough surface showed the lowest proliferation and fewest cells attaching during the initial stage. Calcium deposition and expression of osteoblastic genes were highest on the sub-micro-rough surface. The bone–implant integration in the Sprague–Dawley male rat femur model was the strongest on the micro-rough surface. Thus, the biological effects of titanium surfaces are not necessarily proportional to the degree of roughness in osteoblastic cultures or in vivo. Sub-micro-rough titanium ameliorates the disadvantage of micro-rough titanium by restoring cell attachment and proliferation. However, bone integration and the ability to retain cells are compromised due to its lower interfacial mechanical locking. This is the first report on sub-micron topography on a titanium surface promoting osteoblast function with minimal osseointegration. Full article

A kinetic study was carried out on the solvolysis of o-nitrobenzyl bromide (o-isomer, 1) and p-nitrobenzyl bromide (p-isomer, 3), and o-nitrobenzoyl chloride (o-isomer, 2) in a wide range of solvents under various temperatures. In all of the solvents without aqueous fluoroalcohol, the reactions of 1 were solvolyzed at a similar rate to those observed for 3, and the reaction rates of 2 were about ten times slower than those of the previously studied p-nitrobenzoyl chloride (p-isomer, 4). For solvolysis in aqueous fluoroalcohol, the reactivity of 2 was kinetically more reactive than 4. The l/m values of the extended Grunwald–Winstein (G–W) equation for solvolysis of 1 and 2 in solvents without fluoroalcohol content are all significantly larger than unity while those in all the fluoroalcohol solvents are less than unity. The role of the ortho-nitro group as an intramolecular nucleophilic assistant (internal nucleophile) in the solvolytic reaction of 1 and 2 was discussed. The results are also compared with those reported earlier for o-carbomethoxybenzyl bromide (5) and o-nitrobenzyl p-toluenesulfonate (7). From the product studies and the activation parameters for solvolyses of 1 and 2 in several organic hydroxylic solvents, mechanistic conclusions are drawn. Full article

Heart failure (HF) is a rapidly growing global public health problem. Since HF results in high mortality and re-hospitalization, new effective treatments are desired. Although it remains controversial, omega 3 polyunsaturated fatty acids (n-3 PUFAs), such as the eicosapentaenoic acid and docosahexaenoic acid, have been widely recognized to have benefits for HF. In a large-scale clinical trial regarding secondary prevention of HF by n-3 PUFA (GISSI-HF trial), the supplementation of n-3 PUFA significantly reduced cardiovascular mortality and hospitalization. Other small clinical studies proposed that n-3 PUFA potentially suppresses the ventricular remodeling and myocardial fibrosis, which thereby improves the ventricular systolic and diastolic function both in ischemic and non-ischemic HF. Basic investigations have further supported our understanding regarding the cardioprotective mechanisms of n-3 PUFA against HF. In these reports, n-3 PUFA has protected hearts through (1) anti-inflammatory effects, (2) intervention of cardiac energy metabolism, (3) modification of cardiac ion channels, (4) improvement of vascular endothelial response, and (5) modulation of autonomic nervous system activity. To clarify the pros and cons of n-3 PUFA on HF, we summarized recent evidence regarding the beneficial effects of n-3 PUFA on HF both from the clinical and basic studies. Full article

The culture environment plays an important role for stem cells’ cultivation. Static or dynamic culture preserve differential potentials to affect human mesenchymal stem cells’ (hMSCs) proliferation and differentiation. In this study, hMSCs were seeded on fiber disks and cultured in a bidirectional-flow bioreactor or spinner-flask bioreactor with a supplement of osteogenic medium. The hMSCs’ proliferation, osteogenic differentiation, and extracellular matrix deposition of mineralization were demonstrated. The results showed that the spinner flask improved cell viability at the first two weeks while the bidirectional-flow reactor increased the cell proliferation of hMSCs through the four-week culture period. Despite the flow reactor having a higher cell number, a lower lactose/glucose ratio was noted, revealing that the bidirectional-flow bioreactor provides better oxygen accessibility to the cultured cells/disk construct. The changes of calcium ions in the medium, the depositions of Ca²⁺ in the cells/disk constructs, and alkaline phosphate/osteocalcin activities showed the static culture of hMSCs caused cells to mineralize faster than the other two bioreactors but without cell proliferation. Otherwise, cells were distributed uniformly with abundant extracellular matrix productions using the flow reactor. This reveals that the static and dynamic cultivations regulated the osteogenic process differently in hMSCs. The bidirectional-flow bioreactor can be used in the mass production and cultivation of hMSCs for applications in bone regenerative medicine. Full article

There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast Hansenula polymorpha, lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance. Also, in cells of mutants without the peroxisome inheritance protein Inp2 peroxisome segregation can still occur. In contrast, peroxisome fission and inheritance were not observed in cells of a pex11 inp2 double deletion strain. In buds of cells of this double mutant, new organelles likely appear de novo. Growth of pex11 inp2 cells on methanol, a growth substrate that requires functional peroxisomes, is retarded relative to the wild type control. Based on these observations we conclude that in H. polymorpha de novo peroxisome formation is a rescue mechanism, which is less efficient than organelle fission and inheritance to maintain functional peroxisomes. Full article

The regulation of melatonin secretion in the avian pineal organ is highly complex and shows prominent interspecies differences. The aim of this study was to determine the roles of direct photoreception and the internal oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. The pineal organs were collected from 12-, 13- and 14-week-old female turkeys reared under a 12 L:12 D cycle with the photophase from 07.00 to 19.00, and were incubated in superfusion culture for 3–6 days. The cultures were subjected to different light conditions including 12 L:12 D cycles with photophases between 07.00 and 19.00, 13.00 and 01.00 or 01.00 and 13.00, a reversed cycle 12 D:12 L, cycles with long (16 L:8 D) and short (8 L:16 D) photophases, and continuous darkness or illumination. The pineal organs were also exposed to light pulses of variable duration during incubation in darkness or to periods of darkness during the photophase. The secretion of melatonin was determined by direct radioimmunoassay. The turkey pineal organs secreted melatonin in a well-entrained diurnal rhythm with a very high amplitude. Direct photoreception as an independently acting mechanism was able to ensure quick and precise adaptation of the melatonin secretion rhythm to changes in light-dark conditions. The pineal organs secreted melatonin in circadian rhythms during incubation in continuous darkness or illumination. The endogenous oscillator of turkey pinealocytes was able to acquire and store information about the light-dark cycle and then to generate the circadian rhythm of melatonin secretion in continuous darkness according to the stored data. The obtained data suggest that the turkey pineal gland is highly autonomous in the generation and regulation of the melatonin secretion rhythm. They also demonstrate that the turkey pineal organ in superfusion culture is a valuable model for chronobiological studies, providing a highly precise clock and calendar. This system has several features which make it an attractive alternative to other avian pineal glands for circadian studies. Full article

Pneumonitis is the leading cause of death associated with the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) against non-small cell lung cancer (NSCLC). However, the risk factors and the mechanism underlying this toxicity have not been elucidated. Tumor necrosis factor (TNF) has been reported to transactivate EGFR in pulmonary epithelial cells. Hence, we aimed to test the hypothesis that EGFR tyrosine kinase activity regulates TNF-mediated bronchial epithelial cell survival, and that inhibition of EGFR activity increases TNF-induced lung epithelial cell apoptosis. We used surfactant protein C (SPC)-TNF transgenic (tg) mice which overexpress TNF in the lungs. In this model, gefitinib, an EGFR-TKI, enhanced lung epithelial cell apoptosis and lymphocytic inflammation, indicating that EGFR tyrosine kinase prevents TNF-induced lung injury. Furthermore, IL-17A was significantly upregulated by gefitinib in SPC-TNF tg mice and p38MAPK activation was observed, indicative of a pathway involved in lung epithelial cell apoptosis. Moreover, in lung epithelial cells, BEAS-2B, TNF stimulated EGFR transactivation via the TNF-α-converting enzyme in a manner that requires heparin binding (HB)-EGF and transforming growth factor (TGF)-α. These novel findings have significant implications in understanding the role of EGFR in maintaining human bronchial epithelial cell homeostasis and in NSCLC treatment. Full article

A de novo single-nucleotide mutation in the EGFR gene can cause the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are used for clinical treatment of such lung cancers, but acquired resistance often mitigates their efficacy. Accordingly, monitoring of de novo and acquired nucleotide mutations is essential for clinical treatment of lung cancers with EGFR-TKIs. Previously, we reported that oligoribonucleotide interference-PCR (ORNi-PCR) can accurately and cost-effectively detect single-nucleotide mutations. In this study, we applied ORNi-PCR to simultaneous detection of the de novo L858R and acquired T790M mutations in the EGFR gene in lung cancer cells. First, we established optimal experimental conditions for ORNi-PCR to simultaneously detect the two single-nucleotide mutations in genomic DNA from lung cancer cells. The conditions we established could also be used for ORNi-PCR using complementary DNA reverse-transcribed from extracted RNA. We found that ORNi-PCR could detect lung cancer cells possessing both single-nucleotide mutations among a large number of cells harboring wild-type sequences, even when the cancer cells constituted less than ~0.2% of all cells. Our findings demonstrate that ORNi-PCR can simultaneously detect multiple single-nucleotide mutations in a gene of interest and might therefore be useful for simultaneous detection of EGFR mutations in clinical examinations. Full article

Modulated electrohyperthermia (mEHT), an innovative complementary technique of radio-, chemo-, and targeted oncotherapy modalities, can induce tumor apoptosis and contribute to a secondary immune-mediated cancer death. Here, we tested the efficiency of high-fever range (~42 °C) mEHT on B16F10 melanoma both in cell culture and allograft models. In vivo, mEHT treatment resulted in significant tumor size reduction when repeated three times, and induced major stress response as indicated by upregulated cytoplasmic and cell membrane hsp70 levels. Despite the increased PUMA and apoptosis-inducing factor 1, and moderate rise in activated-caspase-3, apoptosis was not significant. However, phospho-H2AX indicated DNA double-strand breaks, which upregulated p53 protein and its downstream cyclin-dependent kinase inhibitors p21^waf1 and p27^kip. Combined in vitro treatment with mEHT and the p53 activator nutlin-3a additively reduced cell viability compared to monotherapies. Though mEHT promoted the release of damage-associated molecular pattern (DAMP) damage signaling molecules hsp70, HMGB1 and ATP to potentiate the tumor immunogenicity of melanoma allografts, it reduced MHC-I and melan-A levels in tumor cells. This might explain why the number of cytotoxic T cells was moderately reduced, while the amount of natural killer (NK) cells was mainly unchanged and only macrophages increased significantly. Our results suggest that mEHT-treatment-related tumor growth control was primarily mediated by cell-stress-induced p53, which upregulated cyclin-dependent kinase inhibitors. The downregulated tumor antigen-presenting machinery may explain the reduced cytotoxic T-cell response despite increased DAMP signaling. Decreased tumor antigen and MHC-I levels suggest that natural killer (NK) cells and macrophages were the major contributors to tumor eradication. Full article

In this communication, lipase A from Candida antarctica (CALA) was immobilized by covalent bonding on magnetic nanoparticles coated with chitosan and activated with glutaraldehyde, labelled CALA-MNP, (immobilization parameters: 84.1% ± 1.0 for immobilization yield and 208.0 ± 3.0 U/g ± 1.1 for derivative activity). CALA-MNP biocatalyst was characterized by X-ray Powder Diffraction (XRPD), Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetry (TG) and Scanning Electron Microscope (SEM), proving the incorporation of magnetite and the immobilization of CALA in the chitosan matrix. Besides, the immobilized biocatalyst showed a half-life 8–11 times higher than that of the soluble enzyme at pH 5–9. CALA showed the highest activity at pH 7, while CALA-MNP presented the highest activity at pH 10. The immobilized enzyme was more active than the free enzyme at all studied pH values, except pH 7. Full article

This review, one in a series on myotonic dystrophy (DM), is focused on the development and potential use of small molecules as therapeutics for DM. The complex mechanisms and pathogenesis of DM are covered in the associated reviews. Here, we examine the various small molecule approaches taken to target the DNA, RNA, and proteins that contribute to disease onset and progression in myotonic dystrophy type 1 (DM1) and 2 (DM2). Full article

The tight junction (TJ) is an intercellular sealing component found in epithelial and endothelial tissues that regulates the passage of solutes across the paracellular space. Research examining the biology of TJs has revealed that they are complex biochemical structures constructed from a range of proteins including claudins, occludin, tricellulin, angulins and junctional adhesion molecules. The transient disruption of the barrier function of TJs to open the paracellular space is one means of enhancing mucosal and transdermal drug absorption and to deliver drugs across the blood–brain barrier. However, the disruption of TJs can also open the paracellular space to harmful xenobiotics and pathogens. To address this issue, the strategies targeting TJ proteins have been developed to loosen TJs in a size- or tissue-dependent manner rather than to disrupt them. As several TJ proteins are overexpressed in malignant tumors and in the inflamed intestinal tract, and are present in cells and epithelia conjoined with the mucosa-associated lymphoid immune tissue, these TJ-protein-targeted strategies may also provide platforms for the development of novel therapies and vaccines. Here, this paper reviews two TJ-protein-targeted technologies, claudin binders and an angulin binder, and their applications in drug development. Full article

Microglial activation and the resulting neuroinflammation are associated with a variety of brain diseases, such as Alzheimer’s disease and Parkinson’s disease. Thus, the control of microglial activation is an important factor in the development of drugs that can treat or prevent inflammation-related neurodegenerative disorders. Atractylodis Rhizoma Alba (ARA) has been reported to exhibit antioxidant, gastroprotective, and anti-inflammatory effects. However, the effects of ARA ethanolic extract (ARAE) on microglia-mediated neuroinflammation have not been fully elucidated. In this work, we explored the anti-neuroinflammatory properties and underlying molecular mechanisms of ARAE in lipopolysaccharide (LPS)-stimulated microglial BV2 cells. Our results showed that ARAE significantly attenuates the production of nitric oxide (NO) and inflammatory cytokines induced by LPS. ARAE treatment also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 without causing cytotoxicity. ARAE markedly attenuated the transcriptional activities of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPK) phosphorylation, and induced heme oxygenase (HO)-1 expression. High-performance liquid chromatography (HPLC) analysis showed that ARAE contains three main components—atractylenolide I, atractylenolide III, and atractylodin—all compounds that significantly inhibit the production of inflammatory factors. These findings indicate that ARAE may be a potential therapeutic agent for the treatment of inflammation-related neurodegenerative diseases. Full article

TASK-3 is a two-pore domain potassium (K_2P) channel highly expressed in the hippocampus, cerebellum, and cortex. TASK-3 has been identified as an oncogenic potassium channel and it is overexpressed in different cancer types. For this reason, the development of new TASK-3 blockers could influence the pharmacological treatment of cancer and several neurological conditions. In the present work, we searched for novel TASK-3 blockers by using a virtual screening protocol that includes pharmacophore modeling, molecular docking, and free energy calculations. With this protocol, 19 potential TASK-3 blockers were identified. These molecules were tested in TASK-3 using patch clamp, and one blocker (DR16) was identified with an IC₅₀ = 56.8 ± 3.9 μM. Using DR16 as a scaffold, we designed DR16.1, a novel TASK-3 inhibitor, with an IC₅₀ = 14.2 ± 3.4 μM. Our finding takes on greater relevance considering that not many inhibitory TASK-3 modulators have been reported in the scientific literature until today. These two novel TASK-3 channel inhibitors (DR16 and DR16.1) are the first compounds found using a pharmacophore-based virtual screening and rational drug design protocol. Full article

In idiopathic pulmonary fibrosis (IPF) structural properties of the extracellular matrix (ECM) are altered and influence cellular responses through cell-matrix interactions. Scaffolds (decellularized tissue) derived from subpleural healthy and IPF lungs were examined regarding biomechanical properties and ECM composition of proteins (the matrisome). Scaffolds were repopulated with healthy fibroblasts cultured under static stretch with heavy isotope amino acids (SILAC), to examine newly synthesized proteins over time. IPF scaffolds were characterized by increased tissue density, stiffness, ultimate force, and differential expressions of matrisome proteins compared to healthy scaffolds. Collagens, proteoglycans, and ECM glycoproteins were increased in IPF scaffolds, however while specific basement membrane (BM) proteins such as laminins and collagen IV were decreased, nidogen-2 was also increased. Findings were confirmed with histology, clearly showing a disorganized BM. Fibroblasts produced scaffold-specific proteins mimicking preexisting scaffold composition, where 11 out of 20 BM proteins were differentially expressed, along with increased periostin and proteoglycans production. We demonstrate how matrisome changes affect fibroblast activity using novel approaches to study temporal differences, where IPF scaffolds support a disorganized BM and upregulation of disease-associated proteins. These matrix-directed cellular responses emphasize the IPF matrisome and specifically the BM components as important factors for disease progression. Full article