Hyperglycemia Induces Inflammatory Response of Human Macrophages to CD163-Mediated Scavenging of Hemoglobin-Haptoglobin Complexes

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.     

Depletion of Arg1-Positive Microglia/Macrophages Exacerbates Cerebral Ischemic Damage by Facilitating the Inflammatory Response

Stroke is a serious worldwide disease that causes death and disability, more than 80% of which is ischemic stroke. The expression of arginase 1 (Arg1), a key player in regulating nitrogen homeostasis, is altered in the peripheral circulation after stroke. Growing evidence indicates that ischemic stroke also induces upregulated Arg1 expression in the central nervous system, especially in activated microglia and macrophages. This implies that Arg1 may affect stroke progression by modulating the cerebral immune response. To investigate the effect of Arg1⁺ microglia/macrophages on ischemic stroke, we selectively eliminated cerebral Arg1⁺ microglia/macrophages by mannosylated clodronate liposomes (MCLs) and investigated their effects on behavior, neurological deficits, and inflammatory responses in mice after ischemic stroke. More than half of Arg1⁺ cells, mainly Arg1⁺ microglia/macrophages, were depleted after MCLs administration, resulting in a significant deterioration of motility in mice. After the elimination of Arg1⁺ microglia/macrophages, the infarct volume expanded and neuronal degenerative lesions intensified. Meanwhile, the absence of Arg1⁺ microglia/macrophages significantly increased the production of pro-inflammatory cytokines and suppressed the expression of anti-inflammatory factors, thus profoundly altering the immune microenvironment at the lesion site. Taken together, our data demonstrate that depletion of Arg1⁺ microglia/macrophages exacerbates neuronal damage by facilitating the inflammatory response, leading to more severe ischemic injury. These results suggest that Arg1⁺ microglia/macrophages, as a subpopulation regulating inflammation, is beneficial in controlling the development of ischemia and promoting recovery from injury. Regulation of Arg1 expression on microglia/macrophages at the right time may be a potential target for the treatment of ischemic brain injury.     

Impact of Leptin on the Expression Profile of Macrophages during Mechanical Strain In Vitro

Childhood obesity is a growing problem in industrial societies and associated with increased leptin levels in serum and salvia. Orthodontic treatment provokes pressure and tension zones within the periodontal ligament, where, in addition to fibroblasts, macrophages are exposed to these mechanical loadings. Given the increasing number of orthodontic patients with these conditions, insights into the effects of elevated leptin levels on the expression profile of macrophages during mechanical strain are of clinical interest. Therefore, the aim of this in vitro study was to assess the influence of leptin on the expression profile of macrophages during simulated orthodontic treatment. RAW264.7 macrophages were incubated with leptin and lipopolysaccharides (LPS) from Porphyromonas gingivalis (P. gingivalis) or with leptin and different types of mechanical strain (tensile, compressive strain). Expression of inflammatory mediators including tumor necrosis factor (TNF), Interleukin-1-B (IL1B), IL6, and prostaglandin endoperoxide synthase (PTGS2) was assessed by RT-qPCR, ELISAs, and immunoblot. Without additional mechanical loading, leptin increased Tnf, Il1b, Il6, and Ptgs2 mRNA in RAW264.7 macrophages by itself and after stimulation with LPS. However, in combination with tensile or compressive strain, leptin reduced the expression and secretion of these inflammatory factors. By itself and in combination with LPS from P. gingivalis, leptin has a pro-inflammatory effect. Both tensile and compressive strain lead to increased expression of inflammatory genes. In contrast to its effect under control conditions or after LPS treatment, leptin showed an anti-inflammatory phenotype after mechanical stress.     

Anti-Inflammatory Effect of Auranofin on Palmitic Acid and LPS-Induced Inflammatory Response by Modulating TLR4 and NOX4-Mediated NF-κB Signaling Pathway in RAW264.7 Macrophages

Chronic inflammation, which is promoted by the production and secretion of inflammatory mediators and cytokines in activated macrophages, is responsible for the development of many diseases. Auranofin is a Food and Drug Administration-approved gold-based compound for the treatment of rheumatoid arthritis, and evidence suggests that auranofin could be a potential therapeutic agent for inflammation. In this study, to demonstrate the inhibitory effect of auranofin on chronic inflammation, a saturated fatty acid, palmitic acid (PA), and a low concentration of lipopolysaccharide (LPS) were used to activate RAW264.7 macrophages. The results show that PA amplified LPS signals to produce nitric oxide (NO) and various cytokines. However, auranofin significantly inhibited the levels of NO, monocyte chemoattractant protein-1, and pro-inflammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor-α, and IL-6, which had been increased by co-treatment with PA and LPS. Moreover, the expression of inducible NO synthase, IL-1β, and IL-6 mRNA and protein levels increased by PA and LPS were reduced by auranofin. In particular, the upregulation of NADPH oxidase (NOX) 4 and the translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) induced by PA and LPS were suppressed by auranofin. The binding between the toll-like receptor (TLR) 4 and auranofin was also predicted, and the release of NO and cytokines was reduced more by simultaneous treatment with auranofin and TLR4 inhibitor than by auranofin alone. In conclusion, all these findings suggested that auranofin had anti-inflammatory effects in PA and LPS-induced macrophages by interacting with TLR4 and downregulating the NOX4-mediated NF-κB signaling pathway.     

Modulatory Effects of Fractalkine on Inflammatory Response and Iron Metabolism of Lipopolysaccharide and Lipoteichoic Acid-Activated THP-1 Macrophages

Fractalkine (CX3CL1) acts as a chemokine as well as a regulator of iron metabolism. Fractalkine binds CX3CR1, the fractalkine receptor on the surface of monocytes/macrophages regulating different intracellular signalling pathways such as mitogen-activated protein kinase (MAPK), phospholipase C (PLC) and NFκB contributing to the production of pro-inflammatory cytokine synthesis, and the regulation of cell growth, differentiation, proliferation and metabolism. In this study, we focused on the modulatory effects of fractalkine on the immune response and on the iron metabolism of Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (LPS) and Staphylococcus aureus lipoteichoic acid (LTA) activated THP-1 cells to get a deeper insight into the role of soluble fractalkine in the regulation of the innate immune system. Pro-inflammatory cytokine secretions of the fractalkine-treated, LPS/LTA-treated, and co-treated THP-1 cells were determined using ELISArray and ELISA measurements. We analysed the protein expression levels of signalling molecules regulated by CX3CR1 as well as hepcidin, the major iron regulatory hormone, the iron transporters, the iron storage proteins and mitochondrial iron utilization. The results showed that fractalkine treatment alone did not affect the pro-inflammatory cytokine secretion, but it was proposed to act as a regulator of the iron metabolism of THP-1 cells. In the case of two different LPS and one type of LTA with fractalkine co-treatments, fractalkine was able to alter the levels of signalling proteins (NFκB, PSTAT3, Nrf2/Keap-1) regulating the expression of pro-inflammatory cytokines as well as hepcidin, and the iron storage and utilization of the THP-1 cells.     

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.     

Encapsulation Reduces the Deleterious Effects of Salicylic Acid Treatments on Root Growth and Gravitropic Response

The role of salicylic acid (SA) on plant responses to biotic and abiotic stresses is well documented. However, the mechanism by which exogenous SA protects plants and its interactions with other phytohormones remains elusive. SA effect, both free and encapsulated (using silica and chitosan capsules), on Arabidopsis thaliana development was studied. The effect of SA on roots and rosettes was analysed, determining plant morphological characteristics and hormone endogenous levels. Free SA treatment affected length, growth rate, gravitropic response of roots and rosette size in a dose-dependent manner. This damage was due to the increase of root endogenous SA concentration that led to a reduction in auxin levels. The encapsulation process reduced the deleterious effects of free SA on root and rosette growth and in the gravitropic response. Encapsulation allowed for a controlled release of the SA, reducing the amount of hormone available and the uptake by the plant, mitigating the deleterious effects of the free SA treatment. Although both capsules are suitable as SA carrier matrices, slightly better results were found with chitosan. Encapsulation appears as an attractive technology to deliver phytohormones when crops are cultivated under adverse conditions. Moreover, it can be a good tool to perform basic experiments on phytohormone interactions.     

Increased Weight Gain and Insulin Resistance in HF-Fed PLTP Deficient Mice Is Related to Altered Inflammatory Response and Plasma Transport of Gut-Derived LPS

Bacterial lipopolysaccharides (LPS, endotoxins) are found in high amounts in the gut lumen. LPS can cross the gut barrier and pass into the blood (endotoxemia), leading to low-grade inflammation, a common scheme in metabolic diseases. Phospholipid transfer protein (PLTP) can transfer circulating LPS to plasma lipoproteins, thereby promoting its detoxification. However, the impact of PLTP on the metabolic fate and biological effects of gut-derived LPS is unknown. This study aimed to investigate the influence of PLTP on low-grade inflammation, obesity and insulin resistance in relationship with LPS intestinal translocation and metabolic endotoxemia. Wild-type (WT) mice were compared with Pltp-deficient mice (Pltp-KO) after a 4-month high-fat (HF) diet or oral administration of labeled LPS. On a HF diet, Pltp-KO mice showed increased weight gain, adiposity, insulin resistance, lipid abnormalities and inflammation, together with a higher exposure to endotoxemia compared to WT mice. After oral administration of LPS, PLTP deficiency led to increased intestinal translocation and decreased association of LPS to lipoproteins, together with an altered catabolism of triglyceride-rich lipoproteins (TRL). Our results show that PLTP, by modulating the intestinal translocation of LPS and plasma processing of TRL-bound LPS, has a major impact on low-grade inflammation and the onset of diet-induced metabolic disorders.     

酸洗钝化工艺对钛合金氢含量及耐蚀性的影响

研究了酸洗钝化工艺对TC4钛合金的氢含量及其电化学性能的影响,并优化了工艺参数。结果表明,经酸洗钝化后的钛合金的氢含量未显著增加,符合氢质量分数增加不大于0.002%的标准;经过酸洗钝化处理的TC4钛合金的电化学性能有了明显提高,维钝电位范围扩大,维钝电流密度减小,钝化膜转化电位提高,阻抗值增大;研究的酸洗钝化工艺,最佳酸洗t为10～20min,θ为35℃,钝化t为60 min,在此工艺参数控制下生成的钝化膜具有较好的耐蚀性。     

EPA and DHA Exposure Alters the Inflammatory Response but not the Surface Expression of Toll‐like Receptor 4 in Macrophages

Dietary intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and their respective enrichment in cell membranes have been negatively associated with atherosclerotic lesion development. This effect may be mediated, in part, by dampened inflammatory response of macrophages triggered by toll‐like receptor 4 (TLR4) activation. This study investigated the influence of membrane fatty acid profile on TLR4‐mediated inflammation in RAW 264.7 macrophages. Cells pretreated with myristic acid (MA), EPA, DHA or vehicle control for 24 h were stimulated with ultra‐pure LPS, a specific TLR4 agonist, for 6 or 24 h, corresponding to early and late stages of TNFα and IL‐6 protein induction. Treatment significantly increased cell membrane MA, EPA, and DHA by 4.5‐, 20.6‐, and 8.9‐fold, respectively. MA significantly increased IL‐6 secretion 6 h post‐exposure to the fatty acid, but did not change TNFα secretion in response to any other treatment condition. EPA and DHA significantly reduced TNFα secretion by 36 and 41 %, respectively, in cells stimulated for 24 h but not 6 h. In contrast, EPA and DHA significantly reduced IL‐6 secretion at both 6 h (67 and 72 %, respectively) and 24 h (69 and 72 %, respectively). MA or DHA treatment had no significant effect compared to vehicle on factors influencing cellular LPS recognition, including LPS‐cell association, and cell surface expression of TLR4, TLR4‐MD2 complex, and CD14. These data suggest that membrane fatty acid profiles influence the TLR4‐mediated inflammatory response in macrophages, via mechanisms that occur downstream of TLR4 receptor activation.     

In Vivo Analysis of the Biocompatibility and Bone Healing Capacity of a Novel Bone Grafting Material Combined with Hyaluronic Acid

The present in vivo study analyses both the inflammatory tissue reactions and the bone healing capacity of a newly developed bone substitute material (BSM) based on xenogeneic bone substitute granules combined with hyaluronate (HY) as a water-binding molecule. The results of the hyaluronate containing bone substitute material (BSM) were compared to a control xenogeneic BSM of the same chemical composition and a sham operation group up to 16 weeks post implantationem. A major focus of the study was to analyze the residual hyaluronate and its effects on the material-dependent healing behavior and the inflammatory tissue responses. The study included 63 male Wistar rats using the calvaria implantation model for 2, 8, and 16 weeks post implantationem. Established and Good Laboratory Practice (GLP)-conforming histological, histopathological, and histomorphometrical analysis methods were conducted. The results showed that the new hyaluronate containing BSM was gradually integrated within newly formed bone up to the end of the study that ended in a condition of complete bone defect healing. Thereby, no differences to the healing capacity of the control BSM were found. However, the bone formation in both groups was continuously significantly higher compared to the sham operation group. Additionally, no differences in the (inflammatory) tissue response that was analyzed via qualitative and (semi-) quantitative methods were found. Interestingly, no differences were found between the numbers of pro- and anti-inflammatory macrophages between the three study groups over the entire course of the study. No signs of the HY as a water-binding part of the BSM were histologically detectable at any of the study time points, altogether the results of the present study show that HY allows for an optimal material-associated bone tissue healing comparable to the control xenogeneic BSM. The added HY seems to be degraded within a very short time period of less than 2 weeks so that the remaining BSM granules allow for a gradual osteoconductive bone regeneration. Additionally, no differences between the inflammatory tissue reactions in both material groups and the sham operation group were found. Thus, the new hyaluronate containing xenogeneic BSM and also the control BSM have been shown to be fully biocompatible without any differences regarding bone regeneration.     

On the Bioactivity of Echinacea purpurea Extracts to Modulate the Production of Inflammatory Mediators

Inflammatory diseases are the focus of several clinical studies, due to limitations and serious side effects of available therapies. Plant-based drugs (e.g., salicylic acid, morphine) have become landmarks in the pharmaceutical field. Therefore, we investigated the immunomodulatory effects of flowers, leaves, and roots from Echinacea purpurea. Ethanolic (EE) and dichloromethanolic extracts (DE) were obtained using the Accelerated Solvent Extractor and aqueous extracts (AE) were prepared under stirring. Their chemical fingerprint was evaluated by liquid chromatography–high resolution mass spectrometry (LC-HRMS). The pro- and anti-inflammatory effects, as well as the reduction in intracellular reactive oxygen and nitrogen species (ROS/RNS), of the different extracts were evaluated using non-stimulated and lipopolysaccharide-stimulated macrophages. Interestingly, AE were able to stimulate macrophages to produce pro-inflammatory cytokines (tumor necrosis factor -TNF-α, interleukin -IL-1β, and IL-6), and to generate ROS/RNS. Conversely, under an inflammatory scenario, all extracts reduced the amount of pro-inflammatory mediators. DE, alkylamides-enriched extracts, showed the strongest anti-inflammatory activity. Moreover, E. purpurea extracts demonstrated generally a more robust anti-inflammatory activity than clinically used anti-inflammatory drugs (dexamethasone, diclofenac, salicylic acid, and celecoxib). Therefore, E. purpurea extracts may be used to develop new effective therapeutic formulations for disorders in which the immune system is either overactive or impaired.     

Alpha-Lipoic Acid Alleviates Acute Inflammation and Promotes Lipid Mobilization During the Inflammatory Response in White Adipose Tissue of Mice

Recently, white adipose tissue has been shown to exhibit immunological activity, and may play an important role in host defense and protection against bacterial infection. Αlpha‐lipoic acid (α‐LA) has been demonstrated to function as an anti‐inflammatory and anti‐oxidant agent. However, its influence on the inflammatory response and metabolic changes in white adipose tissue remains unknown. We used male C57BL/6 mice as models to study the effect of α‐LA on the inflammatory response and metabolic changes in white adipose tissue after stimulation with lipopolysaccharide (LPS). The non‐esterified fatty acid content was measured by an automatic biochemical analyzer. The expression of inflammation‐, lipid‐ and energy metabolism‐related genes and proteins was determined by quantitative real‐time polymerase chain reaction and western blotting. The results indicated that α‐LA significantly decreased the epididymis fat weight index and the non‐esterified fatty acid content in plasma compared with the control group. LPS significantly increased the expression of inflammation genes and α‐LA reduced their expression. The LPS‐induced expression of nuclear factor‐κB protein was decreased by α‐LA. Regarding lipid metabolism, α‐LA significantly counteracted the inhibitory effects of LPS on the expression of hormone‐sensitive lipase gene and protein. α‐LA evidently increased the gene expression of fatty acid transport protein 1 and cluster of differentiation 36. Regarding energy metabolism, α‐LA significantly increased the expression of most of mitochondrial DNA‐encoded genes compared with the control and LPS group. Accordingly, α‐LA can alleviate acute inflammatory response and this action may be related with the promotion of lipid mobilization in white adipose tissue.     

Trypanosoma brucei rhodesiense Inhibitor of Cysteine Peptidase (ICP) Is Required for Virulence in Mice and to Attenuate the Inflammatory Response

The protozoan Trypanosoma brucei rhodesiense causes Human African Trypanosomiasis, also known as sleeping sickness, and penetrates the central nervous system, leading to meningoencephalitis. The Cathepsin L-like cysteine peptidase of T. b. rhodesiense has been implicated in parasite penetration of the blood–brain barrier and its activity is modulated by the chagasin-family endogenous inhibitor of cysteine peptidases (ICP). To investigate the role of ICP in T. b. rhodesiense bloodstream form, ICP-null (Δicp) mutants were generated, and lines re-expressing ICP (Δicp:ICP). Lysates of Δicp displayed increased E-64-sensitive cysteine peptidase activity and the mutant parasites traversed human brain microvascular endothelial cell (HBMEC) monolayers in vitro more efficiently. Δicp induced E-selectin in HBMECs, leading to the adherence of higher numbers of human neutrophils. In C57BL/6 mice, no Δicp parasites could be detected in the blood after 6 days, while mice infected with wild-type (WT) or Δicp:ICP displayed high parasitemia, peaking at day 12. In mice infected with Δicp, there was increased recruitment of monocytes to the site of inoculation and higher levels of IFN-γ in the spleen. At day 14, mice infected with Δicp exhibited higher preservation of the CD4⁺, CD8⁺, and CD19⁺ populations in the spleen, accompanied by sustained high IFN-γ, while NK1.1⁺ populations receded nearly to the levels of uninfected controls. We propose that ICP helps to downregulate inflammatory responses that contribute to the control of infection.     

An Oligomannuronic Acid-Sialic Acid Conjugate Capable of Inhibiting Aβ42 Aggregation and Alleviating the Inflammatory Response of BV-2 Microglia

Oligomannuronic acid (MOS) from seaweed has antioxidant and anti-inflammatory activities. In this study, MOS was activated at the terminal to obtain three different graft complexes modified with sialic acid moiety (MOS-Sia). The results show that MOS-Sia addition can reduce the β-structure formation of Aβ42, and the binding effect of MOS-Sia3 is more obvious. MOS-Sia conjugates also have a better complexing effect with Ca²⁺ while reducing the formation of Aβ42 oligomers in solutions. MOS-Sia3 (25–50 μg/mL) can effectively inhibit the activation state of BV-2 cells stimulated by Aβ42, whereas a higher dose of MOS-Sia3 (>50 μg/mL) can inhibit the proliferation of BV-2 cells to a certain extent. A lower dose of MOS-Sia3 can also inhibit the expression of IL-1β, IL-6, TNF-α, and other proinflammatory factors in BV-2 cells induced by Aβ42 activation. In the future, the MOS-Sia3 conjugate can be used to treat Alzheimer’s disease.     

In Vitro Framework to Assess the Anti-Helicobacter pylori Potential of Lactic Acid Bacteria Secretions as Alternatives to Antibiotics

Helicobacter pylori is a prevalent bacterium that can cause gastric ulcers and cancers. Lactic acid bacteria (LAB) ameliorate treatment outcomes against H. pylori, suggesting that they could be a source of bioactive molecules usable as alternatives to current antibiotics for which resistance is mounting. We developed an in vitro framework to compare the anti-H. pylori properties of 25 LAB and their secretions against H. pylori. All studies were done at acidic and neutralized pH, with or without urea to mimic various gastric compartments. Eighteen LAB strains secreted molecules that curtailed the growth of H. pylori and the activity was urea-resistant in five LAB. Several LAB supernatants also reduced the urease activity of H. pylori. Pre-treatment of H. pylori with acidic LAB supernatants abrogated its flagella-mediated motility and decreased its ability to elicit pro-inflammatory IL-8 cytokine from human gastric cells, without reverting the H. pylori-induced repression of other pro-inflammatory cytokines. This study identified the LAB that have the most anti-H. pylori effects, decreasing its viability, its production of virulence factors, its motility and/or its ability to elicit pro-inflammatory IL-8 from gastric cells. Once identified, these molecules can be used as alternatives or complements to current antibiotics to fight H. pylori infections.     

Caffeic Acid Reduces the Viability and Migration Rate of Oral Carcinoma Cells (SCC-25) Exposed to Low Concentrations of Ethanol

Alcohol increases the risk of carcinoma originated from oral epithelium, but the biological effects of ultra-low doses of ethanol on existing carcinoma cells in combination with natural substances are still unclear. A role for ethanol (EtOH), taken in small amounts as an ingredient of some beverages or mouthwashes to change the growth behavior of established squamous cell carcinoma, has still not been examined sufficiently. We designed an in vitro study to determine the effect of caffeic acid (CFA) on viability and migration ability of malignant oral epithelial keratinocytes, exposed to ultra-low concentrations (maximum 100 mmol/L) EtOH. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dimethyltetrazolium bromide) and LDH (lactate dehydrogenase) assays were used to assess the cytotoxic effect of EtOH/CFA and the viability of squamous carcinoma SCC-25 cells (ATCC CRL-1628, mobile part of the tongue). Tested EtOH concentrations were: 2.5, 5, 10, 25, 50, and 100 mmol/L, along with an equal CFA concentration of 50 μmol/L. Carcinoma cells’ migration was investigated by monolayer “wound” healing assay. We demonstrated that very low concentrations of EtOH ranging between 2.5 and 10 mmol/L may induce the viability of oral squamous cell carcinoma cells, while the results following addition of CFA reveal an antagonistic effect, attenuating pro-proliferative EtOH activity. The migration rate of oral squamous carcinoma cells can be significantly inhibited by the biological activity of caffeic acid.     

Identification of Novel miRNAs and Their Target Genes in the Response to Abscisic Acid in Arabidopsis

miRNAs are involved in various biological processes, including adaptive responses to abiotic stress. To understand the role of miRNAs in the response to ABA, ABA-responsive miRNAs were identified by small RNA sequencing in wild-type Arabidopsis, as well as in abi1td, mkkk17, and mkkk18 mutants. We identified 10 novel miRNAs in WT after ABA treatment, while in abi1td, mkkk17, and mkkk18 mutants, three, seven, and nine known miRNAs, respectively, were differentially expressed after ABA treatment. One novel miRNA (miRn-8) was differentially expressed in the mkkk17 mutant. Potential target genes of the miRNA panel were identified using psRNATarget. Sequencing results were validated by quantitative RT-PCR of several known and novel miRNAs in all genotypes. Of the predicted targets of novel miRNAs, seven target genes of six novel miRNAs were further validated by 5′ RLM-RACE. Gene ontology analyses showed the potential target genes of ABA-responsive known and novel miRNAs to be involved in diverse cellular processes in plants, including development and stomatal movement. These outcomes suggest that a number of the identified miRNAs have crucial roles in plant responses to environmental stress, as well as in plant development, and might have common regulatory roles in the core ABA signaling pathway.