Dietary limonene promotes gastrointestinal barrier function via upregulating tight/adherens junction proteins through cannabinoid receptor type-1 antagonistic mechanism and alters cellular metabolism in intestinal epithelial cells.

Limonene, a dietary monocyclic monoterpene commonly found in citrus fruits and various aromatic plants, has garnered increasing interest as a gastrointestinal protectant. This study aimed to assess the effects of limonene on intestinal epithelial barrier function and investigate the involvement of cannabinoid receptor type-1 (CB1R) in vitro. Additionally, the study focused on examining the metabolomic changes induced by limonene in the intestinal epithelial cells (Caco-2). Initial analysis of transepithelial electrical resistance (TEER) revealed that both l-limonene and d-limonene, isomers of limonene, led to a dose- and time-dependent increase in TEER in normal cells and those inflamed by pro-inflammatory cytokines mixture (CytoMix). Furthermore, both types of limonene reduced CytoMix-induced paracellular permeability, as demonstrated by a decrease in Lucifer yellow flux. Moreover, d-limonene and l-limonene treatment increased the expression of tight junction molecules (TJs) such as occludin, claudin-1, and ZO-1, at both the transcriptional and translational levels. d-Limonene upregulates E-cadherin, a molecule involved in adherens junctions (AJs). Mechanistic investigations demonstrated that d-limonene and l-limonene treatment significantly inhibited CB1R at the protein, while the mRNA level remained unchanged. Notably, the inhibitory effect of d-limonene on CB1R was remarkably similar to that of pharmacological CB1R antagonists, such as rimonabant and ORG27569. d-limonene also alters Caco-2 cell metabolites. A substantial reduction in ß-glucose and 2-succinamate was detected, suggesting limonene may impact intestinal epithelial cells' glucose uptake and glutamate metabolism. These findings suggest that d-limonene's CB1R antagonistic property could effectively aid in the recovery of intestinal barrier damage, marking it a promising gastrointestinal protectant.

Green synthesis of Piper nigrum copper-based nanoparticles: in silico study and ADMET analysis to assess their antioxidant, antibacterial, and cytotoxic effects.

Nanobiotechnology is a popular branch of science that is gaining interest among scientists and researchers as it allows for the green manufacturing of nanoparticles by employing plants as reducing agents. This method is safe, cheap, reproducible, and eco-friendly. In this study, the therapeutic property of Piper nigrum fruit was mixed with the antibacterial activity of metallic copper to produce copper nanoparticles. The synthesis of copper nanoparticles was indicated by a color change from brown to blue. Physical characterization of Piper nigrum copper nanoparticles (PN-CuNPs) was performed using UV-vis spectroscopy, FT-IR, SEM, EDX, XRD, and Zeta analyzer. PN-CuNPs exhibited potential antioxidant, antibacterial, and cytotoxic activities. PN-CuNPs have shown concentration-dependent, enhanced free radical scavenging activity, reaching maximum values of 92%, 90%, and 86% with DPPH, H2O2, and PMA tests, respectively. The antibacterial zone of inhibition of PN-CuNPs was the highest against Staphylococcus aureus (23 mm) and the lowest against Escherichia coli (10 mm). PN-CuNPs showed 80% in vitro cytotoxicity against MCF-7 breast cancer cell lines. Furthermore, more than 50 components of Piper nigrum extract were selected and subjected to in silico molecular docking using the C-Docker protocol in the binding pockets of glutathione reductase, E. coli DNA gyrase topoisomerase II, and epidermal growth factor receptor (EGFR) tyrosine to discover their druggability. Pipercyclobutanamide A (26), pipernigramide F (32), and pipernigramide G (33) scored the highest Gibbs free energy at 50.489, 51.9306, and 58.615 kcal/mol, respectively. The ADMET/TOPKAT analysis confirmed the favorable pharmacokinetics, pharmacodynamics, and toxicity profiles of the three promising compounds. The present in silico analysis helps us to understand the possible mechanisms behind the antioxidant, antibacterial, and cytotoxic activities of CuNPs and recommends them as implicit inhibitors of selected proteins.

Limonene protects human skin keratinocytes against UVB-induced photodamage and photoaging by activating the Nrf2-dependent antioxidant defense system.

Long term exposure to solar ultraviolet B (UVB) radiation is one of the primary factors of premature skin aging and is referred to as photoaging. Also, mammalian skin exposed to UVB triggers an increase in production of α-melanocyte-stimulating hormone (α-MSH), which is critically involved in the pathogenesis of hyperpigmentary skin diseases. This study investigated the protective effect of limonene on UVB-induced photodamage and photoaging in immortalized human skin keratinocytes (HaCaT) in vitro. Initially, we determined cell viability and levels of reactive oxygen species (ROS) in UVB-irradiated HaCaT cells. Pretreatment with limonene increased cell viability followed by inhibition of intracellular ROS generation in UVB-irradiated HaCaT cells. Interestingly, the antioxidative activity of limonene was directly correlated with an increase in expression of endogenous antioxidants, including heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO-1), and γ-glutamylcysteine synthetase (γ-GCLC), which was associated with enhanced nuclear translocation and activation of NF-E2-related factor-2 (Nrf2). Indeed, Nrf2 knockdown reduced limonene's protective effects. Additionally, we observed that limonene treatment inhibited UVB-induced α-MSH secretion followed by inhibition of proopiomelanocortin (POMC) via suppression of p53 transcriptional activation. Moreover, limonene prevented UVB-mediated depletion of tight junction regulatory proteins, including occludin and zonula occludens-1. On the other hand, limonene treatment significantly decreased matrix metalloproteinase-2 levels in UVB-irradiated HaCaT cells. Based on these results, limonene may have a dermato-protective effect in skin cells by activating the Nrf2-dependent cellular antioxidant defense system.

Strategies for the evaluation of the eye irritation potential of different types of surfactants and silicones used in cosmetic products.

The ocular irritation potential of products that may come into contact with the eyes should be assessed by the combination of different in vitro alternative methods to determine different mechanisms of toxicity previously evaluated by the Draize in vivo assay. Thus, this study proposed to apply two strategies for the prediction of the eye irritation potential of different concentrations of surfactants and silicones, the first one involving evaluation Hen's Egg Test - Chorioallantoic Membrane (HET-CAM), and the other one using Bovine Corneal Opacity and Permeability (BCOP) followed by histopathological. HET-CAM was considered important in assessing the ocular irritation potential and, despite classifying almost all surfactants as "severe irritants", it could discriminate moderate and slight irritant SLES concentrations as well as Cocoamidopropyl Betaine as a severe irritant, when the coagulation score was taken into consideration. The BCOP assay alone also did not offer a good prediction of the irritant potential of surfactants, since almost all of them were classified as "no prediction can be made". However, the histopathological evaluation of the BCOP corneas was very important for establishing the degree and depth of damage related to reversibility. The present study also showed those strategies are sensitive to small variations in the studied anionic, cationic amphoteric surfactant concentrations and can be used for predicting their toxicity in the final product and can be used depending on the focus of the analysis.

Antcins from Antrodia cinnamomea and Antrodia salmonea Inhibit Angiotensin-Converting Enzyme 2 (ACE2) in Epithelial Cells: Can Be Potential Candidates for the Development of SARS-CoV-2 Prophylactic Agents.

Antcins are newly identified steroid-like compounds from Taiwan's endemic medicinal mushrooms Antrodia cinnamomea and Antrodia salmonea. Scientific studies of the past two decades confirmed that antcins have various pharmacological activities, including potent anti-oxidant and anti-inflammatory effects. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease-2019 (COVID-19) pandemic and is characterized as a significant threat to global public health. It was recently identified that SARS-CoV-2 required angiotensin converting enzyme 2 (ACE2), a receptor which supports host cell entry and disease onset. Here, we report a novel function of antcins, in which antcins exhibit inhibitory effects on ACE2. Compared to the untreated control group, treatment with various antcins (antcin-A, antcin-B, antcin-C, antcin-H, antcin-I, and antcin-M) significantly inhibited ACE2 activity in cultured human epithelial cells. Indeed, among the investigated antcins, antcin-A, antcin-B, antcin-C, and antcin-I showed a pronounceable inhibition against ACE2. These findings suggest that antcins could be novel anti-ACE2 agents to prevent SARS-CoV-2 host cell entry and the following disease onset.

Essential Oils of Alpinia nantoensis Retard Forskolin-Induced Melanogenesis via ERK1/2-Mediated Proteasomal Degradation of MITF.

The anti-melanogenic activity of essential oils of Alpinia nantoensis and their bioactive ingredients were investigated in vitro. Treatment with leaf (LEO) and rhizome (REO) essential oils of A. nantoensis, significantly reduced forskolin-induced melanin production followed by down-regulation of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1) expression at both transcriptional and translational levels. Further studies revealed that down-regulation TYR and TRP-1 were caused by LEO/REO-mediated suppression of Microphthalmia-associated transcription factor (MITF), as evidenced by reduced nuclear translocation of MITF. Also, we found that LEO/REO induce the sustained activation of ERK1/2, which facilitate subsequent proteasomal degradation of MITF, as confirmed by that LEO/REO failed to inhibits MITF activity in ERK1/2 inhibitor treated cells. In addition, a significant increase of ubiquitinated MITF was observed after treatment with LEO and REO. Furthermore, the chemical composition of LEO and REO were characterized by gas chromatography-mass spectrometry (GC-MS) resulted that camphor, camphene, α-pinene, ß-pinene, isoborneol and D-limonene were the major compounds in both LEO and REO. Further studies revealed that α-pinene and D-limonene were the active components responsible for the anti-melanogenic properties of LEO and REO. Based on the results, this study provided a strong evidence that LEO and REO could be promising natural sources for the development of novel skin-whitening agents for the cosmetic purposes.

Geranium and Lemon Essential Oils and Their Active Compounds Downregulate Angiotensin-Converting Enzyme 2 (ACE2), a SARS-CoV-2 Spike Receptor-Binding Domain, in Epithelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease-2019 (COVID-19), is a pandemic disease that has been declared as modern history's gravest health emergency worldwide. Until now, no precise treatment modality has been developed. The angiotensin-converting enzyme 2 (ACE2) receptor, a host cell receptor, has been found to play a crucial role in virus cell entry; therefore, ACE2 blockers can be a potential target for anti-viral intervention. In this study, we evaluated the ACE2 inhibitory effects of 10 essential oils. Among them, geranium and lemon oils displayed significant ACE2 inhibitory effects in epithelial cells. In addition, immunoblotting and qPCR analysis also confirmed that geranium and lemon oils possess potent ACE2 inhibitory effects. Furthermore, the gas chromatography-mass spectrometry (GC-MS) analysis displayed 22 compounds in geranium oil and 9 compounds in lemon oil. Citronellol, geraniol, and neryl acetate were the major compounds of geranium oil and limonene that represented major compound of lemon oil. Next, we found that treatment with citronellol and limonene significantly downregulated ACE2 expression in epithelial cells. The results suggest that geranium and lemon essential oils and their derivative compounds are valuable natural anti-viral agents that may contribute to the prevention of the invasion of SARS-CoV-2/COVID-19 into the human body.

A mechanistic and empirical review of antcins, a new class of phytosterols of formosan fungi origin.

Antcins are unique phytosterols isolated from A. cinnamomea and A. salmomea, which are the endemic fungus of Taiwan. A. cinnamomea has long been highly valued medicinal mushroom in Taiwan and traditionally used as a folk remedy for various human illness. Recent scientific explorations claimed that the pharmacological activities of A. cinnamomea and A. salmomonea are gone beyond their original usage. The therapeutic efficacy of these medicinal mushrooms was attributed to their high content of unique bioactive secondary metabolites, including terpenoids, benzenoids, ubiquinol derivatives, polysaccharides, lignans, nucleic acids, steroids, and maleic/succinic acid derivatives. Antcins is a group of steroids in Antrodia spp. with ergostane skeleton received much attention in Taiwan's academic circle due to their broad-spectrum of biological activities. At present, twelve antcins, i.e. antcin A, B, C, D, E, F, G, H, I, K, M, and N along with twelve derivatives/epimers (25R/S-antcin A, B, C, H, I and K) and seven analogs (methyl antcinate A, B, G, H, K, L and N) were identified. Several studies have demonstrated that antcins possessed anti-cancer, anti-inflammation, anti-oxidant, anti-diabetic, anti-aging, immunomodulation, hepatoprotection, and hypolipedimic activities. The main goal of this review is to define the chemistry, isolation, advances in production, and biological activities of antcins and their derivatives/analogs. Special attention has been given to a detail view of their biological activities in vitro and in vivo and their pharmacological potentials.

Antcin-A Modulates Epithelial-to-Mesenchymal Transition and Inhibits Migratory and Invasive Potentials of Human Breast Cancer Cells via p53-Mediated miR-200c Activation.

Antcin-A (ATA) is a steroid-like phytochemical isolated from the fruiting bodies of a precious edible mushroom Antrodia cinnamomea. We previously showed that ATA has strong anti-inflammatory and anti-tumor effects; however, other possible bioactivities of this unique compound remain unexplored. In the present study, we aimed to investigate the modulation of epithelial-to-mesenchymal transition (EMT), anti-migration, and anti-invasive potential of ATA against human breast cancer cells in vitro. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were incubated with ATA for 24 h. Wound healing, trans-well invasion, western blot, q-PCR, F-actin staining, and immunofluorescence assays were performed. We found that treatment with ATA significantly blocked EMT processes, as evidenced by upregulation of epithelial markers (E-cadherin and occludin) and downregulation of mesenchymal markers (N-cadherin and vimentin) via suppression of their transcriptional repressor ZEB1. Next, we found that ATA could induce miR-200c, which is a known player of ZEB1 repression. Further investigations revealed that ATA-mediated induction of miR-200c is associated with transcriptional activation of p53, as confirmed by the fact that ATA failed to induce miR-200c or suppress ZEB1 activity in p53 inhibited cells. Further in vitro wound healing and trans-well invasion assays support that ATA could inhibit migratory and invasive potentials of breast cancer cells, and the effect was likely associated with induced phenotypic modulation. Taken together, the present study suggests that antcin-A could be a lead phyto-agent for the development of anti-metastatic drug for breast cancer treatment.

MicroRNA-708 activation by glucocorticoid receptor agonists regulate breast cancer tumorigenesis and metastasis via downregulation of NF-κB signaling.

Therapeutic administration of glucocorticoids (GCs) is frequently used as add-on chemotherapy for palliative purposes during breast cancer treatment. Recent studies have shown that GC treatment induces microRNA-708 in ovarian cancer cells, resulting in impaired tumor cell proliferation and metastasis. However, the regulatory functions of GCs on miR-708 and its downstream target genes in human breast cancer cells (BCCs) are poorly understood. In this study, we found that treatment with either the synthetic GC dexamethasone (DEX) or the natural GC mimic, antcin A (ATA) significantly increased miR-708 expression by transactivation of glucocorticoid receptor alpha (GRα) in MCF-7 and MDA-MB-231 human BCCs. Induction of miR-708 by GR agonists resulted in inhibition of cell proliferation, cell-cycle progression, cancer stem cell (CSC)-like phenotype and metastasis of BCCs. In addition, GR agonist treatment or miR-708 mimic transfection remarkably inhibited IKKß expression and suppressed nuclear factor-kappaB (NF-κB) activity and its downstream target genes, including COX-2, cMYC, cyclin D1, Matrix metalloproteinase (MMP)-2, MMP-9, CD24, CD44 and increased p21CIP1 and p27KIP1 that are known to be involved in proliferation, cell-cycle progression, metastasis and CSC marker protein. BCCs xenograft models indicate that treatment with GR agonists significantly reduced tumor growth, weight and volume. Overall, our data strongly suggest that GR agonists induced miR-708 and downstream suppression of NF-κB signaling, which may be applicable as a novel therapeutic intervention in breast cancer treatment.

Activation of Nrf2-mediated anti-oxidant genes by antrodin C prevents hyperglycemia-induced senescence and apoptosis in human endothelial cells.

In the present study, we investigated the effects of antrodin C (ADC), a maleimide derivative isolated from mycelia of Antrodia cinnamomea, on high glucose (HG, 30 mM)-accelerated endothelial dysfunction in vitro. HG-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) was significantly ameliorated by ADC. In addition, treatment with ADC significantly prevented HG-induced senescence, growth arrest at the G1-S transition phase and apoptosis in HUVECs. Moreover, the increased level of intracellular reactive oxygen species (ROS) under HG condition was significantly ameliorated by ADC. Further analysis revealed that ADC-mediated anti-oxidant effects were due to up-regulation of cellular anti-oxidant genes, such as HO-1 and NQO-1 via promotion of the transcriptional activity of Nrf2, which was further confirmed by the failure of ADC to protect HUVECs from HG-induced dysfunction under HO-1 inhibition or Nrf2 silencing. Furthermore, hyperosmotic glucose (HOG, 60 mM)-induced uncontrolled production of ROS, rapid apoptotic cell death and HUVEC injury were significantly prevented by ADC, whereas these preventive effects were barely observed in HO-1 inhibited or Nrf2 silenced cells. Taken together, these results suggest that ADC may represent a promising intervention in diabetic-associated cardiovascular diseases by activating the Nrf2-dependent cellular anti-oxidant defense system.

Metastatic Renal Cell Carcinoma Causing Small Intestinal Polyps with Intussusception: A Report of Two Cases.

Renal cell carcinomas have diverse clinical presentations. Patients may present with metastatic symptoms at the time of diagnosis or years after resection of primary tumour. Small intestine is a rare site for metastatic renal cell carcinoma. Presentation as polypoid metastasis with intussusception is extremely rare. Here, we report two cases with this rare presentation. Case 1 was a 65-year-old male with multiple polypoid metastasis causing intussusception while on palliative chemotherapy for renal cell carcinoma. Case 2 was a 68-year-old female with history of nephrectomy presenting with single polypoid metastasis causing intussusception.

Jejunal Gastric Heterotopia causing Multiple Strictures and Perforation Peritonitis- A Case Report with Review of Literature.

Gastric heterotopias beyond the ligament of Treitz though rare, should be thought of in the differential diagnosis of polypoid lesions presenting with gastrointestinal bleed or obstructive symptoms especially in children and in the young. Here is a 24-year-old male with multifocal jejunal gastric heterotopias causing multiple strictures and perforation peritonitis. Patient presented with acute abdomen pain and an emergency laparotomy was performed revealing jejunum with multiple strictures and perforation, followed by jejunal resection. On gross examination polypoid mucosa was noted at the stricture sites which showed heterotopic gastric mucosa on microscopy. Jejunal gastric heterotopias are extremely rare with less than ten reported cases and those presenting with multiple strictures are even rarer. To our knowledge this is the second case of jejunal gastric heterotopia presenting with multiple strictures.

Legionella longbeachae Is Immunologically Silent and Highly Virulent In Vivo.

BACKGROUND: Legionella longbeachae (Llo) and Legionella pneumophila (Lpn) are the most common pneumonia-causing agents of the genus. Although both species can be lethal to humans and are highly prevalent, little is known about the molecular pathogenesis of Llo infections. In murine models of infection, Lpn infection is self-limited, whereas Llo infection is lethal. METHODS: We used mouse macrophages, human macrophages, human epithelial cells, and mouse infections in vivo to evaluate multiple parameters of the infection. RESULTS: We determined that the Llo Dot/Icm secretion system is critical for virulence. Different than Lpn, Llo disseminates and the animals develop a severe pulmonary failure, as demonstrated by lung mechanics and blood oxygenation assays. As compared to Lpn, Llo is immunologically silent and fails to trigger the production of cytokines in human pulmonary epithelial cells and in mouse and human macrophages. Infections in Tnfr1-/-, Ifng-/-, and Il12p40-/- mice supported the participation of cytokines for the resistance phenotype. CONCLUSIONS: Both Lpn and Llo require the Dot/Icm system for pathogenesis, but the infection outcome is strikingly different. Llo is immunologically silent, highly virulent, and lethal. The differences reported herein may reflect unappreciated clinical differences in patients infected with Lpn or Llo.

A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1.

The present study revealed the anti-aging properties of antcin M (ANM) and elucidated the molecular mechanism underlying the effects. We found that exposure of human normal dermal fibroblasts (HNDFs) to high-glucose (HG, 30 mM) for 3 days, accelerated G0/G1 phase arrest and senescence. Indeed, co-treatment with ANM (10 µM) significantly attenuated HG-induced growth arrest and promoted cell proliferation. Further molecular analysis revealed that ANM blocked the HG-induced reduction in G1-S transition regulatory proteins such as cyclin D, cyclin E, CDK4, CDK6, CDK2 and protein retinoblastoma (pRb). In addition, treatment with ANM eliminated HG-induced reactive oxygen species (ROS) through the induction of anti-oxidant genes, HO-1 and NQO-1 via transcriptional activation of Nrf2. Moreover, treatment with ANM abolished HG-induced SIPS as evidenced by reduced senescence-associated ß-galactosidase (SA-ß-gal) activity. This effect was further confirmed by reduction in senescence-associated marker proteins including, p21CIP1, p16INK4A, and p53/FoxO1 acetylation. Also, the HG-induced decline in aging-related marker protein SMP30 was rescued by ANM. Furthermore, treatment with ANM increased SIRT-1 expression, and prevented SIRT-1 depletion. This protection was consistent with inhibition of SIRT-1 phosphorylation at Ser47 followed by blocking its upstream kinases, p38 MAPK and JNK/SAPK. Further analysis revealed that ANM partially protected HG-induced senescence in SIRT-1 silenced cells. A similar effect was also observed in Nrf2 silenced cells. However, a complete loss of protection was observed in both Nrf2 and SIRT-1 knockdown cells suggesting that both induction of Nrf2-mediated anti-oxidant defense and SIRT-1-mediated deacetylation activity contribute to the anti-aging properties of ANM in vitro. Result of in vivo studies shows that ANM-treated C. elegens exhibits an increased survival rate during HG-induced oxidative stress insult. Furthermore, ANM significantly extended the life span of C. elegans. Taken together, our results suggest the potential application of ANM in age-related diseases or as a preventive reagent against aging process.

Antrodin C inhibits epithelial-to-mesenchymal transition and metastasis of breast cancer cells via suppression of Smad2/3 and ß-catenin signaling pathways.

Epithelial-to-mesenchymal transition (EMT) is a crucial event involved metastasis of certain tumors. Thus, identifying chemical agents that can block EMT is highly warranted for the development of anti-cancer chemoprevention/chemotherapies. In this study, we found that Antrodin C (ADC), a maleimide derivative isolated from Antrodia cinnamomea health food product inhibits TGF-ß1-induced EMT and breast cancer cell metastasis in vitro. Pretreatment of MCF-7 cells with ADC significantly blocked TGF-ß1-induced phenotypic changes and actin cytoskeleton remodeling. In addition, ADC was able to up-regulate epithelial markers such as E-cadherin and occludin, whereas mesenchymal markers including N-cadherin and vimentin were significantly inhibited, possibly through the modulation of transcriptional regulators Smad/Smad3. ADC blocked TGF-ß1-induced migration and invasion of MCF-7 cells through the down-regulation of matrix-metalloproteinases (MMP-2, -9) and urokinase plasminogen activator (uPA). The inhibition of MMPs and uPA activity by ADC was reasoned by suppression of its corresponding transcription factor ß-catenin. Taken together, our data suggested that ADC attenuates the TGF-ß1-induced EMT, migration and invasion of human breast carcinoma through the suppression of Smad2/3 and ß-catenin signaling pathways.

Spread of Oropouche virus into the central nervous system in mouse.

Oropouche virus (OROV) is an important cause of arboviral illness in Brazil and other Latin American countries, with most cases clinically manifested as acute febrile illness referred to as Oropouche fever, including myalgia, headache, arthralgia and malaise. However, OROV can also affect the central nervous system (CNS) with clinical neurological implications. Little is known regarding OROV pathogenesis, especially how OROV gains access to the CNS. In the present study, neonatal BALB/c mice were inoculated with OROV by the subcutaneous route and the progression of OROV spread into the CNS was evaluated. Immunohistochemistry revealed that OROV infection advances from posterior parts of the brain, including the periaqueductal gray, toward the forebrain. In the early phases of the infection OROV gains access to neural routes, reaching the spinal cord and ascending to the brain through brainstem regions, with little inflammation. Later, as infection progresses, OROV crosses the blood-brain barrier, resulting in more intense spread into the brain parenchyma, with more severe manifestations of encephalitis.

Thee-component, one-pot synthesis of hexahydroazepino[3,4-b]indole and tetrahydro-1H-pyrido[3,4-b]indole derivatives and evaluation of their cytotoxicity.

A three-component, four-center Ugi reaction has been developed to produce a novel class of 2-aryl-3-oxo-hexahydroazepino[3,4-b]indole and 2-aryl-3-oxo-tetrahydro-1H-pyrido[3,4-b]indole derivatives in good to high yields. A few of them exhibit moderate cytotoxicity against various cancer cell lines such as HeLa (human epithelial cervical cancer), A549 (human lung carcinoma epithelial), DU145 (human prostate carcinoma epithelial) and MCF-7 (human breast adenocarcinoma).

Differences in collagen distribution of healthy and regenerated periodontium. Histomorphometric study in dogs.

Previous studies have shown that there is a relationship between periodontal disease and the distribution of collagen fibers. This study evaluated the distribution of collagen types I and III in regenerated bone and periodontal ligament, comparing them to the tissues near the regenerated area and to the healthy periodontium. In the third (P3) and fourth (P4) mandibular premolars of 5 healthy mongrel dogs, bilaterally, buccal class 2 furcation lesions were surgically created and chronified for 3 weeks. After that, full flaps were elevated and expanded polytetrafluoroethylene (e-PTFE) membranes were adapted, sutured and recovered by the flaps. Two weeks after surgery, two membranes on the same side were removed and the other membranes were removed four weeks after surgery. The dogs were euthanized at 12 weeks following placement of the e-PTFE membranes. P3 and P4 teeth as well as the second premolars (healthy control teeth) and their periodontal tissues were removed and histologically processed for Collagen Quantification (COLQ). The amount of type III collagen was higher in native bone compared to the regenerated area. For periodontal ligament, COLQ for type I collagen showed statistically significant differences (Tukeys's Multiple Comparison, p⟨0.05) between the regenerated groups and the control group. These differences were not found for type III COLQ. There are significant differences in collagen distribution among the regenerated, native and control tissues. Membrane removal 2 or 4 weeks postoperatively did not influence the collagen composition.

In vitro and in vivo studies disclosed the depigmenting effects of gallic acid: a novel skin lightening agent for hyperpigmentary skin diseases.

Gallic acid (GA) is a phenolic compound, which has been reported to suppress melanogenesis in melanoma cells. However, the molecular mechanism underlying this inhibitory effect was poorly understood. In this article, we revealed that GA down-regulated melanogenic regulatory genes including tyrosinase, tyrosinase related protein-1 (TRP-1), and dopachrome tatamerase (Dct) expression at transcriptional and translational level. In addition, GA effectively suppressed the microphthalmia-associated transcription factor (MITF) expression by down-regulating the cAMP-mediated PKA/CREB signaling cascades. To delineate the inhibition of MITF by GA, the activation of extracellular signal-regulated protein kinase (ERK) and AKT was investigated. GA caused significant increase of ERK and AKT phosphorylation, while ERK (PD98059) or AKT (LY294002) inhibitor prevents their phosphorylation and increased melanin biosynthesis. In addition, pre-treatment of MITF-siRNA significantly reduced melanin production from 100 to 40%, and even decreased into 10% by combination treatment with GA. Furthermore, UVB-induced hyperpigmentation in the mice skin was significantly rescued by topical application of GA for 4 weeks. Immunohistochemical analyses also confirmed that GA significantly inhibited melanin production followed by the down-regulation of MITF, tyrosinase and their regulatory proteins. In addition, when compared with control zebrafish, GA caused a remarkable inhibition on the endogenous pigmentation in the zebrafish. Results presented in this study strongly suggest that GA is an effective de-pigmenting or skin lightening cosmetics for topical application.