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Objectives: This study aims to evaluate the pharmacological role of indigo extract in accelerating the wound healing in a rat model. Methods: Female Sprague-Dawley rats were anesthetized with ketamine (30 mg/kg, i.p.) and the full thickness of the marked skin was then cut carefully and wounds were left undressed. Indigo extract (5%) in PBS was applied topically twice daily until healing was complete. A control group of rats was treated with povidone-iodide (Betadine®). Rats treated with phosphate buffer saline were used as a negative control group. The rate of wound healing was assessed daily. Histopathological examination of skin sections were qualitatively assessed by independent evaluators. The inflammatory and apoptotic markers were assessed in skin tissue homogenates using ELISA. Results: Histopathology data showed that applying indigo to skin wounds enhanced the healing process, resulting in a significant decrease in dermal inflammation in comparison to untreated rats. Topical application of indigo significantly increased antioxidant enzyme activities with reduced malondialdehyde (MDA) levels in wound tissues. The levels of matrix metalloproteases-2 and -9 were significantly lower with an accompanied increase in the level of TGF-β1 in skin tissues from rats treated with indigo compared to the control group treated with PBS. Conclusions: The antioxidant and anti-inflammatory properties of indigo leaf extract accelerate the healing of skin injuries.

Bladder cancer (BC) is the 10th most common cancer worldwide. Genetic studies estimated 30% heritability in BC risk. Adiponectin is an adipocytokine that has important roles in the regulation of energy metabolism. Recent evidence suggests dysregulation of adiponectin levels in BC tissues. Serum level of adiponectin is influenced by single nucleotide polymorphisms (SNPs) in the ADIPOQ gene. However, limited evidence is available regarding the association between adiponectin serum levels or SNPs in ADIPOQ and BC risk. This study aimed to assess whether adiponectin serum levels or SNPs in ADIPOQ may modify BC risk. In this case-control study, 114 BC patients were recruited along with 114 controls. Study subjects were genotyped for variations in ADIPOQ SNPs, namely rs17300539, rs266729, rs2241766, and rs1501299. Adiponectin levels were measured from the serum of study subjects. Our analysis showed that the G allele and the GG genotype of rs1501299 were significantly more frequent in BC patients compared to those in the control group (p-value < 0.05). Moreover, two ADIPOQ haplotypes containing the above G allele were associated with increased BC risk (p-value < 0.05). Multivariate analysis showed that increased serum adiponectin, smoking or age were all significant predictors of BC (p-value < 0.05). The data supports use of serum adiponectin and the G allele of rs1501299 SNP in ADIPOQ as potential biomarkers and/or targets in BC. To further validate findings in this study, larger populations of various ethnicities and/or genetic backgrounds are required. More investigations on the functional role of adiponectin in BC will also provide better understanding of potential targeting adiponectin for BC treatment.

Periodontal regenerative techniques are performed to accomplish the restitution of soft and hard teeth-supporting tissues that have been lost due to trauma or inflammatory disease. Periodontal membranes are used for these techniques to provide support and a framework for cell growth and tissue regeneration. They act as a temporary and selective barrier to cell proliferation. Easy clinical handling, biomechanical specifications, high biocompatibility, cell-occlusivity, and satisfactory bioresorption rate are essential properties a membrane needs to be effective. The creation and maintenance of a secluded space is also a fundamental rule in periodontal regenerative techniques. The use of barrier membranes in the field of restorative dentistry has progressed toward the use of minimally invasive approaches optimizing wound closure and limiting patient morbidity. This review intends to provide an overview of the major cellular events in the surgical wound and membrane surface. It was also performed to assess, from literature data, the pertinence of using non-resorbable and resorbable membranes for this regenerative purpose. Special attention will be given to collagen membranes.

Statins are primary drugs in the treatment of hyperlipidemias. This group of drugs is known for its beneficial pleiotropic effects (e.g., reduction of inflammatory state). However, a growing body of evidence suggests its diabetogenic properties. The culpable mechanism is not completely understood and might be related to the damage to pancreatic beta cells. Therefore, we conceived an in vitro study to explore the impact of atorvastatin on pancreatic islet beta cells line (1.1.E7). We evaluated the influence on viability, insulin, low-density lipoprotein (LDL) receptor, and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. A significant drop in mRNA for proinsulin and insulin expression was noted. Concurrently, a rise in LDL receptor at the protein level in cells exposed to atorvastatin was noted. Further experiments have shown that exenatide - belonging to glucagon-like peptide 1 (GLP-1) analogs that are used in a treatment of diabetes and known for its weight reducing properties - can alleviate the observed alterations. In this case, the mechanism of action of exenatide was dependent on a protein kinase A pathway. In conclusion, our results support the hypothesis that statin may have diabetogenic properties, which according to our study is related to reduced insulin expression. The concomitant use of GLP-1 receptor agonist seemed to successfully revert insulin expression.

Background: Interferon-gamma (IFN-γ) is a chief proinflammatory cytokine with a significant role in the immune response against viral infections. Today there is increasing evidence about the association between individual genetic polymorphisms and cytokines in predicting HBV infection susceptibility. Aim: This study aimed to investigate the association between IFN-γ gene polymorphisms and susceptibility to hepatitis B viral infection (HBV), and the impact of these genetic polymorphisms on IFN-γ production. IFN-γ (+874A/T, rs2430561, and +2109A/G, rs1861494) was genotyped by single-stranded polymorphism-polymerase chain reaction (SSP-PCR) in 126 Egyptians with chronic HBV infection and in 100 healthy control subjects. The plasma levels of IFN-γ were measured by Enzyme-linked immunosorbent assay (ELISA). Results: Compared to the control subjects there was a slight increase in +874TT genotype frequency in HBV patients. However, no statistical significance in IFN-γ (+874A/T and +2109A/G) genotype/allele distribution was demonstrated, indicating the lack of association between these SNPs and susceptibility to HBV infection. In +2109A/G, only AG genotype was observed with a complete abrogation of GG and AA genotypes. Haplotypes between different loci on selected genes showed insignificant changes in their frequency in patients and control subjects. HBV patients had a significantly higher level of IFN-γ (P < 0.001) compared to controls. The maximum significant increase in IFN-γ production was observed in subjects harboring the +874TA genotype. Conclusions: As no association could be characterized between the polymorphism in IFN-γ (+874A/T and +2109A/G) and susceptibility to chronic HBV infection, our data support the concept that IFN-γ gene polymorphisms are not predictors of HBV susceptibility in this segment of the Egyptian population.

Introduction: Lung cancer is the leading cause of cancer-related deaths worldwide. For this reason, huge efforts are being invested in discovering suitable blood biomarkers that would allow early diagnosis and treatment. One of the possible promising candidates for this role are microRNA molecules (miRNAs). The aim of the study was to identify individual blood miRNAs that could be used as potential biomarkers for early diagnosis of lung cancer. Methods: This prospective study analyzed blood samples of 60 patients with early-stage lung cancer, and blood samples of 60 healthy individuals. All study patients with lung cancer had undergone radical pulmonary resection at the University Hospital Ostrava within the study period (2015-2017). Definitive diagnosis of lung cancer was confirmed by histopathology examination of the resected pulmonary specimen. We investigated relative expressions in selected 13 blood miRNAs; the examined miRNAs were miR-126, miR-155, miR-221, miR-21, miR-143, miR-145, miR-133a, let-7a, miR-146a, miR-31, miR-182, let-7g and miR-19b. Results: The outcome of this study showed that the levels of the majority of the tested circulating miRNA in lung cancer patients are significantly altered. The most significant serum miRNA biomarkers for the early detection of lung cancer are as follows: miR-143, let-7g, miR-126, let-7a, and miR-145 (miR-143 and miR-145 have oncogene functions, while miR-126, let-7g and let-7a have suppressor functions). Conclusions: We have demonstrated the excellent diagnostic value of several miRNAs (miR-126, miR-143, miR-145, let-7a and let7g). These have an estimated sensitivity and specificity of 75-85% and 0.90-0.93 AUC. However, these individual miRNA biomarkers require further validation in larger prospective cohorts.

Background: Despite the many benefits that follow antiretroviral therapy (ART) initiation, its chronic use contributes to the early aging of people living with HIV/AIDS (PLWHA). The aim of this cross-sectional study was to trace the prevalence of and investigate possible renal, bone and metabolic changes, as well as cardiovascular risk in 94 asymptomatic PLWHA, relating them to the duration of ART use. Methods: Four groups were evaluated according to ART use: G1 (n = 21), ART-naïve individuals; G2 (n = 17), <2 years; G3 (n = 40), 2-10 years; and G4 (n = 16) on ART for more than 10 years. Results: Our results showed a high prevalence of dyslipidemic individuals (64%), especially in those under ART. Lower creatine phosphokinase levels were observed in G1 as compared to the others (p < 0.05). Regarding the Framingham score, 12.1% of PLWHA showed moderate and high risk, and the highest proportion (38.5%) occurred in G4 (p = 0.003). A decrease in glomerular filtration rates occurred in 20% of patients, which was also more significant in G3 and G4 (p = 0.007). High prevalences of osteopenia and osteoporosis (53.2%) were found, especially in G1 and G4; however, G1 showed the lowest means for alkaline phosphatases (AP, p = 0.04 and BAP, p = 0.005) and osteocalcin (p = 0.005), in addition to higher vitamin-D concentrations (p = 0.04). Conclusions: Our study showed the possible contributory role of ART in these changes, which leads us to reflect on the need for specific conducts and patient care, pointing out the importance of individualized care in an attempt to increase life expectancy.

Rosmarinic acid is a natural polyphenolic compound that is found in different plant species and used for different medicinal purposes. This study aimed to investigate the chemo-preventive effect of rosmarinic acid and evaluate its antitumor efficacy alone or in combination with Paclitaxel in breast cancer mice model. Ehrlich induced mice mammary solid tumor model was used in the study. Mice were treated with oral rosmarinic acid and intraperitoneal Paclitaxel. Inflammation, angiogenesis, and apoptosis were checked. Enzyme linked immunosorbent assay (ELISA), quantitative real time PCR, and immunohistochemical methods were performed. Rosmarinic acid used prior to tumor induction suppressed NF-κB, TNF-α, vascular endothelial growth factor (VEGF) serum levels, and VEGF receptors. It also triggered apoptosis by restoring the levels of P53, Bcl-2, Bax, and caspase-3. Furthermore, in Ehrlich solid tumor mice, rosmarinic acid, and/or Paclitaxel significantly suppressed tumor growth with an increase in apoptotic markers P53 and Caspase-3 levels, and suppressed the Bcl2/Bax ratio. Rosmarinic acid exerted chemo-preventive and therapeutic potential alone or in combination with Paclitaxel. Moreover, rosmarinic acid targets numerous signaling pathways associated with breast cancer.

Aims: To test the hypothesis that spinal cord stimulation (SCS) acutely improves heart rate variability (HRV) and baroreceptor sensitivity (BRS) in patients with heart failure (HF). Methods: SCS (15 minutes) was delivered in four different settings: 90% of maximal tolerated stimulation amplitude (MTA) targeting the T1-T4 spinal cord segments (SCS90T1-4), 60% of MTA (SCS60T1-4), 90% of MTA with cranial (SCS90CR) and caudal (SCS90CA) electrode configuration. HRV and BRS were recorded continuously and stimulation was compared to device off. Results: Fifteen HF patients were included. SCS90T1-4 did not change the standard deviation of intervals between normal beats (SDNN, p = 0.90), BRS (p = 0.55) or other HRV parameters. In patients with baseline SDNN <50 ms, SCS90T1-4 significantly increased SDNN (p = 0.004). Conclusions: Acute SCS at 60-90% of MTA targeting upper thoracic spinal cord segments does not improve autonomic balance or baroreceptor sensitivity in unselected patients with heart failure but may improve HRV in patients with low SDNN.

This study evaluates the protective effect of Echinacoside on acute liver toxicity induced by acetaminophen in mice and the mechanism behind it. Echinacoside and N-Acetyl Cysteine were intragastrically administrated for 7 days, and acetaminophen was intraperitoneally injected into mice 1 h after the last treatment on day 7. At the end of the experimental period, histological examination, parameters for the level of oxidative damage, hepatic malondialdehyde, serum pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1β), UDP-glucuronosyltransferases, and sulfotransferases changes were examined using enzyme-linked immunosorbent assay and standard biochemical procedures. The expression of cytochrome P450 2E1 protein was assessed by western blot, followed by in silico molecular docking. Acetaminophen treatment obviously increased the levels of ALT and AST, changed hepatic histopathology, promoted oxidative stress, decreased antioxidant enzyme activities, and elevated the pro-inflammatory cytokines. Echinacoside significantly attenuated Acetaminophen-induced liver damage in a dose-dependent manner, with the most effective dose at 100 mg/kg. The pretreatments of Echinacoside in different concentrations altered the Acetaminophen-induced hepatotoxicity levels by decreasing the level of liver enzymes, reducing the liver necrosis with vacuolization, decreasing the hepatic malondialdehyde formation, increasing hepatic antioxidants activities, suppressing the pro-inflammatory cytokines (Tumor Necrosis Factor, Interleukin-6 and Interleukin-1beta), inhibiting Nitric Oxide production, enhancing sulfotransferases and UDP-glucuronosyltransferases activities. Notably, the expression of cytochrome P450 2E1 was inhibited by Echinacoside in a dose-dependent manner and the binding energy was -214.3 MeV. Echinacoside showed a significant protective effect against Acetaminophen-induced hepatotoxicity through the inhibition of oxidative stress, the expression of pro-inflammatory cytokines and cytochrome P450 2E1 protein expression.

Context: Clausena excavata Burm. f is a plant used in folklore medicine for the treatment of various ailments in South East Asia. The plant parts contain chemical components that are cytotoxic to many cancer cells. Objective: The study investigated the cytotoxic effects of ethyl acetate, methanol and chloroform C. excavata leaf extracts on the non-small-lung cancer, NCI-H460, cell line. Methods: Based on the 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay, among extracts, ethyl acetate C. excavata leaf extract (EACE) was the most potent anti-NCI-H460 cells, with IC50 value of 47.1 ± 6.1 μg/ml. The effects of EACE on NCI-H460 cells were also determined by clonogenic, 4', 6-diamidino-2-phenylindole (DAPI), and annexin-V-fluorescein isothiocyanate/propidium iodide-PI flow cytometric assays. Reactive oxygen species (ROS) production and apoptotic gene expressions was determined via flow cytometry and real-time quantitative PCR, respectively. Results: EACE-treated NCI-H460 cells after 48 h underwent apoptosis as evident by loss of cell viability, cell shrinkage, and chromatin condensation. The results also showed EACE mediated increase in ROS production by the NCI-H460 cells. After 48 h treatment, EACE increased the pro-apoptotic BAX and decreased the anti-apoptotic Bcl-2, Survivin and c-Myc gene expressions. Conclusions: EACE is a potential anti-lung cancer by increasing cancer cell ROS production and apoptosis.

Backgrounds: Adiponectin, adipocyte-fatty acid binding protein (A-FABP), and Wnt1 inducible signaling pathway protein-1 (WISP-1) are adipokines closely associated with insulin resistance. The aim of the study was to compare their levels in women with gestational diabetes (GDM), type 2 diabetes mellitus (T2DM) and healthy controls and determine their relation to metabolic parameters. Methods: Women with GDM, T2DM and healthy women were included in this cross-sectional study. In addition to adipokines, anthropometric, lipid parameters, markers of insulin resistance and glucose control were assessed in all participants. Results: Compared to healthy controls (n = 35) significantly lower levels of adiponectin were detected in women with GDM (n = 50), whereas in women with T2DM (n = 50) higher levels of A-FABP and WISP-1 and lower levels of adiponectin were found. Women with T2DM had also lower levels of adiponectin and higher levels of A-FABP compared to women with GDM. A-FABP and adiponectin were independently associated with levels of triglycerides, HDL-cholesterol and C-peptide insulin resistance index. WISP-1 correlated only with waist circumference. Conclusions: Adverse adipokines production reflecting dysfunctional fat tissue is less presented in women with GDM than in women with T2DM, but more expressed compared to healthy women.

Carvacrol presents action in Salmonella Typhimurium biofilms, however the antibiofilm mechanism of this compound has not been fully established yet. In the present study, the aim was to evaluate protein profile changes in S. Typhimurium biofilm treated with carvacrol. Proteomic analysis of treated versus untreated biofilm showed several changes in proteins involved with S. Typhimurium biofilm and antioxidant activity. The proteins DsbA (thiol: disulfide interchange protein DsbA), LuxS (S-ribosylhomocysteine lyase), DksA (RNA polymerase binding transcription factor DksA), and SODs (superoxide dismutases) A, B and C had their synthesis decreased after treatment with carvacrol. These proteins play a key role in S. Typhimurium biofilm formation, demonstrating the dynamic antibiofilm action of carvacrol. The differentially expressed proteins identified provide possible action targets for future studies in order to gain more insight into the mechanism of action of carvacrol on S. Typhimurium biofilm.

Health status is determined by the balance of oxidants and antioxidants which protects healthy cells against the threat of internal and external risk factors. Antioxidants such as ascorbate (vitamin C, ascorbic acid) are of fundamental importance in this respect. Ascorbate neutralizes potential damage caused by cellular oxidative stress which may be the greatest risk of damage to healthy tissue. Cellular oxidative stress is mediated by external factors (e.g. psychological stress, physical exertion, drugs, various diseases, environmental pollution, preservatives, smoking, and alcohol) and internal factors (products of cellular metabolism including reactive oxygen species). When the products of oxidative stress are not sufficiently neutralized, healthy cells are at risk for both mitochondrial and DNA damage. In the short term, cell function may deteriorate, while an increased production of proinflammatory cytokines over time may lead to the development of chronic inflammatory changes and diseases, including cancer. Although pharmaceutical research continues to bring effective chemotherapeutic agents to the market, a limiting factor is often the normal tissue and organ toxicity of these substances, which leads to oxidative stress on healthy tissue. There is increasing interest and imperative to protect healthy tissues from the negative effects of radio-chemotherapeutic treatment. The action of ascorbate against the development of oxidative stress may justify its use not only in the prevention of carcinogenesis, but as a part of supportive or complementary therapy during treatment. Ascorbate (particularly when administered parentally at high doses) may have antioxidant effects that work to protect healthy cells and improve patient tolerability to some toxic radio-chemotherapy regimens. Additionally, ascorbate has demonstrated an immunomodulatory effect by supporting mechanisms essential to anti-tumor immunity. Intravenous administration of gram doses of vitamin C produce high plasma levels immediately, but the levels drop rapidly. Following oral vitamin C administration, plasma levels increase slowly to relatively low values, and then gradually decay. With an oral liposomal formulation, significantly higher levels are attainable than with standard oral formulations. Therefore, oral administration of liposomal vitamin C appears to be an optimal adjunct to intravenous administration. In this review, the basic mechanisms and clinical benefits of ascorbate as an antioxidant that may be useful as complementary therapy to chemotherapeutic regimens will be discussed.

Examination of semen characteristics is routinely performed for fertility status investigation of the male partner of an infertile couple as well as for evaluation of the sperm donor candidate. A useful tool for preliminary assessment of semen characteristics might be an artificial neural network. Thus, the aim of the present study was to construct an artificial neural network, which could be used for predicting the result of semen analysis based on the basic questionnaire data. On the basis of eleven survey questions two models of artificial neural networks to predict semen parameters were developed. The first model aims to predict the overall performance and profile of semen. The second network was developed to predict the concentration of sperm. The network to evaluate sperm concentration proved to be the most efficient. 92.93% of the patients in the learning process were properly qualified for the group with a correct or incorrect result, while the result for the test set was 85.71%. This study suggests that an artificial neural network based on eleven survey questions might be a valuable tool for preliminary evaluation and prediction of the semen profile.

In traditional medicine, natural silk is regarded as a cognitive enhancer and a cure for ameliorating the symptoms of heart disease, atherosclerosis, and metabolic disorders. In this review, general characteristics of both silk proteins, fibroin and sericin, extracted from silkworm Bombyx mori and their potential use in the neuronal disorders was discussed. Evidence shows that silk proteins exhibit neuroprotective effects in models of neurotoxicity. The antioxidant, neuroprotective, and acetylcholinesterase inhibitory mechanisms of silk proteins could prove promising in the treatment of neurodegenerative diseases. Owing to their excellent neurocompatibility and physicochemical properties, silk proteins have been used as scaffolds and drug delivery materials in the neuronal tissue engineering. These data support the potential of silk proteins as an effective complementary agent for central and peripheral neurological disorders.

Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability.

The purpose of this review is to discuss rare neurological disorders with respect to communication difficulties typical of children. Firstly, communication disorders with special focus on rare communication neurological disorders are discussed. Secondly, on the basis of literature review, the authors explore clinical studies on the most typical rare children's communication neurological disorders. Thirdly, on the basis of the findings from the clinical studies, they set a few recommendations for their medical therapies and management. The methodology was based on the literature review of research studies exploring the research issue. The findings show that the intervention strategies appear to have positive effects on the improvement of speech and language production among children suffering from Landau-Kleffner syndrome and childhood apraxia of speech. Nevertheless, randomized control trials are needed in order to accelerate and facilitate an early and relevant diagnosis and treatment management. In addition, a multidisciplinary approach seems to be the most appropriate for the accurate diagnosis and comprehensive treatment.

Cobalt is an essential trace element that is known to mimic hypoxia and hypoxic training. Inorganic Co compounds are capable of Hypoxia-inducible factor-1 (HIF-1) activation, resulting in up-regulation of gene expression including erythropoietin (Epo). Experimental studies have demonstrated that Co treatment may increase hypoxic tolerance of different tissues, improve muscle metabolism and exercise performance. Other mechanisms may also involve modulation of steroid hormone and iron metabolism. Based on these experimental studies, in 2017 inorganic cobalt compounds were added into the World Anti-Doping Agency (WADA) prohibited list as doping agents. However, the existing data on beneficial effects of cobalt on exercise performance in athletes are scarce. Similarly, only experimental studies demonstrated exercise-induced decrease in tissue Co levels, whereas human data are inconsistent. In addition, multiple studies have demonstrated that excessive Co intake may be toxic due to prooxidant, proinflammatory, and proapoptotic activity. Therefore, monitoring of Co deficiency and overload is required to prevent potential health hazards in athletes. At the same time, modulation of Co status should be performed through supplementation avoiding excessive doses of inorganic cobalt that are used for doping and are accompanied by adverse health effects of metal toxicity.

Since last decade, biogenic synthesis of metal or metal-oxide nanoparticles is emerging as an alternative method, which is environment friendly, simple and safe to use. In this article, fruit waste (peel) extract (FWE) of three citrus fruits viz. Citrus limon, Citrus sinensis, and Citrus limetta were used as bio-reductant for green and sustainable synthesis of silver nanoparticles (AgNPs). As-synthesised AgNPs were characterized by using UV-vis spectroscopy, Dynamic light scattering, and High Resolution Transmission Electron Microscopy. TEM studies revealed 9-46 nm size range of synthesized AgNPs. The antimicrobial and antioxidant activities were also studied by using Agar well diffusion method and DPPH Assay, respectively. Nanoparticles showed good antimicrobial activity against both Gram positive (S. aureus) and Gram negative (E. coli) bacteria. Further, bioactivity assays revealed selective cytotoxicity (anticancer) of the nanoparticles against human lung cancer cell line A549. The nanoparticles are able to induce cancer cell specific apoptosis at G0/G1 phase of cell cycle. The results showed potential mechanism of action of nanoparticles via augmentation of antioxidant system in cancer cells. Over all, this study show multifaceted potential bioactivities of nanoparticles generated from fruit waste.

The urokinase-type plasminogen activator (uPA) and PA inhibitor 1 (PAI-1) play important roles in breast cancer metastasis through cell migration and invasion. They are clinically applicable prognostic and predictive markers. High levels of uPA and PAI-1 are associated with high risk of recurrence and adjuvant chemotherapy provides substantial benefit for this breast cancer population. The current sole validated method for quantifying uPA level in breast tumour tissue is ELISA assay. It requires 50-300 mg of fresh or frozen tissue, which is the main limitation for routine use. In this study, we evaluated the performances of customized antibody microarray to quantify uPA concentration from reduced extraction solution of breast tumour tissue and compared it with standard ELISA kit. We firstly optimized the elaboration of customized antibody microarray in order to sensitively detect and quantify uPA standard solutions. In the best conditions, we analysed uPA concentration in 16 cytosolic extracts from breast tumour tissue. Results showed that our customized antibody microarray could correctly quantify uPA concentration while consuming 100 times less volume of tumour tissue extraction solution than ELISA. Our antibody microarray is a powerful and promising tool for the miniaturization of the immunoassay quantification of uPA from breast tumour tissue extracts.

The purpose of this study is to evaluate current legislative policies and regulations with respect to the use of orphan drugs, to emphasize strengths and weaknesses of these policies in the period of 2010-2016 in European countries, especially in relation to the objectives set by the European Council in 2009. This was done by the method of literature search in the databases PubMed, Scopus, ScienceDirect, and Web of Science from 2010 to 2016. The findings show a lack of polices for the appraisal and reimbursement of orphan drugs, a lack of uniform European legislation and different orphan drugs expenditure.

Background/Objectives
To determine effects of gender, age, diet, and smoking status on circadian rhythm characteristics of ascorbic acid (vitamin C).
Subjects/Methods
Ascorbic acid was measured spectrophotometrically in serum collected from 162 healthy volunteers (103 males and 59 females; 7-75 years) every 6 h for 24 h (4 samples). Data were analyzed by single and population mean cosinor. Effects of gender, age, diet (vegetarian vs. omnivore), and smoking status on the rhythm-adjusted mean (MESOR) and circadian amplitude were examined by multiple analysis of variance.
Results
A circadian rhythm is documented with statistical significance by population mean cosinor. In addition to effects of gender and age, the MESOR is affected by diet and smoking status. The circadian amplitude changes nonlinearly as a function of age. The circadian acrophase advances with increasing age.
Conclusion
The present observations confirm a definite circadian rhythm in ascorbic acid concentrations with significant effects of age, diet and smoking status in clinical health. Mapping the circadian rhythm of serum ascorbic acid in health can help explore its role in different pathophysiological conditions as pre-disease conditions may be characterized by alterations in the circadian amplitude and/or phase before there is a change in mean value.

Tibial pseudarthrosis often features deficient bone formation, excessive bone resorption, and extensive pathological fibrosis, particularly in individuals with Neurofibromatosis type I (NF1). It was hypothesized that overactive NF1-Ras-JNK signalling may underlie the pathological fibrosis, and that this could be treated via a JNK antagonist. CC-930, a small molecule JNK inhibitor, was trialed in closed fractures in wild type mice CC-930 (25 mg/kg/twice daily) was dosed throughout fracture healing (D2-21) and during the latter stages of repair (D11-21). All fractures healed by D21, regardless of treatment, with some of the CC-930 (D11-21) treatment group showing early bridging. CC-930 (D11-21) was tested in an Nf1-null fracture model where Nf1 was inactivated by Ad-Cre virus injection in Nf1^flox/flox mice; these mice also possessed a Cre-responsive tdTomato transgene. CC-930 resulted in a significant decrease in non-unions (93% vehicle vs. 64% CC-930, p < 0.01). Local treatment with the bone anabolic rhBMP-2 (10 μg) increased union and callus bone volume, but also increased the fibrotic tissue at the fracture site. Fractures treated with a combination of rhBMP-2 (10 μg) and CC-930 were all partially or fully bridged by D21 (p < 0.01 vs. vehicle) and there was a significant decrease in fibrosis vs. rhBMP-2 alone (p < 0.01). In untreated Nf1-null fractures, the tdTomato transgene was expressed in fibrous tissue at the fracture site, but not in the newly forming bone. These data suggest that JNK inhibition may be an effective therapeutic approach for reducing pathological fibrosis in NF1 tibial pseudarthrosis, however other adjunctive strategies may be required to augment bone formation.

Plants offer unlimited source of bioactive compounds that have tremendous applications in pharmaceutical industry. To find new sources of antioxidants and antimicrobial agents against pan-drug resistant (PDR) pathogens of skin burn infections, methanolic extracts of nine Egyptian plants were evaluated. Total phenolic content varied from 19.48 to 65.48mg GAE/g dry weight (dw). Total flavonoid content varied from 2.90 to 11.09mg QE/g dw, while total alkaloid content varied from 18 to 60mg/g dw. Suaeda vermiculata, Varthemia candicans and Arthrocnemum glaucum showed the highest content of phenolics, flavonoids and alkaloids, respectively. The results of antioxidant (DPPH assay) activity were varied to a great extent and Varthemia candicans (90.6%) and Suaeda vermiculata (90.5%) showed the highest antioxidant activities. Antimicrobial activity was assessed against four PDR bacterial and fungal strains; namely Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans and Aspergillus flavus. Among all examined extracts, Salsola vermiculata and Suaeda vermiculata showed significant antimicrobial activity. The fatty acid composition of Salsola vermiculata and Suaeda vermiculata leaf extracts was detected using GC-MS analysis. Overall, Salsola vermiculata and Suaeda vermiculata could be used as an alternative source for the exploration of new antioxidant and antimicrobial agents that are potentially valued for food and biomedical applications.

Purpose
Cadmium (Cd) is a classic cumulative nephrotoxicant and literature suggests that its toxicity is associated with oxidative stress and inflammation which contribute to pathologies in various tissues. We sought to investigate whether polyphenols isolated from virgin coconut oil (VCO) would modulate nephrotoxicity and inflammation induced by Cd in rats.
Methods
Rats were administered polyphenols prior to and along with Cd (5 mg/kg, orally) for 7 weeks. Serum markers of renal damage, interleukin-6 (IL-6), C-reactive protein (CRP) and nitric oxide (NO) were evaluated; renal activities of antioxidant enzymes, as well as malondialdehyde (MDA) and reduced glutathione (GSH) content were determined. Histopathologic alterations were evaluated to define kidney damage.
Results
Cadmium exposure induced nephrotoxicity and oxidative stress evident by significantly increased serum levels of creatinine, urea, and uric acid along with remarkable depression in renal activities of antioxidant enzymes and GSH with prominent increase in MDA. Inflammatory markers - IL-6, CRP and NO were significantly increased and confirmed by histopathology. Sub-chronic administration of VCO polyphenols attenuated the Cd-induced biochemical alterations compared to Cd control with remarkably improved histopathological observations.
Conclusion
The findings showed that VCO polyphenol supplementation protects against Cd-induced nephrotoxicity via its antioxidant and anti-inflammatory mechanisms in rats.

In the present study the effect of pramipexole alginate nanodispersion (PAND) was studied on the transgenic Drosophila exhibiting the PD symptoms. The PD flies were allowed to feed on the diet having PAND at final concentration of 1, 2 and 3 μM. A dose dependent significant delay in the loss of climbing ability and improvement in the activity was observed in PD flies. A dose dependent significant change in the oxidative stress markers and dopamine content was also observed in the PD flies exposed to various doses PAND. The improvement in the PD symptoms was more in PD flies exposed to PAND compared to PD flies exposed to pramipexole alone.

Objective
There is an increasing incidence of Helicobacter pylori eradication therapy failure secondary to antibiotic resistance. In this study, we aimed to assess the P-glycoprotein expression levels among subjects who were H. pylori-positive and received multiple courses of eradication therapy to understand factors that may associate with their non-responses.
Methods
Eleven resistant subjects were recruited during their hospital visit for upper gastrointestinal endoscopies. H. pylori infection status was confirmed by rapid urease test and bacterial culture. P-glycoprotein expressions from the antral and duodenal biopsies were measured by Western blot. The data was compared with H. pylori-negative (n = 54) and H. pylori-positive-treatment naïve (n = 22) that were recruited earlier in another study.
Results
The resistant group showed higher relative antral P-glycoprotein expression compared to the H. pylori-negative group (p = 0.0361). Most subjects demonstrated resistance to clarithromycin (72%), metronidazole (63.6%) or both (54.5%). No significant difference was observed between the P-glycoprotein expression in H. pylori-treatment resistant and H. pylori-positive-treatment naïve groups (p = 0.319).
Conclusion
H. pylori infection induces the expression of P-glycoprotein in the antrum. This study shows a potential protective mechanism which may be exploited through the use of P-glycoprotein inducers, such as rifabutin, may be beneficial in eradication regimens.

The aim of the presented research was to formulate, prepare and evaluate novel film wound dressings containing lidocaine hydrochloride. The conversion of partially substituted fibrous sodium carboxymethylcellulose (CMC) to an acidic form of CMC enabled the formation of an insoluble matrix which consequently provided the prepared films with excellent handling properties in their wet state. The drug concentration which was incorporated into an external layer of the film was 5 mg/cm². The films demonstrated satisfactory mass and drug content uniformity as well as an acidic surface pH advantageous for wound application. An in vitro drug release test proved that the insoluble CMC matrix served as a reliable carrier without slowing down the release of lidocaine hydrochloride - more than 90% of the drug was released during the first 15 min, indicating a quick rate of anesthetic action. The prepared films could be potential wound dressings for comfortable and efficient topical anesthesia before/after procedures on the wound.

Cannabinoids have been indicated for the treatment of glaucoma in humans. However, pharmacological studies in other species are lacking. Healthy Beagle dogs were treated with 0.1% cannabinoid eyedrops for 3 or 7 days. Intraocular pressure (IOP) and pupillary diameter (PD) were measured. To evaluate whether the topical cannabinoid formulation affects the motor and central nervous systems, a parallel study was performed. Male Swiss mice received ocular or intraperitoneal (i.p.) cannabinoid solution for 1 day (acute) or 7 days (subacute) and were tested in the open field (OF), elevated plus maze (EPM), open field habituation test (HT), and marble burying test (MBT). The treated dogs exhibited a significant reduction of IOP and PD. Acute i.p. cannabinoid administration reduced locomotion in mice in the OF and increased the number of entries into the open arms of the EPM. Subacute ocular cannabinoid administration increased the time spent on the closed arms and reduced the time spent on the open arms of the EPM. The cannabinoid i.p. and ocular did not exert anxiogenic effects in the MBT. These results indicate that the cannabinoid reduced IOP when used topically, and psychotropic effects occurred only with systemic administration.