Role of Matrix Metalloproteinase-9 in Neonatal Hypoxic-Ischemic Encephalopathy.

BACKGROUND: Neonatal encephalopathy is a heterogeneous syndrome characterised by signs of central nervous system dysfunction in the newborn. Matrix metalloproteinase-9(MMP-9) increases the blood-brain barrier permeability, and their inhibitors can reduce its damage. MMP-9 has been implicated specifically in cerebral ischemia. AIM: To measure serum MMP-9 in neonatal hypoxic-ischemic encephalopathy and evaluate its correlation to the severity of early prediction and treatment. METHODS: its case-control study. The serum concentration of MMP-9 was determined by ELISA in 100 hypoxic neonates and 50 healthy neonates of matched age and sex who served as controls. RESULTS: In our present study the serum MMP-9 level was significantly higher at p = 0.0001 in hypoxic-ischemic full-term newborns (176.7 ± 68.7 ng/ml)as compared to control newborn (69.4 ± 34.85 ng/ml)and it was significantly higher at p = 0.0075 in hypoxic-ischemic preterm newborn (171.2 ± 132.9 ng/ml) when compared to control newborn (72.54 ± 36.74 ng/ml), also MMP-9 was significantly higher at Sarnat stage III at p = 0.0001. CONCLUSION: Serum MMP-9 level was significantly higher in hypoxic-ischemic newborns, and significantly increased with severity, so we suggest that serum MMP-9 level is important for predicting neurological sequel and severity in neonatal encephalopathy.

Epitope-Based Peptide Vaccine Against Fructose-Bisphosphate Aldolase of Madurella mycetomatis Using Immunoinformatics Approaches.

BACKGROUND: Mycetoma is a distinct body tissue destructive and neglected tropical disease. It is endemic in many tropical and subtropical countries. Mycetoma is caused by bacterial infections (actinomycetoma) such as Streptomyces somaliensis and Nocardiae or true fungi (eumycetoma) such as Madurella mycetomatis. To date, treatments fail to cure the infection and the available marketed drugs are expensive and toxic upon prolonged usage. Moreover, no vaccine was prepared yet against mycetoma. AIM: The aim of this study is to predict effective epitope-based vaccine against fructose-bisphosphate aldolase enzymes of M. mycetomatis using immunoinformatics approaches. METHODS AND MATERIALS: Fructose-bisphosphate aldolase of M. mycetomatis sequence was retrieved from NCBI. Different prediction tools were used to analyze the nominee's epitopes in Immune Epitope Database for B-cell, T-cell MHC class II and class I. Then the proposed peptides were docked using Autodock 4.0 software program. RESULTS AND CONCLUSIONS: The proposed and promising peptides KYLQ show a potent binding affinity to B-cell, FEYARKHAF with a very strong binding affinity to MHC I alleles and FFKEHGVPL that shows a very strong binding affinity to MHC II and MHC I alleles. This indicates a strong potential to formulate a new vaccine, especially with the peptide FFKEHGVPL which is likely to be the first proposed epitope-based vaccine against fructose-bisphosphate aldolase of M. mycetomatis. This study recommends an in vivo assessment for the most promising peptides especially FFKEHGVPL.

Effect of GO nanosheets on spectrophotometric determination of tyrosine in urine and serum using nitrosonaphthol.

Here, we aimed to use graphene oxide to improve the selectivity and sensitivity of Tyr determination via the reaction with 1-nitroso-2-naphthol as a selective reagent of Tyr. The reaction between Tyr and 1-nitroso-2-naphthol in absence and presence of GO was studied spectrophotometrically. Different parameters such as concentrations, temperature, incubation time were optimized. The obtained data showed that the maximum absorbance was achieved by using 2 mL of 0.03% 1-nitroso-2-naphthol at temperature 60 °C for 10 min. On the basis of calibration curve of various concentrations of Tyr in the presence of 20 µg mL-1 GO, the limit of detection was 6.4 × 10-6 M (1.15 µg mL-1), where in absence of GO was 1.1 × 10-5 M (19.9 µg mL-1). The selectivity of Tyr in presence of other amino acids and phenols was studied with and without GO. The data obtained revealed that the selectivity of Tyr in presence of GO with respect to some amino acids and phenols was improved. The proposed method has been applied for the determination of Tyr in urine and serum samples. Therefore, GO is a powerful catalytic surface for the sensitive and selective determination of Try in biological fluids.

Uptake of Tyrosine Amino Acid on Nano-Graphene Oxide.

Graphene oxide (GO) is emerging as a promising nanomaterial with potential application in the detection and analysis of amino acids, DNA, enzymes, and proteins in biological fluid samples. So, the reaction of GO with amino acids should be characterized and determined before using it in biosensing methods and devices. In this study, the reaction of tyrosine amino acid (Tyr) with GO was characterized using FT-IR, UV-vis spectrophotometry, and scanning electron microscopy (SEM) before its use. The optimum conditions for GO's interaction with Tyr amino acid have been studied under variable conditions. The optimum conditions of pH, temperature, shaking time, and GO and tyrosine concentrations for the uptaking of tyrosine amino acid onto the GO's surface from aqueous solution were determined. The SEM analysis showed that the GO supplied was in a particle size range between 5.4 and 8.1 nm. A pH of 8.4-9.4 at 25 °C and 5 min of shaking time were the optimum conditions for a maximum uptake of 1.4 µg/mL of tyrosine amino acid onto 0.2 mg/mL of GO.

Validity of a sponge trainer as a simple training model for percutaneous renal access.

OBJECTIVE: To evaluate the efficacy of our simply designed trainer for junior urologists to acquire the initial skills for percutaneous renal access (PRA). SUBJECTS AND METHODS: Three sponge sheets (60 × 50 × 10 cm) were arranged horizontally over each other. A rectangular groove was made in the middle sheet to accommodate an inflated balloon of a Foley catheter, radio-opaque metal balls, metal rings, or a plastic tube that were sequentially placed for the four training tasks. In each session, 18 trainees were asked to pass a fluoroscopically guided puncture needle from a surface point to the placed object in middle sheet. Clinical impact of training was evaluated by an experience survey on a 5-piont Likert scale (for model usefulness, tactile and fluoroscopic-guidance feedback) and success rate in further mentored practice. RESULTS: There was a gradual increase in tasks' and sessions' scores over the training sessions. According to the experience survey after first clinical practice, the mean (SD) score for overall model usefulness by trainees was 3.8 (0.9) with high fluoroscopic guidance reality [3.6 (1.1)] but poor tactile realism [2.3 (0.9)]. On mentored PRA, the success rate for trainees was 78.3%. CONCLUSION: Our early evaluation showed our novel, cost-effective and reproducible sponge trainer could be an effective training model for PRA with a beneficial impact on subsequent clinical practice.

Transferrin conjugated nontoxic carbon dots for doxorubicin delivery to target pediatric brain tumor cells.

Among various cancers, pediatric brain tumors represent the most common cancer type in children and the second most common cause of cancer related deaths. Anticancer drugs and therapies, such as doxorubicin (Dox), have severe side effects on patients during chemotherapy, especially for children as their bodies are still under development. These side effects are believed to be due to the lack of a delivery system with high efficacy and targeting selectivity, resulting in serious damages of normal cells. To improve the efficacy and selectivity, the transferrin (Trans) receptor mediated endocytosis can be utilized for drug delivery system design, as transferrin receptors are expressed on the blood brain barrier (BBB) and often over expressed in brain tumor cells. Carbon dots (C-Dots) have recently emerged as benign nanoparticles in biomedical applications owing to their good water solubility, tunable surface functionalities and excellent biocompatibility. The unique characteristics of C-Dots make them promising candidates for drug delivery development. In this study, carbon dots-transferrin-doxorubicin covalent conjugate (C-Dots-Trans-Dox) was synthesized, characterized by different spectroscopic techniques and investigated for the potential application as a drug delivery system for anticancer drug doxorubicin to treat pediatric brain tumors. Our in vitro results demonstrate greater uptake of the C-Dots-Trans-Dox conjugate compared to Dox alone presumably owing to the high levels of transferrin receptors on these tumor cells. Experiment showed that C-Dots-Trans-Dox at 10 nM was significantly more cytotoxic than Dox alone, reducing viability by 14-45%, across multiple pediatric brain tumor cell lines.

Crossing the blood-brain-barrier with transferrin conjugated carbon dots: A zebrafish model study.

Drug delivery to the central nervous system (CNS) in biological systems remains a major medical challenge due to the tight junctions between endothelial cells known as the blood-brain-barrier (BBB). Here we use a zebrafish model to explore the possibility of using transferrin-conjugated carbon dots (C-Dots) to ferry compounds across the BBB. C-Dots have previously been reported to inhibit protein fibrillation, and they are also used to deliver drugs for disease treatment. In terms of the potential medical application of C-Dots for the treatment of CNS diseases, one of the most formidable challenges is how to deliver them inside the CNS. To achieve this in this study, human transferrin was covalently conjugated to C-Dots. The conjugates were then injected into the vasculature of zebrafish to examine the possibility of crossing the BBB in vivo via transferrin receptor-mediated endocytosis. The experimental observations suggest that the transferrin-C-Dots can enter the CNS while C-Dots alone cannot.

Enhancing selectivity in spectrofluorimetric determination of tryptophan by using graphene oxide nanosheets.

Reaction of formaldehyde with amino acids followed by oxidation with hydrogen peroxide to produce a fluorophore Norharman product is well known and was used for the spectrofluorimetric determination of l-tryptophan (Trp). This study aimed to use graphene oxide (GO) to enhance the selectivity and sensitivity of Trp in presence of other amino acids and possible interfering compounds. Different parameters such as pH, temperature, incubation time, and concentrations of formaldehyde, H2O2 and GO were studied to optimize the condition of determination. Experimental data showed that the maximum fluorescence intensity was achieved in pH 7.0-9.0 phosphate buffer mixed with 7-10% (v/v) formaldehyde and 1-2% (v/v) H2O2 as oxidizing agent at 60°C for 1h. On the basis of calibration curve of various concentrations of Trp in the presence of 20 µg mL(-1) GO, the lower limit of detection (LOD) of Trp was determined as 0.092 nmol mL(-1) and the lower limit of quantification (LOQ) was 0.3 nmol mL(-1). The selectivity of Trp in presence of other amino acids and possible interfering compounds were studied with and without GO. The data obtained after inner filter effect corrections revealed that the selectivity of Trp in presence of amino acids and other possible interfering agents was improved in the range of 76-96%, compared with that in absence of GO. The enhancement of selectivity in the presence of GO indicates that the Trp and other amino acid and possible interfering compounds were adsorbed by GO, and the selective uptaking of Trp-by the reaction with formaldehyde followed by oxidation with H2O2 at 60°C with high selectivity and sensitivity was achieved successfully.

Potentiometric determination of α-L-fucosidase enzyme by using 2-chloro-4-nitrophenol-rhodamine B ion pair chemical recognition in PVC membrane sensor.

The activity of the α-L-fucosidase (AFU) enzyme represents an excellent test for diagnosis of hepatocellular carcinoma (HCC) and fucosidosis recognized in inborn disorder of metabolism and increases the sensitivity of detection to 95.5% in patients with HCC. Therefore, the determination of the activity of AFU enzyme is very important and can be used as a screening tool for the early diagnosis of tumors for HCC patients. A simple, accurate, and sensitive potentiometric method was developed for measuring the activity of AFU. The method was based upon measuring the concentration of 2-chloro-4-nitrophenol (2-chloro-4-NP) using a 2-chloro-4-NP-rhodamine B ion pair in a PVC membrane sensor. The electrode shows a linear, reproducible, and stable potentiometric response with an anionic Nernstian slope of -51.13 ± 0.6 mV/decade over a wide range of concentrations 10(-5)-10(-2) M and a detection limit of 1.0 × 10(-6) M of 2-chloro-4-NP. The membrane exhibits a fast response time of 30 s, over a pH range of 4.0-6.5. The selectivity coefficients indicate excellent selectivity for 2-chloro-4-NP over a number of interfering species, e.g., chloride, nitrate, sulfate, chromate urea, albumin, glucose, uric acid, and total protein. The prepared sensor has been used successfully for the determination of 2-chloro-4-NP produced from the hydrolysis of 2-chloro-4-NP-α-L-fucopyranoside substrate. It was also applied for the determination α-L-fucosidase enzyme of 33 serum samples of healthy subjects and patients. The average recoveries ± RSD for the healthy subjects, cirrhosis of chronic hepatitis C and B, and HCC serum samples were 102.6 ± 1.01%, 101.5 ± 0.95%, and 100.1 ± 1.1%, respectively. The results obtained are in good agreement with those obtained by standard methods.

52-Week oral gavage chronic toxicity study with ubiquinone in rats with a 4-week recovery.

Potential ubiquinone (CoQ10; a natural fermentation product) toxicity was assessed in rats administered CoQ(10) by oral gavage for 1 year at 100, 300, 600, and 1200 mg/(kg day). No adverse changes in mortality, clinical signs, body weight, food consumption, or clinical pathology results occurred. CoQ(10) had elimination half-lives ranging from 10.7 to 15.2 h. At 1200 mg/(kg day), a high incidence of orange, granular, lumenal exudate in nasal turbinates occurred; microscopically, findings similar to those in the turbinates were occasionally observed in small granulomas within lung alveoli. A dose-related increased incidence of vacuolated macrophages (mesenteric lymph nodes) and vacuolated hepatic periportal cells was noted. Neither were associated with tissue damage or organ dysfunction, so they were not considered to be adverse. The nasal turbinate and lung findings were probably secondary to incidental exposure to crystallized test material. Overall, CoQ(10) was well tolerated by male and female rats at dose levels up to 1200 mg/(kg day).

The pharmacokinetics and safety profile of oral ganciclovir combined with zalcitabine or stavudine in asymptomatic HIV- and CMV-seropositive patients.

Two open-label, randomized, multiple-dose, three-way crossover studies were performed to assess the pharmacokinetics and safety of oral ganciclovir 1000 mg q8h in asymptomatic patients seropositive for human immunodeficiency virus and cytomegalovirus. Ganciclovir was administered alone and in combination with zalcitabine 0.75 mg q8h (study 1) or stavudine 40 mg q12h (study 2). In the presence of zalcitabine, the only statistically significant change in the pharmacokinetic parameters of ganciclovir was a 22.2% mean increase in AUC0-8. However, there was no significant change in the renal clearance of ganciclovir when coadministered with zalcitabine, suggesting that the increase in serum ganciclovir concentration cannot be attributed to competition for active renal tubular secretion. No change in zalcitabine pharmacokinetics was observed in combination with ganciclovir. There were no significant changes in the pharmacokinetics of ganciclovir or stavudine when coadministered. Ganciclovir was well tolerated when given alone and in combination with either zalcitabine or stavudine.

The pharmacokinetics and safety profile of oral ganciclovir in combination with trimethoprim in HIV- and CMV-seropositive patients.

AIMS: We investigated the pharmacokinetics and safety profile of oral ganciclovir coadministered with trimethoprim in HIV-and CMV-seropositive patients. METHODS: In an open-label, randomized, 3-way crossover study, 12 adult males received oral ganciclovir 1000 mg every 8h, oral trimethoprim 200 mg once daily, or both drugs concomitantly in a sequence of three 7-day treatment periods. Pharmacokinetic parameters were determined and adverse events recorded for each treatment. RESULTS: The presence of trimethoprim significantly decreased CLr (12.9%, P=0.0068) and increased t1/2 (18.1%, P=0.0378) of ganciclovir. However, these changes are unlikely to be clinically meaningful. There were no statistically significant changes in trimethoprim pharmacokinetic parameters in the presence of ganciclovir, with the exception of a 12.7% increase in Cmin. Ganciclovir was well tolerated when administered alone or in combination with trimethoprin. CONCLUSIONS: There was no clinically significant pharmacokinetic interaction between oral ganciclovir and trimethoprim when coadministered.

Intrahepatic distribution of hydrochlorothiazide and quinidine in rats: implications in pharmacokinetics.

The intrahepatic distributions of a nonmetabolized drug, hydrochlorothiazide (HCTZ) and a highly metabolized drug, quinidine (QD), were studied separately in five anesthetized rats during steady-state intravenous infusion. Liver samples (20-24) from various parts (0.1-0.2 g each) at the end of infusion were collected from each rat and analyzed for drug concentration by HPLC. The distribution of HCTZ in various macro regions appears quite "homogeneous," with a CV of drug concentration for each rat ranging from 2.6 to 4.7% (grand mean, 3.7 +/- 0.8%), whereas that of QD seems less "homogeneous," with a CV ranging from 8.5 to 28.3% (grand mean, 15.6 +/- 7.7%). The above results indicate that drugs that are not complicated by hepatic metabolism may tend to show more "homogeneous" distribution and those that are highly metabolized and/or known to strongly bind to hepatic tissue component(s) may show less "homogeneous" distribution. The results from the present QD study are in contrast with the general, much more heterogeneous distribution found in an isolated in situ perfusion study reported earlier. The implication of the present study in physiological pharmacokinetic and hepatic modeling is discussed.

Erythrocytes as a total barrier for renal excretion of hydrochlorothiazide: slow influx and efflux across erythrocyte membranes.

The potential barrier effect of erythrocytes (RBC) on renal excretion (mainly by tubular secretion) of hydrochlorothiazide (HCTZ) was evaluated in nine anesthetized rats during steady-state iv infusion. Drug concentrations in plasma and blood from the carotid artery and renal vein were assayed by a simple modified HPLC method. Renal extraction ratios were concentration-independent with a mean of 0.17 +/- 0.05 (SD). The renal excretion was found to occur primarily from the drug in plasma; the mean net fractional removal from plasma was 0.57 +/- 0.12, while that from RBC was less than 0.008 +/- 0.041. The virtual total unavailability of HCTZ from RBC (containing approximately 70% of drug in arterial blood) for renal excretion is attributed to relatively slow efflux of drug from RBC to plasma during each passage through the kidney compared with the blood transit time (in seconds). Preliminary in vitro influx and efflux kinetics of HCTZ across RBC membranes were studied using rat and human blood. The flux data could be adequately described by a linear, reversible, closed two-component system model, and the mean equilibration half-times (ET1/2) in rat and human blood were 10.9 and 20.5 min, respectively. The mean residence time of drug in blood circulation of rats was estimated to be 8.32 +/- 1.06 min, which is shorter than the ET1/2. This is consistent with data indicating that distribution equilibrium of HCTZ in arterial blood might not be reached in vivo even at steady state. Other implications of slow transport kinetics of drugs across RBC membranes are discussed.

Stability of diltiazem hydrochloride in aqueous sugar solutions.

The accelerated stability of diltiazem in aqueous sugar solutions, with or without sodium dihydrogen phosphate, was investigated using a high-pressure liquid chromatographic method. The degradation of diltiazem in all sugar solutions followed pseudo first-order kinetics. The shelf-life of diltiazem in the pure sugar solutions (0.28 M) was arranged in the following order: fructose greater than dextrose greater than sucrose = sorbitol = mannitol greater than lactose. Incorporation of 0.1 or 0.2 M sodium dihydrogen phosphate in the sugar solutions resulted in a decrease in the shelf-life of diltiazem. The reduction was most pronounced with fructose. The use of the above sugars (with the exception of lactose) would make it possible to prepare liquid dosage forms of diltiazem extemporaneously which will retain their potencies for a minimum of 50 days.