Anti-methicillin resistance Staphylococcus aureus and in vitro toxicology evaluation of corilagin-loaded gelatin/agar microspheres with potential biotextile applications.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged since the early 1960s. The increasing resistance of pathogens to currently used antibiotics requires the urgent discovery of new antimicrobials effective in combating drug-resistant bacteria. From past to present, medicinal plants are useful to cure human diseases. Corilagin (ß-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose), commonly found in Phyllanthus species, exerts potentiating effect on ß-lactams against MRSA. However, its biological effect may not be fully utilized. Therefore, incorporating microencapsulation technology with the delivery of corilagin would be more effective in utilizing the potential effect on biomedical applications. This work reports the development of a safe micro-particulate system which combined agar with gelatin as wall matrix materials for topical delivery of corilagin in order to eliminate the potential toxicity of the crosslinker formaldehyde. The optimal parameters for microsphere preparation were identified and the particle size of optimal microspheres was 20.11 µm ± 3.58. Antibacterial studies revealed that micro-trapped corilagin (minimum bactericidal concentration, MBC = 0.5 mg/mL) possessed a higher potency against MRSA than free corilagin (MBC = 1 mg/mL). The in vitro skin cytotoxicity showed the safety of the corilagin-loaded microspheres for topical applications, with approximately 90 % of HaCaT cell viability. Our results demonstrated the potential of corilagin-loaded gelatin/agar microspheres for the applicable bio-textile products to treat drug-resistant bacterial infections.

A new mild hyperthermia device to treat vascular involvement in cancer surgery.

Surgical margin status in cancer surgery represents an important oncologic parameter affecting overall prognosis. The risk of disease recurrence is minimized and survival often prolonged if margin-negative resection can be accomplished during cancer surgery. Unfortunately, negative margins are not always surgically achievable due to tumor invasion into adjacent tissues or involvement of critical vasculature. Herein, we present a novel intra-operative device created to facilitate a uniform and mild heating profile to cause hyperthermic destruction of vessel-encasing tumors while safeguarding the encased vessel. We use pancreatic ductal adenocarcinoma as an in vitro and an in vivo cancer model for these studies as it is a representative model of a tumor that commonly involves major mesenteric vessels. In vitro data suggests that mild hyperthermia (41-46 °C for ten minutes) is an optimal thermal dose to induce high levels of cancer cell death, alter cancer cell's proteomic profiles and eliminate cancer stem cells while preserving non-malignant cells. In vivo and in silico data supports the well-known phenomena of a vascular heat sink effect that causes high temperature differentials through tissues undergoing hyperthermia, however temperatures can be predicted and used as a tool for the surgeon to adjust thermal doses delivered for various tumor margins.

Preclinical in vivo and in vitro comparison of the translocator protein PET ligands [18F]PBR102 and [18F]PBR111.

PURPOSE: To determine the metabolic profiles of the translocator protein ligands PBR102 and PBR111 in rat and human microsomes and compare their in vivo binding and metabolite uptake in the brain of non-human primates (Papio hamadryas) using PET-CT. METHODS: In vitro metabolic profiles of PBR102 and PBR111 in rat and human liver microsomes were assessed by liquid chromatography-tandem mass spectrometry. [18F]PBR102 and [18F]PBR111 were prepared by nucleophilic substitution of their corresponding p-toluenesulfonyl precursors with [18F]fluoride. List mode PET-CT brain imaging with arterial blood sampling was performed in non-human primates. Blood plasma measurements and metabolite analysis, using solid-phase extraction, provided the metabolite profile and metabolite-corrected input functions for kinetic model fitting. Blocking and displacement PET-CT scans, using PK11195, were performed. RESULTS: Microsomal analyses identified the O-de-alkylated, hydroxylated and N-de-ethyl derivatives of PBR102 and PBR111 as the main metabolites. The O-de-alkylated compounds were the major metabolites in both species; human liver microsomes were less active than those from rat. Metabolic profiles in vivo in non-human primates and previously published rat experiments were consistent with the microsomal results. PET-CT studies showed that K1 was similar for baseline and blocking studies for both radiotracers; VT was reduced during the blocking study, suggesting low non-specific binding and lack of appreciable metabolite uptake in the brain. CONCLUSIONS: [18F]PBR102 and [18F]PBR111 have distinct metabolic profiles in rat and non-human primates. Radiometabolites contributed to non-specific binding and confounded in vivo brain analysis of [18F]PBR102 in rodents; the impact in primates was less pronounced. Both [18F]PBR102 and [18F]PBR111 are suitable for PET imaging of TSPO in vivo. In vitro metabolite studies can be used to predict in vivo radioligand metabolism and can assist in the design and development of better radioligands.

Non-toxic agarose/gelatin-based microencapsulation system containing gallic acid for antifungal application.

Aspergillus niger (A. niger) is a common species of Aspergillus molds. Cutaneous aspergillosis usually occurs in skin sites near intravenous injection and approximately 6% of cutaneous aspergillosis cases which do not involve burn or HIV-infected patients are caused by A. niger. Biomaterials and biopharmaceuticals produced from microparticle-based drug delivery systems have received much attention as microencapsulated drugs offer an improvement in therapeutic efficacy due to better human absorption. The frequently used crosslinker, glutaraldehyde, in gelatin-based microencapsulation systems is considered harmful to human beings. In order to tackle the potential risks, agarose has become an alternative polymer to be used with gelatin as wall matrix materials of microcapsules. In the present study, we report the eco-friendly use of an agarose/gelatin-based microencapsulation system to enhance the antifungal activity of gallic acid and reduce its potential cytotoxic effects towards human skin keratinocytes. We used optimal parameter combinations, such as an agarose/gelatin ratio of 1:1, a polymer/oil ratio of 1:60, a surfactant volume of 1% w/w and a stirring speed of 900 rpm. The minimum inhibitory concentration of microencapsulated gallic acid (62.5 µg/ml) was significantly improved when compared with that of the original drug (>750 µg/ml). The anti-A. niger activity of gallic acid -containing microcapsules was much stronger than that of the original drug. Following 48 h of treatment, skin cell survival was approximately 90% with agarose/gelatin microcapsules containing gallic acid, whereas cell viability was only 25-35% with free gallic acid. Our results demonstrate that agarose/gelatin-based microcapsules containing gallic acid may prove to be helpful in the treatment of A. niger-induced skin infections near intravenous injection sites.

Generalised involuntary limb twitching after ingestion of Mesobuthus martensii Karsch (Quanxie) powder.

Mesobuthus martensii Karsch, commonly known as the Chinese scorpion or Manchurian scorpion, has been used in traditional Chinese medicine as Quanxie to treat chronic pain, tetanus, tremors, convulsion, and paralysis for more than a thousand years. We report a case of poisoning after ingestion of a teaspoon of Quanxie powder. The patient presented with chest pain, dizziness, diaphoresis, generalised involuntary limb twitching, and hypertonia around 15 minutes post-ingestion. The patient recovered uneventfully after supportive management. Intravenous diazepam appeared to be effective in alleviating limb twitching. Failure to accurately measure the dose and to boil before consumption may have contributed to his clinical toxicities.

D-Glucose as a modifying agent in gelatin/collagen matrix and reservoir nanoparticles for Calendula officinalis delivery.

Gelatin/Collagen-based matrix and reservoir nanoparticles require crosslinkers to stabilize the formed nanosuspensions, considering that physical instability is the main challenge of nanoparticulate systems. The use of crosslinkers improves the physical integrity of nanoformulations under the-host environment. Aldehyde-based fixatives, such as formaldehyde and glutaraldehyde, have been widely applied to the crosslinking process of polymeric nanoparticles. However, their potential toxicity towards human beings has been demonstrated in many previous studies. In order to tackle this problem, D-glucose was used during nanoparticle formation to stabilize the gelatin/collagen-based matrix wall and reservoir wall for the deliveries of Calendula officinalis powder and oil, respectively. In addition, therapeutic selectivity between malignant and normal cells could be observed. The C. officinalis powder loaded nanoparticles significantly strengthened the anti-cancer effect towards human breast adenocarcinoma MCF7 cells and human hepatoma SKHep1 cells when compared with the free powder. On the contrary, the nanoparticles did not show significant cytotoxicity towards normal esophageal epithelial NE3 cells and human skin keratinocyte HaCaT cells. On the basis of these evidences, D-glucose modified gelatin/collagen matrix nanoparticles containing C. officinalis powder might be proposed as a safer alternative vehicle for anti-cancer treatments.

A randomized, double-blind, placebo-controlled study of the effect of a Chinese herbal medicine preparation (Dang Gui Buxue Tang) on menopausal symptoms in Hong Kong Chinese women.

OBJECTIVE: Many complementary or alternative medicines are being used for the treatment of menopausal symptoms but most have not been properly tested for efficacy or for safety. This study examined the effect of a Chinese herbal preparation (Dang Gui Buxue Tang) on menopausal symptoms in Hong Kong Chinese women. METHODS: A 6-month randomized, double-blind, placebo-controlled study of the effect of Dang Gui Buxue Tang (a 1 : 5 combination of Dang Gui (Angelicae sinensis) and Huang Qi (Astragalus membranaceus)) on acute menopausal symptoms. A total of 103 symptomatic women were enrolled. Three failed to meet inclusion criteria, leaving 50 subjects for inclusion in each group. RESULTS: Overall, mild hot flushes were reported more frequently than either moderate or severe flushes. In analysis by severity of flushes, there was a significant reduction in the number of mild hot flushes per month in the treatment group but not in the placebo group (from 18.9 +/- 23.5 at baseline to 8.6 +/- 17.1 at 6 months in the treatment group (p < 0.01) and from 26.0 +/- 43.5 to 12.4 +/- 17.6 in the placebo group (p = 0.062)). For moderate flushes, there was a significant reduction in the placebo group compared with the treatment group (from 18.9 +/- 28.7 at baseline to 11.1 +/- 29.9 at 6 months in the placebo group (p < 0.05) and from 10.5 +/- 22.3 to 6.0 +/- 16.0 in the treatment group (p = 0.107)). There was no significant change in either treatment or placebo groups in the reporting of severe hot flushes. Episodes of night sweats decreased significantly in the placebo but not in the treatment group (from 6.8 +/- 10.0 at baseline to 1.9 +/- 5.7 at 6 months in the placebo group (p < 0.05) and from 5.4 +/- 8.9 to 3.2 +/- 8.5 in the treatment group (p = 0.471)). In the vasomotor domain of the Menopause Specific Quality of Life, there was a significant reduction in scoring in the placebo group (from 2.8 +/- 1.6 to 1.7 +/- 1.3, p < 0.01) but not in the treatment group (from 2.8 +/- 2.1 to 2.3 +/- 1.6, p = 0.247). CONCLUSIONS: This study found overall no significant difference between Dang Gui Buxue Tang and placebo in the treatment of vasomotor symptoms in Hong Kong Chinese women. The frequency of mild, moderate and severe hot flushes decreased in both treatment and placebo groups, but Dang Gui Buxue Tang was statistically superior to placebo only in the treatment of mild hot flushes. There were no serious adverse events attributable to treatment during the study period.

Effects of the intensity of downregulation on outcome of in vitro fertilization and embryo transfer.

Gonadotropin releasing hormone agonists (GnRHa) are commonly used during in vitro fertilization and embryo transfer (IVF/ET) treatment cycles to downregulate the hypothalamic-pituitary-ovarian axis prior to ovarian stimulation with gonadotropins. It has been suggested that profound downregulation may have an adverse effect on IVF/ET outcome. The aim of this study was to examine the relationship between the degree of downregulation and IVF/ET outcome. A retrospective analysis was performed on 151 IVF/ET cycles conducted over a six month period. Intensity of downregulation was assessed using measurements of serum concentrations of luteinizing hormone (LH) and estradiol (E2) made at the end of a two week downregulation period. There was no correlation between serum concentration of LH (whether used alone or in combination with E2) and IVF/ET pregnancy rates. However, those subjects who were more suppressed according to the E2 concentration (< 148 pmol/l, [median]) required significantly more gonadotropins (3306 IU versus 2863 IU, p < 0.05) and took longer for follicles to reach maturity (10.9 days versus 9.7 days, p < 0.05). They also had a lower pregnancy rate per embryo transfer (10.4% versus 28.6%, p < 0.05) compared with those having a higher basal E2 concentration. We conclude from this study that the basal serum E2 concentration rather than the LH concentration is a more sensitive indicator of the intensity of downregulation by GnRHa and it may be a better predictor of IVF outcome.

Bioenergetics and RNA/DNA ratios in the common carp (Cyprinus carpio) under hypoxia.

Hypoxia caused by eutrophication occurs over large areas in aquatic systems worldwide. Common carp (Cyprinus carpio) exposed to hypoxia (1 mg.O2.l(-1) and 2 mg.O2.l(-1)) for 1 week showed a significant reduction in feeding rate, respiration rate, faecal production and nitrogenous excretion compared to those maintained at normoxia (7 mg.O2.l(-1)). Fish exposed to hypoxia showed negative scope for growth (SfG), but no significant difference in the specific growth rate was revealed after 1 week in both hypoxic groups. A significant reduction in RNA/DNA ratio was, however, clearly evident in the white muscle of the 1 mg.O2.l(-1) treatment group, but not in the 2 mg.O2.l(-1) treatment group. Both specific growth rate and RNA/DNA ratio were significantly reduced when fish were exposed to severe hypoxia (0.5 mg.O2.l(-1)) for 4 weeks. At all levels of hypoxia, growth reduction was accompanied by a significant decrease in RNA/DNA ratio in white muscle. Covariance analysis showed no significant difference between the slope of RNA/DNA ratio and growth rate under normoxic conditions and 0.5 mg. O2.l(-1) for 4 weeks (F= 1.036, P > 0.326), as well as 1.0 mg.O2.l(-1) and 2.0 mg.O2.l(-1) for 1 week (F = 0.457, P > 0.5), indicating that the RNA/DNA ratio serves as a biomarker of growth under all oxygen levels, at least under controlled experimental conditions. SfG also appears to be more sensitive than the RNA/DNA ratio in responding to hypoxia in fish.

Rational design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors.

Mechanistic information and structure-based design methods have been used to design a series of nonpeptide cyclic ureas that are potent inhibitors of human immunodeficiency virus (HIV) protease and HIV replication. A fundamental feature of these inhibitors is the cyclic urea carbonyl oxygen that mimics the hydrogen-bonding features of a key structural water molecule. The success of the design in both displacing and mimicking the structural water molecule was confirmed by x-ray crystallographic studies. Highly selective, preorganized inhibitors with relatively low molecular weight and high oral bioavailability were synthesized.

The role of transcutaneous electrical nerve stimulation in management of labour in obstetric patients.

The study evaluated the effect of transcutaneous electrical nerve stimulation (TENS) in the management of labour. It comprised of 125 subjects with 93 primiparae and 32 multiparae. They were divided into 3 groups. The first group received electrical stimulation from the TENS unit (TENS); the second group (placebo group) had the TENS machine connected but with no current from the TENS unit (TPL); whereas the third group had no machine application (control group). Analgesic requirements, pain assessment, labour duration, Obstetric outcome, and the comments from mother and midwife were analysed. No negative effects on the mothers and babies were reported. Neither was there any objective clinical significant differences demonstrated among the 3 groups.

Fluorescence-based viability assay for studies of reactive drug intermediates.

Studies of drug toxicity, toxicologic structure-function relationships, screening of idiosyncratic drug reactions, and a variety of cytotoxic events and cellular functions in immunology and cell biology require the sensitive and rapid processing of often large numbers of cell samples. This report describes the development of a high-sensitivity, high-throughput viability assay based on (a) the carboxyfluorescein derivative 2'-7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as a vital dye, (b) instrumentation capable of processing multiple small (less than 100 cells) samples, and (c) a 96-well unidirectional vacuum filtration plate. Double staining of cultured peripheral blood mononuclear cells with BCECF and propidium iodide (PI) showed no overlap between PI+ (nonviable) and BCECF+ (viable) cells by flow cytometric analysis. Optimal conditions were developed for dye loading and minimizing physical cell damage and fluorescence quench during the assay procedure. The ratio of BCECF fluorescence to internal standard fluorescent particles was linear from 40 to greater than 20,000 cells with a signal:noise ratio of approximately 3 at 40 cells/well. Sulfamethoxazole hydroxylamine (SMX-HA) was used as a model toxic drug metabolite to explore the validity of the BCECF procedure. SMX-HA, but not its parent compound sulfamethoxazole, resulted in a dose dependent loss of cellular fluorescence and the parallel accumulation of PI+ nonviable cells. When compared to the currently used tetrazolium dye reduction viability assay, the BCECF method was 3-fold more sensitive, greater than 10-fold faster, and required 1/10-1/100 the cell numbers.

Resistance to Millettia molluscicide in Biomphalaria glabrata: a quantitative genetical approach.

This paper demonstrates the use of a quantitative genetical technique to assess genetic variance in molluscicide resistance in a Millettia thonningii-Biomphalaria glabrata system. Results indicated that there were significant genetic variances in both snail size and survival time in 10 p.p.m. molluscicide suspension. The possibility of wider applications of this technique in examining the genetic basis of molluscicide or pesticide resistance in target species is discussed.

Potentiation of genotoxicity by concurrent application of compounds found in betel quid: arecoline, eugenol, quercetin, chlorogenic acid and Mn2+.

5 components of the betel quid were examined for their clastogenic activities individually and in various combinations. They included the alkaloid, arecoline, from the betel nut (Areca catechu L.), eugenol, from the betel vine (Piper belle L.), chlorogenic acid, from tobacco leaves (Nicotiana tabacum), quercetin, from fennel seeds (Foeniculus vulgare Mill.) and the ubiquitous transition metal Mn2+. The clastogenic effects of the concurrent applications of arecoline plus eugenol, arecoline plus quercetin and arecoline plus chlorogenic acid were greater than the sum of the action of each individual component. Similarly, the combinations of arecoline, chlorogenic acid and Mn2+ induced frequencies of chromosome aberrations which exceeded the sum of the clastogenic activities of individually applied compounds or the sum of the clastogenic activities of 2 jointly applied compounds (arecoline plus Mn2+, or chlorogenic acid plus Mn2+). The clastogenic activity was estimated as the frequency of metaphase plates with at least 1 chromatid break or chromatid exchange, or the average number of chromatid breaks and exchanges per Chinese hamster ovary (CHO) cell. A potentiating (enhancing) action was also evident when 2 clastogens were used at doses which would not lead to a detectable increase in the frequency of chromosome aberrations when applied individually. It may be useful to distinguish between a "genotoxic range", which would be applicable to individually assayed compounds, and a "cogenotoxic range", which may include concentrations at which a chemical exerts a potentiating effect when combined with other genotoxic or non-genotoxic compounds.

The need for a mammalian test system for mutagens: action of some reducing agents.

Reducing agents and cysteine, cysteamine, glutathione, ascorbic acid and H2O2 with and without the addition of Cu2+ did not increase significantly the frequency of mutations in the Salmonella test at non-toxic concentrations but triggered a marked DNA repair synthesis and induced a relatively high frequency of chromosome aberrations in cultured mammalian cells. Both latter effects were reduced by the addition of catalase to solutions of the reducing agents plus Cu2+. To avoid 'False Negatives' in mutagenicity screening the use of several test subjects including mammalian cells seems to be required.

Possible use of short-term tests for carcinogens in experimental epidemiology.

This brief overview pointed out the feasibility of applying the recently developed short-term bioassays for carcinogens/mutagens to the field of epidemiology. The simplicity and economy of these test systems permitted their adaptation for large-scale screening for intrinsic and extrinsic carcinogens with an eye to detecting subpopulations with elevated sensitivity to particular carcinogens.

The search for relevant short term bioassays for chemical carcinogens: the tribulation of a modern Sisyphus.

Based on a good correlation between carcinogenicity and mutagenic activity several rapid microbial bioassays for chemical carcinogens have been recently developed. We would like to suggest, that these microbial tests should be followed by bioassays using cultured human cells of the "average" man, and of persons with elevated cancer risk or increased susceptibility to carcinogenic agents. The main objective of using DNA repair (unscheduled uptake of 3HTdR) and DNA fragmentation (shift in sedimentation profiles) of cultured human cells was to design a test system that can simulate conditions found in man and thus provide information relevant to the human population. A trial on 98 different carcinogens, precarcinogens and noncarcinogens showed the suitability of DNA repair synthesis as a rapid, economic and relevant assay for detection of chemical carcinogens. To check the adaptability of DNA repair synthesis of human cells as a bioassay for chemical carcinogens we examined carcinogenic nitrosation products which are formed from the interaction of nitrite and nitrosatable compounds, carcinogenic or mutagenic photosensitizing chemicals, and the effect of complex interactions. Organotropic carcinogens can be detected by measuring DNA fragmentation and DNA repair in various target organs following the in vivo application of chemical carcinogens. The pros and cons of several bioassays and their usefulness in judging a carcinogenic or mutagenic hazard to human populations is discussed.