Intriguing role of novel ionic liquids in stochastic degradation of chitosan.

Green technique for hydrolysis of chitosan was developed using novel Brønsted Acidic Ionic Liquids (BAILs) as homogenous reusable catalysts. Efficiency of BAILs in controlling stochastic and irregular breakdown of chitosan was compared with that of mineral acids. Structural elucidation of the novel BAILs was performed using H1-NMR evaluation and supplemented using mass spectroscopy. Additionally, thermal characterization was conducted using TGA-DTA analysis, while acidity was estimated by deriving the Hammet acidity function. BAILs investigated in this work enabled consistent production of LMWCS variants, with minimum formation of residual impurities. Around 80 % reduction in molecular weight was noted as compared to original under extreme conditions employed. Further, Box-Behnken Design (BBD) was implemented to optimize effect of processing parameters for conversion of chitosan to low molecular weight congeners.

Mechanistic insights into controlled depolymerization of Chitosan using H-Mordenite.

Kinetics of chitosan depolymerization were studied in dilute acetic acid solution, in presence of H-Mordenite (H-MOR). Rate constants for chitosan depolymerization were determined by measurement of molecular weight, using Gel permeation Chromatography (GPC). Depolymerization rate of chitosan was altered in presence of an acidic, porous material like H-MOR. Maximum concentration of H-MOR studied during process led to minimal increase in energy of activation, from 20.54 kJ/moL to 23.25 kJ/moL. Infra-red spectroscopy, adsorption studies and rheological assessment indicated adsorption /grafting of chitosan onto porous H-MOR surface as the possible mechanism for facilitation of the depolymerization process. Under extreme conditions investigated during process optimization, H-MOR resulted in a three-fold reduction in 5-Hydroxy Methyl Furfural (5-HMF) formation and over ten times decrease in glucosamine content, as compared to reactions conducted without H-MOR. Therefore, presence of H-MOR is imperative to cleave chitosan in controlled manner and obtain products of desired molecular weight, with fewer impurities.

Probing synergistic interplay between bio-inspired peptidomimetic chitosan-copper complexes and doxorubicin.

The current investigation reports a novel and facile method for modification of low molecular weight chitosan (Cs) with guanidine moieties, aimed at enhancing its cellular interaction and thus augmenting its cellular internalization. Guadinylated chitosan-copper (Cs-Gn-Cu) chelates, based on copper-nitrogen co-ordination, were established. Characterization of chelates was conducted using 1H NMR, 13C NMR, XPS, XRD, TGA-DTA, and GPC techniques. Anticancer activity of formed chelates was confirmed against A549 cells using MTT assay. Experimental outcomes, for the first time, have provided an empirical evidence for synergistic interaction between the chelated polymer (Cs-Gn-Cu) and the established anti-cancer agent, Doxorubicin (Dox), based on analysis by the Chou Talalay method and estimation of their combination indices. ROS induction was demonstrated as the mechanism of action of the chelated polymer, which supplemented rapid destruction of cancerous cells by Dox. These findings strongly advocate the need for harnessing unexplored potential of these innovative metal polymer chelates in cases of Dox resistant lung cancer, wherein the polymeric system itself would serve as an anti-cancer agent.

Drug-conjugated polymers as gene carriers for synergistic therapeutic effect.

The ability to safely and effectively transfer gene into cells is the fundamental goal of gene delivery. In spite of the best efforts of researchers around the world, gene therapy has limited success. This may be because of several limitations of delivering gene which is one of the greatest technical challenges in the modern medicine. To address these issues, many efforts have been made to bind drugs and genes together by polymers for co-delivery to achieve synergistic effect. Usually, binding interaction of drugs with polymers is either physical or chemical. In case of drug-polymer physical interaction, the efficiency of drugs generally decreases because of separation of drugs from polymers in vivo whenever it comes in contact with charged biofluid/s or cells. While chemical interaction of drug-polymer overcomes the aforementioned obstacle, several problems such as steric hindrance, solubility, and biodegradability hinder it to develop as gene carrier. Considering these benefits and pitfalls, the objective of this review is to discuss the possible extent of drug-conjugated polymers as safe and efficient gene delivery carriers for achieving synergistic effect to combat various genetic disorders.

Enhanced uptake and siRNA-mediated knockdown of a biologically relevant gene using cyclodextrin polyrotaxane.

Ideal cationic polymers for siRNA delivery could result in its enhanced cellular internalization, escape from endosomal degradation, and rapid release in cell cytoplasm, to facilitate knockdown of the target gene. In this study, we have investigated the ability of an in-house synthesized cationic polyrotaxane to bind siRNA into nanometric complexes. This polymer, which had earlier shown improved transfection of model siRNA (luciferase), was used to improve the cellular internalization of the siRNA molecule with therapeutic implications. In cellular assays, the polymer enhanced the knockdown of a gene involved in the pathogenesis of tuberculosis, when the nanocomplexes were compared with free siRNA. The efficacy and cellular non-toxicity of this polymer encourage its further exploitation in animal models of tuberculosis and other intracellular bacterial infections.

Adjunctive growth hormone during ovarian hyperstimulation increases levels of insulin-like growth factor binding proteins in follicular fluid: a randomized, placebo-controlled, cross-over study.

GH increases circulating insulin-like growth factor I (IGF-I), which can promote the growth and differentiated function of ovarian granulosa and theca cells. Reported studies of GH as an adjunct to menotropin stimulation in women, largely those with ovarian dysfunction, have not consistently shown a benefit of GH, despite increases in serum and follicular fluid IGF-I. We hypothesized that changes in intrafollicular IGF-binding proteins (IGFBPs), which can antagonize IGF actions on granulosa cells, may underlie the inconsistent effects of GH. In the present study of GH, administered in double-blind, placebo-controlled, cross-over fashion to regularly cycling women undergoing in vitro fertilization, we found that follicular fluid levels of IGFBP-1, -3, and -4 and serum levels of IGFBP-3, as well as follicular fluid and serum IGF-I, were significantly increased in the GH-treated cycles, when compared with the placebo cycle of the same patient. We suggest that the net increase in intrafollicular IGFBPs in GH cycles may mitigate the potential beneficial effect of increased IGF-I.

Laboratory setup for human in vitro fertilization.

None of the procedures utilized in human IVF are unique or technically very difficult to perform. However, when considering the nature of the material being handled, it is essential that all laboratory procedures, equipment, and supplies are optimal for IVF . The individuals handling the gametes and embryos must be suitably trained and experienced in handling mammalian gametes and embryos before working with human tissues. In addition, the technologists must be trained in proper tissue culture techniques. Above and beyond the training and experience of the technologist, it is necessary that all laboratory equipment and chemicals for medium preparation be of the highest quality and known consistency, and that periodic checks of the culture conditions be performed using the bioassay of mouse embryos. The functioning of the equipment must be constantly monitored and backup systems be in place wherever possible in order to minimize the chances that laboratory accidents will result in loss or damage of the embryos. Meticulous attention to all aspects of the laboratory procedures will result in the patient's having the best possible chance to obtain a pregnancy.

Polypronuclear embryos after in vitro fertilization.

Triploidy occurs in 1% to 3% of recognized conceptions in vivo. In vitro fertilization (IVF), which places large numbers of sperm in proximity to the oocyte, may increase the risk of triploidy. The risk also could be influenced by stimulation protocols that differentially affect egg quality. We found no significant difference in triploidy when cycles were stimulated with clomiphene citrate/human menopausal gonadotropin (3.2%) compared with gonadotropin-releasing hormone analog/human menopausal gonadotropin stimulation (4.4%). Triploidy was less common with male factor infertility (0.7%) compared with other causes of infertility (3.4% to 3.8%). Triploid embryos can develop to cleavage stages indistinguishable from those generated by diploid embryos. Thus, identifying and discarding embryos with greater than 2 pronuclei is a vital function of an IVF laboratory.

Optimizing the sperm penetration assay with human follicular fluid.

Major limitations of the conventional sperm penetration assay are the inability to assess several aspects of sperm function (zona binding and penetration) and the absence of human ovulatory products known to influence fertilization. We therefore modified the sperm penetration assay by the addition of human follicular fluid (FF) to induce the acrosome reaction in an attempt to improve the false-negative rate of the conventional technique. In 26 patients with negative results, results became positive in 20 with human FF and the acrosome reaction increased fourfold. In 19 different men, we compared the results of the conventional and modified assays with outcome of in vitro fertilization. The false-positive rate was the same, but the false-negative rate was reduced from 40% to 7% with the addition of human FF.

Maturation of immature oocytes by coculture with granulosa cells.

To increase the number of embryos available for transfer, immature human oocytes were cocultured with granulosa cells from preovulatory follicles. Greater numbers of immature oocytes incubated with granulosa cells had dispersion of the cumulus and corona cells compared with immature oocytes cultured in media alone. Fifty-four percent of immature oocytes were fertilized after coculture with granulosa cells compared with 20% fertilization of immature oocytes cultured without granulosa cells. There were no cases in which only embryos developed from immature oocytes were transferred, and thus we could not determine if the immature oocytes could contribute to a pregnancy.

Polyovular follicles associated with human in vitro fertilization.

Polyovular follicles were found in 61 (24%) of 251 laparoscopies performed for in vitro fertilization. Of 898 follicles that contained at least one oocyte, 76 (8%) were polyovular. When the oocytes from polyovular follicles were assessed by morphologic criteria, it was found that 46 of the follicles contained oocytes that were discordant in maturity. Thus, oocytes can develop at different rates even though they are exposed to the same follicular fluid. It is possible that the microenvironment provided by the cellular investments of oocytes may correlate more precisely with oocyte maturity.

Insulin-like growth factor I (IGF-I) levels in follicular fluid from human preovulatory follicles: correlation with serum IGF-I levels.

Insulin-like growth factor I (IGF-I) levels were measured in both serum and fluid of preovulatory follicles (n = 156) in 43 women undergoing in vitro fertilization (IVF). The mean IGF-I level in follicular fluid (FF) was significantly lower than in serum (0.52 +/- 0.02 IU/L versus 0.66 +/- 0.23 IU/L), and FF levels were significantly correlated with individual serum IGF-I levels as well as with follicular size and FF volume but not with oocyte maturity, granulosa cell appearance, or IVF. This suggests that FF IGF-I levels cannot serve as a clinical indicator for the degree of oocyte/granulosa cell differentiation or a predictor for IVF. Serum IGF-I levels were inversely correlated with the number of human menopausal gonadotropin ampules administered during treatment, suggesting that IGF-I might enhance ovarian gonadotropic stimulation.

Elevated serum progesterone levels on the day of human chorionic gonadotropin administration do not predict outcome in assisted reproduction cycles.

OBJECTIVE: To determine if the level of serum P drawn on the day of hCG administration predicts assisted reproductive technology (ART) outcome in patients undergoing stimulation with hMG under GnRH agonist (GnRH-a) suppression. DESIGN: Retrospective P assay of stored serum. PATIENTS: One hundred seventy-one patients (189 cycles) who had undergone GnRH-a suppression (leuprolide acetate or nafarelin) and stimulation with hMG for an ART procedure. MAIN OUTCOME MEASURES: Progesterone RIA of serum obtained on the day of hCG administration. Measurement of sequential serum LH values by RIA in those patients with the highest P levels. RESULTS: Pregnancy rates per oocyte retrieval were not correlated with the P level before hCG administration. There were 18 of 54 (33.3%) clinical pregnancies in those cycles with P < 0.9 ng/mL (conversion factor to SI unit, 3.180) and 42 of 135 (31.1%) clinical pregnancies in cycles with a P > or = 0.9 ng/mL. Significantly higher serum E2 levels and numbers of of follicles and oocytes obtained were observed in the high P cycles. There were no differences in the number of oocytes fertilized, the number of embryos transferred, or the implantation rate. However, a significantly higher percentage of mature oocytes were fertilized in the low P cycles (73%), as compared with the high P cycles (60%). CONCLUSIONS: Serum P levels before hCG administration do not predict the outcome of ART cycles in patients suppressed with GnRH-a before hMG stimulation. Lower fertilization rates observed in the high P cycles did not have an effect on clinical outcome.

Follicular size and number in human in vitro fertilization.

In an attempt to maximize the success of in vitro fertilization (IVF) and embryo transfer (ET) as a treatment for human infertility, we have examined the relationship of follicular size and number to the rates of oocyte recovery, fertilization, cleavage, and ET in clomiphene citrate-stimulated cycles. The recovery of oocytes from follicles less than 20 mm in diameter was significantly reduced over that from larger follicles, and those oocytes that were obtained from smaller follicles showed a significantly lower rate of fertilization and cleavage. In addition, the overall chance that a patient would undergo ET was greater in a cycle in which more than one follicle 20 mm or larger was developing than in a cycle in which a single large follicle was developing. This latter observation suggests that attempts at laparoscopic oocyte retrieval should be confined to cycles in which more than one accessible large follicle is developing, thereby maximizing the success rate while minimizing the risk and expense for the patient.

Elevated ovarian follicular fluid stem cell factor concentrations are associated with improved pregnancy rates in in-vitro fertilization cycles.

OBJECTIVE: To determine whether ovarian follicular fluid (FF) stem cell factor concentrations are associated with successful IVF pregnancies. DESIGN: Nested case-control design evaluation of stem cell factor levels from the FF of oocytes fertilized and transferred after controlled ovarian hyperstimulation. SETTING: University-based ART program. PATIENT(S): Infertile women undergoing IVF in a university-based ART program. INTERVENTION(S): Fifty-seven FF samples from a cohort of patients (n = 13) with tubal factor and unexplained infertility were stored at -80 degrees C and subsequently evaluated for stem cell factor concentration. Patients with endometriosis, polycystic ovary disease, and male factor infertility were excluded. Stem cell factor concentrations were measured using a commercially available ELISA kit according to the manufacturer's specifications. The groups were analyzed using a one-way analysis of variance, and significance was determined using the chi2 analysis of contingency table, the unpaired t-test, or the Mann-Whitney rank-sum test. MAIN OUTCOME MEASURE(S): FF stem cell factor concentration, pregnancy. RESULT(S): Stem cell factor concentrations were significantly higher in the FF of the patients who achieved successful pregnancies than in those who did not (641.7+/-75.2 pg/mL versus 475.5+/-50.58 pg/mL). CONCLUSION(S): Elevated FF stem cell factor concentrations are associated with an increased likelihood of IVF success. Therefore, stem cell factor may play a role in human follicular and oocyte development, and increasing infrafollicular stem cell factor concentrations may improve pregnancy rates after oocyte retrieval, fertilization, and ET.

Antral histopathological changes in acid peptic disease associated with Helicobacter pylori.

The histopathology of the antral mucosa of patients with acid peptic disease was studied in relation to Helicobacter pylori infection. Three hundred and fifty-five patients underwent gastroscopy and biopsy on 443 occasions. During each gastroscopy, two antral samples were taken for Rapid Urease Test (RUT) for H. pylori and two antral samples for histopathology. Haematoxylin and Eosin and modified Giemsa stained sections were studied. Histopathological changes in the antrum and the density of H. pylori were graded according to the Sydney System criteria. There was a significant association between the RUT and histology results for detection of H. pylori. The overall prevalence of H. pylori was 61.4% with a maximum incidence in the third and fourth decades of life, and an equal sex distribution. H. pylori colonisation was seen in 90.7% of patients with duodenal ulcer, 66.7% with gastric ulcer and 44.3% with non-ulcer dyspepsia. H. pylori colonisation was associated with more severe antral chronic active gastritis, lymphoid follicles, intestinal metaplasia and dysplasia. Elimination of H. pylori by treatment with anti-H. pylori regimens resulted in regression of the changes.

Challenges in integrating 18FDG PET-CT into radiotherapy planning of head and neck cancer.

Radiotherapy forms one of the major treatment modalities for head and neck cancers (HNC), and precision radiotherapy techniques, such as intensity-modulated radiotherapy require accurate target delineation to ensure success of the treatment. Conventionally used imaging modalities, such as X-ray computed tomography (CT) and magnetic resonance imaging are used to delineate the tumor. Imaging, such as positron emission tomography (PET)-CT, which combines the functional and anatomic modalities, is increasingly being used in the management of HNC. Currently, 18-fluorodeoxyglucose is the most commonly used radioisotope, which is accumulated in areas of high glucose uptake, such as the tumor tissue. Because most disease recurrences are within the high-dose radiotherapy volume, defining a biological target volume for radiotherapy boost is an attractive approach to improve the results. There are many challenges in employing the PET-CT for radiotherapy planning, such as patient positioning, target edge definition, and use of new PET tracers, which represent various functional properties, such as hypoxia, protein synthesis, and proliferation. The role of PET-CT for radiotherapy planning is ever expanding and more clinical data underlining the advantages and challenges in this approach are emerging. In this article, we review the current clinical evidence for the application of functional imaging to radiotherapy planning and discuss some of the current challenges and possible solutions that have been suggested to date.

Intrapulmonary concentrations of telithromycin: clinical implications for respiratory tract infections due to Streptococcus pneumoniae.

BACKGROUND: Antimicrobial efficacy is dependent on the ability of the agent to reach the site of infection. To assess the bronchopulmonary drug disposition of a novel ketolide, telithromycin (TEL), the epithelial lining fluid (ELF) and alveolar macrophage (AM) concentrations were utilized as a surrogate marker for lung penetration. METHODS: Adult subjects scheduled for diagnostic bronchoscopy received oral TEL 800 mg once daily for 5 days. Plasma and bronchoalveolar lavage (BAL) samples were collected 2, 8, 12, or 24 h after the last TEL dose. TEL concentrations in the ELF and AM were determined using a validated HPLC assay. ELF drug concentrations were calculated using the urea dilution method. RESULTS: Seventeen subjects with a mean age 65 +/- 13 years and a mean weight of 81 +/- 25 kg completed this open-label study. The median (range) TEL concentrations in plasma and ELF, respectively, were 1.09 mg/l (1.00-4.81) and 3.91 mg/l (2.64-9.59) at 2 h (n = 6), 0.48 and 1.09 mg/l at 8 h (n = 1), 0.65 mg/l (0.18-1.55) and 1.81 mg/l (0.61-10.0) at 12 h (n = 5), and 0.11 mg/l (0.09-0.24) and 0.69 mg/l (0.15-1.58) at 24 h (n = 5). The median AM concentrations obtained from these subjects were 53.35 mg/l at 2 h, 32.55 mg/l at 8 h, 65.96 mg/l at 12 h, and 26.43 mg/l at 24 h. Overall TEL was well tolerated. No discontinuation was required due to an adverse event. CONCLUSIONS: TEL displayed high intrapulmonary penetration with ELF concentrations exceeding that of plasma at all time points. AM intracellular concentrations were multiple times higher than in the ELF and plasma. These data support the clinical efficacy of TEL against intracellular and extracellular pathogens, particularly with Streptococcus pneumoniae having an MIC(90 )well below achievable concentrations at the site of infection.

Perivitelline space of mammalian oocytes: extracellular matrix of unfertilized oocytes and formation of a cortical granule envelope following fertilization.

Extracellular matrices (ECM) present around unfertilized and fertilized mammalian oocytes were studied ultrastructurally in samples prepared in the presence of ruthenium red to facilitate stabilization of extracellular materials. Unfertilized mouse, hamster, and human oocytes have an ECM comprising granules and filaments in their perivitelline spaces (PVS). This matrix is more abundant in the human than in hamsters and mice. The granule/filament matrix appears identical to the matrix seen between cumulus and corona radiata cells following ruthenium red processing and previously shown to comprise protein and hyaluronic acid. By including ruthenium red during fixation, it is possible to demonstrate the existence of cortical granule exudate in the PVS of fertilized oocytes from hamsters, mice, and humans. Much of the cortical granule exudate is trapped in the PVS and forms a new coat around the fertilized oocyte. This material is particulate when stained with ruthenium red and appears to be uniformly dispersed around the entire oocyte surface. We refer to this new coat as the cortical granule envelope. This envelope is observed in the PVS of all developmental stages up to and including blastocysts in all three species. Following hatching of mouse and hamster blastocysts, the cortical granule envelope is no longer present. Possible functions of this envelope are discussed.