Synthesis of the antigen bovine serum albumin‐ergosterol and its immunocharacterization

Fungal contamination of agricultural commodities leads to their spoilage and renders them unfit for human consumption. Ergosterol, a predominant sterol in most fungi and a major constituent of the cell membrane, has been established as a reliable biochemical marker for fungal growth. Various chemical and physico‐chemical methods to quantify ergosterol as an index of fungal contamination are in practice. Yet, immunoassays are the methods of choice in food analysis due to their increased specificity, sensitivity and rapidity. This paper reports the synthesis of an antigen, bovine serum albumin‐ergosterol conjugate, and its immunocharacterization. Ergosterol was converted to ergosterol hemisuccinate (EHS) by succinylation. Subsequently, EHS was conjugated to bovine serum albumin by the mixed anhydride reaction. The molar ratio was found to be 1:28. Antisera raised against the synthesized antigen in rabbits was characterized by the Ouchterlony double‐diffusion technique and an antibody capture assay. Ouchterlony analysis showed a titre of 1:2. Further, characterization by an antibody capture assay, using 50 ng well (10 ng equivalent of ergosterol) of the antigen, gave a titre of 1:4000 dilution of antiserum, with an absorbance of 1.0 at 405 nm. The synthesized antigen may find an application in the development of an immunoanalytical method for ergosterol quantification as a measure of food quality in relation to fungal contamination.     

An AscI boundary library for the studies of genetic and epigenetic alterations in CpG islands

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.     

Sequential parametric optimization of lipase production by a mutant strain Rhizopus sp. BTNT-2

Lipase production by the mutant strain Rhizopus sp. BTNT-2 was optimized in submerged fermentation. Different chemical and physical parameters such as carbon sources, nitrogen sources, oils, inoculum level, pH, incubation time, incubation temperature and aeration have been extensively studied to increase lipase productivity. Potato starch (1.25% w/v) as a carbon source, corn steep liquor (1.5% w/v) as a nitrogen source and olive oil (0.5% v/v) as lipid source were found to be optimal for lipase production. The optimal levels of other parameters are 4 ml of inoculum (2.6x10(8) spores/ml), initial pH of 5.5, incubation time of 48 hours, incubation temperature of 28 degrees C and aeration rate of 120 rpm. With the optimized parameters, the highest production of lipase was 59.2 U/ml while an yield of only 28.7 U/ml was obtained before optimization resulting in 206% increase in the productivity.     

An assessment of a urinary biomarker for total human environmental exposure to benzo[a]pyrene

Urinary banzo[a]pyrene (BaP) metabolite levels were compared to human environmental exposure to BaP through inhalation and dietary ingestion to assess the predictive validity of the exposure biomarker. These measurements were made for 14 adult volunteers over 14 consecutive days, once during summer/fall, again during winter periods. Based on personal air monitoring, median potential inhalation doses of 11.0 and 2.3 ng/day were estimated for the winter and summer/fall studies, respectively. A median potential ingested dose of 176 ng/day, estimated from duplicate plate sampling, exceeded inhalation by 6-and 122-fold for the winter and summer/fall studies, respectively. Total urinary BaP metabolites were measured using a published reverse metabolism (BaP) method of analysis. Median rates of urinary BaP metabolite elimination for the winter and summer/fall studies were 121 and 129 ng/day, respectively. The changes in inhaled and ingested potential doses were regressed on the change in urinary metabolite elimination from week 1 to week 2 to test the predictive validity of the biomarker measurement. The regression was statistically significant (r = 0.620, p = 0.015, n = 25) when body weight was included and two extreme values were removed. Consistent with the exposure measurements showing diet as the dominant route of exposure, most of the variation in urinary metabolite elimination was explained by the ingested dose. It is concluded that the measurement of urinary BaP by reverse metabolism is qualitative and of marginal predictive validity as an exposure biomarker due to the method's low recoveries and the large unexplained variance.     

Immunohistochemical localization of receptors for progesterone and oestradiol-17 beta in the implantation site of the rhesus monkey

The aim of the present study was to examine the cellular basis of the involvement of oestradiol and progesterone in blastocyst implantation in the primate. To this end, the cellular distribution of receptors for oestradiol (ER) and progesterone (PR) in fetal trophoblast cells and in endometrial compartments of timed lacunar (pre-villous) and villous stages of placentation in primary implantation sites collected on days 13-22 of gestation were investigated in rhesus monkeys. Both in pre-villous stage tissue and in villous stage tissue, cytotrophoblast cells and syncytiotrophoblast cells and other trophoblast derived cells were PR positive, while they were generally ER negative. Maternal endometrial cells were ER negative, while epithelial cells, stromal cells and vascular endothelial cells in maternal endometrium showed heterogeneous staining patterns for PR depending on their relative location; these patterns, however, correlated well with glandular hyperplasia and differentiation, stromal-decidual transformation and vascular response seen during blastocyst implantation.     

Sevoflurane output from the Oxford Miniature Vaporizer in drawover mode

The sevoflurane output from the Oxford Miniature Vaporizer (OMV) was measured in a series of bench tests in a drawover system. Using a range of settings on two OMVs in series, output ranged from 0.5 to 5.9% with a 600 ml tidal volume at a rate of 12 breaths/min produced by a Penlon Multivent. The OMVs' output was steady over 40 minutes. In tests with tidal volumes ranging from 300 ml to 1200 ml, the OMVs' output was lower at higher minute volumes. When continuous flow of carrier gas at 7.2 l/min was used, the output was lower than at the equivalent intermittent flow rate. Using two OMVs with a drawover system appears to be a feasible technique for the induction and maintenance of sevoflurane anaesthesia. Clinical tests of sevoflurane drawover anaesthesia are now required so that the advantages of sevoflurane can gain wider application in field and military anaesthesia.     

Can trained volunteers provide psychosocial support to patients undergoing radiotherapy? The perspective of patients and volunteers

PurposeClinic-based psychosocial interventions, including volunteer-based ones, may be a cost-efficient and acceptable means of integrating psychosocial support into cancer care during radiotherapy. The present study evaluated a new psychosocial volunteer support program in a large radiotherapy clinic.Methods and MaterialsPatients were asked to complete a demographic and satisfaction with care questionnaire. Clinic volunteers were asked to report their interactions with patients on shift logs.ResultsOf the 182 participating patients, 93 (51%) recalled meeting a volunteer in the clinic, with the 2 most common support types provided being the following: “listening and caring,” and “information on services.” Analysis of 224 volunteers' shift logs indicated that almost all interactions (94%) were initiated by the volunteers, and almost half (47%) involved the patients' companions in the clinic. The most common support type documented was “information and navigation” (71%), followed by “emotional” (47%), “diversional” (21%), and “physical/practical” (17%) support.ConclusionsTrained volunteers can effectively provide clinic-based psychosocial support and information to a high proportion of radiotherapy patients. These findings demonstrate that volunteer support is a feasible means of meeting the psychosocial needs of patients with cancer attending outpatient radiotherapy clinics, who may not require or want professional psychosocial support.     

Exposure–response analyses of blood pressure and heart rate changes for methylphenidate in healthy adults

The aim of the study was to evaluate the exposure–response (E–R) relationships of blood pressure (BP) and heart rate (HR) changes in healthy adults taking methylphenidate (MPH). Intensive time profiles of BP and HR from healthy adults in placebo and MPH treatment arms of seven clinical trials from the FDA internal database were utilized for this analysis. The analysis model contains a circadian component for placebo effect and an E–R component to describe drug effect. Internal validation was performed using goodness-of-fit plots and visual predictive check. A meta-database based on a systemic literature search was constructed and used for external validation of the developed models. We found that circadian models could quantify the time profiles of BP/HR in placebo arms. Linear models could describe the correlations between MPH concentrations, and BP/HR changes. The BP and HR changes were highly dependent on the shapes of MPH pharmacokinetic (PK) profiles without an apparent time delay. MPH has the greatest effect on HR, followed by systolic BP, and diastolic BP. Internal validation revealed that the developed models could adequately describe the circadian rhythms of HR and BP in placebo arms and the E–R relationships of MPH. External validation showed the models had good predictive capability of the literature data. In conclusion, the developed models adequately characterized the circadian rhythm and the MPH induced effects on BP and HR. The changes in BP and HR were highly correlated with MPH blood levels with no apparent delay. The time courses of BP and HR are similar to the MPH PK profiles. As a result, the immediate-release formulation may yield larger maximum BP and HR effect than the extended-release formulation under similar dose.