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.     

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.     

Identification of predictive MRI and functional biomarkers in a pediatric piglet traumatic brain injury model

Traumatic brain injury(TBI) at a young age can lead to the development of long-term functional impairments. Severity of injury is well demonstrated to have a strong influence on the extent of functional impairments; however, identification of specific magnetic resonance imaging(MRI) biomarkers that are most reflective of injury severity and functional prognosis remain elusive. Therefore, the objective of this study was to utilize advanced statistical approaches to identify clinically relevant MRI biomarkers and predict functional outcomes using MRI metrics in a translational large animal piglet TBI model. TBI was induced via controlled cortical impact and multiparametric MRI was performed at 24 hours and 12 weeks post-TBI using T1-weighted, T2-weighted, T2-weighted fluid attenuated inversion recovery, diffusion-weighted imaging, and diffusion tensor imaging. Changes in spatiotemporal gait parameters were also assessed using an automated gait mat at 24 hours and 12 weeks post-TBI. Principal component analysis was performed to determine the MRI metrics and spatiotemporal gait parameters that explain the largest sources of variation within the datasets. We found that linear combinations of lesion size and midline shift acquired using T2-weighted imaging explained most of the variability of the data at both 24 hours and 12 weeks post-TBI. In addition, linear combinations of velocity, cadence, and stride length were found to explain most of the gait data variability at 24 hours and 12 weeks post-TBI. Linear regression analysis was performed to determine if MRI metrics are predictive of changes in gait. We found that both lesion size and midline shift are significantly correlated with decreases in stride and step length. These results from this study provide an important first step at identifying relevant MRI and functional biomarkers that are predictive of functional outcomes in a clinically relevant piglet TBI model. This study was approved by the University of Georgia Institutional Animal Care and Use Committee(AUP: A2015 11-001) on December 22, 2015.     

Endovascular Therapy for Ischemic Stroke

The utility of intravenous tissue plasminogen activator (IV t-PA) in improving the clinical outcomes after acute ischemic stroke has been well demonstrated in past clinical trials. Though multiple initial small series of endovascular stroke therapy had shown good outcomes as compared to IV t-PA, a similar beneficial effect had not been translated in multiple randomized clinical trials of endovascular stroke therapy. Over the same time, there have been parallel advances in imaging technology and better understanding and utility of the imaging in therapy of acute stroke. In this review, we will discuss the evolution of endovascular stroke therapy followed by a discussion of the key factors that have to be considered during endovascular stroke therapy and directions for future endovascular stroke trials.