Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine–threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [³²P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and V_max of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 700 and 1150 pmol with a linear regression R ² = 0.956.     

LC–MS/MS-based metabolome analysis detected changes in the metabolic profiles of small and large intestinal adenomatous polyps in <Emphasis Type="Italic">Apc</Emphasis><Superscript><Emphasis Type="Italic">Min</Emphasis>/+</Superscript> mice

Introduction

The adenomatous polyposis coli (APC) gene is a tumor suppressor gene that is inactivated in the initiation of colorectal neoplasia. Apc ^Min/+ mice, which possess a heterozygous APC mutation, develop numerous adenomatous polyps, which are similar to those observed in familial adenomatous polyposis (FAP) in humans. However, unlike FAP patients, Apc ^Min/+ mice predominantly develop adenomatous polyps in the small intestine. The metabolic changes associated with the development of polyps in the small and large intestine remain to be investigated.

Objectives

The objective of this study was to elucidate the metabolic changes associated with intestinal polyp formation.

Methods

We compared the metabolite levels of pairs of polyp and non-polyp tissues obtained from the small intestines (n = 12) or large intestines (n = 7) of Apc ^Min/+ mice. To do this, we analyzed the tissue samples using two methods, liquid chromatography-tandem mass spectrometry (1) with a pentafluorophenylpropyl column for cation analysis, and (2) with a C18 reversed phase column coupled to an ion-pair reagent for anion analysis.

Results

Pathway mapping of the metabolites whose levels were significantly altered revealed that the polyp tissue of the small intestine contained significantly higher levels of intermediates involved in glycolysis, the pentose phosphate pathway, nucleotide metabolism, or glutathione biosynthesis than in the equivalent non-polyp tissue. In addition, significantly higher levels of methionine cycle intermediates were detected in the polyp tissues of both the large and small intestines. Organ-dependent (small vs. large intestine) differences were also detected in the levels of most amino acids and urea cycle intermediates.

Conclusion

Our results indicate that various metabolic changes are associated with polyp development, and understanding these alterations could make it possible to evaluate the treatment response of colorectal cancer earlier.

     

Metabolite targeting: development of a comprehensive targeted metabolomics platform for the assessment of diabetes and its complications

Biomarker studies for metabolic disorders like diabetes mellitus (DM) are an important approach towards a better understanding of the underlying pathophysiological mechanisms of diseases (Roberts and Gerszten in Cell Metab 18:43–50, 2013; Wilson et al. in Proteome Res 4:591–598, 2005). Furthermore, screening of potential metabolic biomarkers opens the opportunity of early diagnosis as well as therapy and drug monitoring of metabolic disorders (Rhee et al. in J Clin Invest 10:1–10, 2011; Wang et al. in Nat Med 17:448–458, 2011; Wenk in Nat Rev Drug Discov 4:594–610, 2005). The aim of the present study was to develop methods for the quantitative determination of 74 potential metabolite biomarkers for DM and diabetic nephropathy (DN) in serum. Several studies have shown that the concentrations of many polar metabolites like amino or organic acids are changed in subjects suffering from diabetes (Wang et al. in Nat Med 17:448–458, 2011; Yuan et al. in J Chromatogr B 813:53–58, 2007). Analyzing polar analytes presents a challenge in liquid chromatography (LC) coupled with ESI–MS/MS (Gika et al. in J Sep Sci 31:1598–1608, 2008; Spagou et al. in J Sep Sci 33:716–727, 2010). Considering those reasons we decided to develop a specific HILIC–ESI–QqQ–MS/MS-method for quantitative determination of these polar metabolites. A subsequent method validation was carried out for both HILIC and RP chromatography with respect to the guidelines of the Food and Drug Administration (FDA in Food and Drug Administration: Guidance for industry, bioanalytical method validation, 2001). The HILIC and RP LC–MS methods were successfully validated. Furthermore, the HILIC method presented here was applied to serum samples of GIPR^dn transgenic mice, a diabetic strain developing DN, and non transgenic littermate controls. Significant, diabetes-associated changes were observed for the concentrations of 21 out of 62 metabolites. The new methods described here accurately quantify 74 metabolites known to be regulated in diabetes, allowing for direct comparison between studies and laboratories. Thus, these methods may be highly adoptable in clinical research, providing a starting point for early diagnosis and metabolic screening.     

Evaluation of metabolomics profiles of grain from maize hybrids derived from near-isogenic GM positive and negative segregant inbreds demonstrates that observed differences cannot be attributed unequivocally to the GM trait

Introduction

Past studies on plant metabolomes have highlighted the influence of growing environments and varietal differences in variation of levels of metabolites yet there remains continued interest in evaluating the effect of genetic modification (GM).

Objectives

Here we test the hypothesis that metabolomics differences in grain from maize hybrids derived from a series of GM (NK603, herbicide tolerance) inbreds and corresponding negative segregants can arise from residual genetic variation associated with backcrossing and that the effect of insertion of the GM trait is negligible.

Methods

Four NK603-positive and negative segregant inbred males were crossed with two different females (testers). The resultant hybrids, as well as conventional comparator hybrids, were then grown at three replicated field sites in Illinois, Minnesota, and Nebraska during the 2013 season. Metabolomics data acquisition using gas chromatography–time of flight-mass spectrometry (GC–TOF-MS) allowed the measurement of 367 unique metabolite features in harvested grain, of which 153 were identified with small molecule standards. Multivariate analyses of these data included multi-block principal component analysis and ANOVA-simultaneous component analysis. Univariate analyses of all 153 identified metabolites was conducted based on significance testing (α = 0.05), effect size evaluation (assessing magnitudes of differences), and variance component analysis.

Results

Results demonstrated that the largest effects on metabolomic variation were associated with different growing locations and the female tester. They further demonstrated that differences observed between GM and non-GM comparators, even in stringent tests utilizing near-isogenic positive and negative segregants, can simply reflect minor genomic differences associated with conventional back-crossing practices.

Conclusion

The effect of GM on metabolomics variation was determined to be negligible and supports that there is no scientific rationale for prioritizing GM as a source of variation.

     

Health seeking behavior and use of medicinal plants among the Hamer ethnic group,South Omo zone,southwestern Ethiopia

Background

Health seeking behavior of people around the globe is affected by different socio-cultural and economic factors. In Ethiopia, people living in rural areas in particular, are noted for their use of medicinal plants as a major component of their health care option. This study was conducted to document ethnopharmacological information of the Hamer semi-pastoralists ethnic group in southwestern Ethiopia.

Methods

A cross-sectional study was carried out whereby information on demographic characteristics, prevalence of perceived illnesses, factors associated with preference of health care seeking options, medicinal plants used and hoarded as well as some healers’ socio-economic characteristics were collected using two sets of semi-structured questionnaires – one for household (HH) heads and the other for traditional healers supplemented by focus group discussions (FGDs). Households were selected using a cluster sampling followed by systematic sampling techniques; whereas healers and FGD participants were purposively selected with the assistance of local leaders and elders from the community.

Results

The study revealed that the use of traditional medicine among the Hamer ethnic group is very high. Females preferred traditional medicine more than males. The main reasons for this preference include effectiveness, low cost and ease of availability. Malaria (gebeze) was the most frequently occurring illness in the area identified by all FGD participants. A total of 60 different medicinal plants were reported [34 by HH respondents, 14 by traditional healers and 12 by both]. Fifty-one medicinal plants were fully identified, 3 at generic level and 6 have not yet been identified.

Conclusion

It can be concluded that traditional medical practices, particularly herbal aspect, is widely used by the Hamer ethnic group, although health seeking behavior of the community is affected by different socio-economic and cultural factors.

     

Radiation dose of digital radiography (DR) versus micro-dose x-ray (EOS) on patients with adolescent idiopathic scoliosis: 2016 SOSORT- IRSSD “John Sevastic Award” Winner in Imaging Research

Background

Patients with adolescent idiopathic scoliosis (AIS) frequently receive x-ray imaging at diagnosis and subsequent follow monitoring. The ionizing radiation exposure has accumulated through their development stage and the effect of radiation to this young vulnerable group of patients is uncertain. To achieve the ALARA (as low as reasonably achievable) concept of radiation dose in medical imaging, a slot-scanning x-ray technique by the EOS system has been adopted and the radiation dose using micro-dose protocol was compared with the standard digital radiography on patients with AIS.

Methods

Ninety-nine participants with AIS underwent micro-dose EOS and 33 underwent standard digital radiography (DR) for imaging of the whole spine. Entrance-skin dose was measured using thermoluminescent dosimeters (TLD) at three regions (i.e. dorsal sites at the level of sternal notch, nipple line, symphysis pubis). Effective dose and organ dose were calculated by simulation using PCXMC 2.0. Data from two x-ray systems were compared using independent-samples t-test and significance level at 0.05. All TLD measurements were conducted on PA projection only. Image quality was also assessed by two raters using Cobb angle measurement and a set of imaging parameters for optimization purposes.

Results

Entrance-skin dose from micro-dose EOS system was 5.9–27.0 times lower at various regions compared with standard DR. The calculated effective dose was 2.6?±?0.5 (μSv) and 67.5?±?23.3 (μSv) from micro-dose and standard DR, respectively. The reduction in the micro-dose was approximately 26 times. Organ doses at thyroid, lung and gonad regions were significantly lower in micro-dose (p?<?0.001). Data were further compared within the different gender groups. Females received significantly higher (p?<?0.001) organ dose at ovaries compared to the testes in males. Patients with AIS received approximately 16–34 times lesser organ dose from micro-dose x-ray as compared with the standard DR. There was no significant difference in overall rating of imaging quality between EOS and DR. Micro-dose protocol provided enough quality to perform consistent measurement on Cobb angle.

Conclusions

Entrance-skin dose, effective dose and organ dose were significantly reduced in micro-dose x-ray. The effective dose of a single micro-dose x-ray (2.6 μSv) was less than a day of background radiation. As AIS patients require periodic x-ray follow up for surveillance of curve progression, clinical use of micro-dose x-ray system is beneficial for these young patients to reduce the intake of ionizing radiation.