We recently reported using non-targeted metabolic profiling that serum indolepropionic acid (IPA), a microbial metabolite of tryptophan, was associated with a lower likelihood of developing type 2 diabetes (T2D). In the present study, we established a targeted quantitative method using liquid chromatography with mass spectrometric detection (HPLC-QQQ-MS/MS) and measured the serum concentrations of IPA in all the participants from the Finnish Diabetes Prevention Study (DPS), who had fasting serum samples available from the 1-year study follow-up (n = 209 lifestyle intervention and n = 206 control group). Higher IPA at 1-year study was inversely associated with the incidence of T2D (OR [CI]: 0.86 [0.73–0.99], P = 0.04) and tended to be directly associated with insulin secretion (β = 0.10, P = 0.06) during the mean 7-year follow-up. Moreover, IPA correlated positively with dietary fiber intake (g/day: r = 0.24, P = 1 × 10−6) and negatively with hsCRP concentrations at both sampling (r = − 0.22, P = 0.0001) and study follow-up (β = − 0.19, P = 0.001). Thus, we suggest that the putative effect of IPA on lowering T2D risk might be mediated by the interplay between dietary fiber intake and inflammation or by direct effect of IPA on β-cell function.

Populations of Noccaea caerulescens show tremendous differences in their capacity to hyperaccumulate and hypertolerate metals. To explore the differences that could contribute to these traits, we undertook SOLiD high-throughput sequencing of the root transcriptomes of three phenotypically well-characterized N. caerulescens accessions, i.e., Ganges, La Calamine, and Monte Prinzera. Genes with possible contribution to zinc, cadmium, and nickel hyperaccumulation and hypertolerance were predicted. The most significant differences between the accessions were related to metal ion (di-, trivalent inorganic cation) transmembrane transporter activity, iron and calcium ion binding, (inorganic) anion transmembrane transporter activity, and antioxidant activity. Analysis of correlation between the expression profile of each gene and the metal-related characteristics of the accessions disclosed both previously characterized (HMA4, HMA3) and new candidate genes (e.g., for nickel IRT1, ZIP10, and PDF2.3) as possible contributors to the hyperaccumulation/tolerance phenotype. A number of unknown Noccaea-specific transcripts also showed correlation with Zn(2+), Cd(2+), or Ni(2+) hyperaccumulation/tolerance. This study shows that N. caerulescens populations have evolved great diversity in the expression of metal-related genes, facilitating adaptation to various metalliferous soils. The information will be helpful in the development of improved plants for metal phytoremediation.

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Metabolomics analysis generates vast arrays of data, necessitating comprehensive workflows involving expertise in analytics, biochemistry and bioinformatics in order to provide coherent and high-quality data that enable discovery of robust and biologically significant metabolic findings. In this protocol article, we introduce notame, an analytical workflow for non-targeted metabolic profiling approaches, utilizing liquid chromatography–mass spectrometry analysis. We provide an overview of lab protocols and statistical methods that we commonly practice for the analysis of nutritional metabolomics data. The paper is divided into three main sections: the first and second sections introducing the background and the study designs available for metabolomics research and the third section describing in detail the steps of the main methods and protocols used to produce, preprocess and statistically analyze metabolomics data and, finally, to identify and interpret the compounds that have emerged as interesting.

Conjunctiva occupies most of the ocular surface area, and conjunctival permeability affects ocular and systemic drug absorption of topical ocular medications. Therefore, the aim of this study was to obtain a computational in silico model for structure-based prediction of conjunctival drug permeability. This was done by employing cassette dosing and quantitative structure-property relationship (QSPR) approach. Permeability studies were performed ex vivo across fresh porcine conjunctiva and simultaneous dosing of a cassette mixture composed of 32 clinically relevant drug molecules with wide chemical space. The apparent permeability values were obtained using drug concentrations that were quantified with liquid chromatography tandem-mass spectrometry. The experimental data were utilized for building a QSPR model for conjunctival permeability predictions. The conjunctival permeability values presented a 17-fold range (0.63-10.74 × 10 cm/s). The final QSPR had a Q value of 0.62 and predicted the external test set with a mean fold error of 1.34. The polar surface area, hydrogen bond donor, and halogen ratio were the most relevant descriptors for defining conjunctival permeability. This work presents for the first time a predictive QSPR model of conjunctival drug permeability and a comprehensive description on conjunctival isolation from the porcine eye. The model can be used for developing new ocular drugs.

Thlaspi caerulescens is increasingly acknowledged as one of the best models for studying metal hyperaccumulation in plants. In order to study the mechanisms underlying metal hyperaccumulation, we used proteomic profiling to identify differences in protein intensities among three T. caerulescens accessions with pronounced differences in tolerance, uptake and root to shoot translocation of Zn and Cd. Proteins were separated using two-dimensional electrophoresis and stained with SYPRO Orange. Intensity values and quality scores were obtained for each spot by using PDQuest software. Principal component analysis was used to test the separation of the protein profiles of the three plant accessions at various metal exposures, and to detect groups of proteins responsible for the differences. Spot sets representing individual proteins were analysed with the analysis of variance and non-parametric Kruskal-Wallis test. Clearest differences were seen among the Thlaspi accessions, while the effects of metal exposures were less pronounced. The 48 tentatively identified spots represent core metabolic functions (e.g. photosynthesis, nitrogen assimilation, carbohydrate metabolism) as well as putative signalling and regulatory functions. The possible roles of some of the proteins in heavy metal accumulation and tolerance are discussed.

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F3 families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F3 sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance.

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