Transport of asymmetric dimethylarginine (ADMA) by cationic amino acid transporter 2 (CAT2), organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1)

Asymmetric dimethylarginine (ADMA), inhibiting the nitric oxide (NO) synthesis from l-arginine, is a known cardiovascular risk factor. Our aim was to investigate if ADMA and/or l-arginine are substrates of the human cationic amino acid transporters 2A (CAT2A, SLC7A2A) and 2B (CAT2B, SLC7A2B), the organic cation transporter 2 (OCT2, SLC22A2), and the multidrug and toxin extrusion protein 1 (MATE1, SLC47A1). We systematically investigated the kinetics of ADMA and l-arginine transport in human embryonic kidney (HEK293) cells stably overexpressing CAT2A, CAT2B, OCT2, or MATE1. Vector-only transfected HEK293 cells served as controls. Compared to vector control cells, uptake of ADMA and l-arginine was significantly higher (p < 0.05) in cells expressing CAT2B and OCT2 at almost all investigated concentrations, while cells expressing CAT2A only showed a significant uptake at concentrations above 300 μM. Uptake of MATE1 overexpressing cells was significantly (p < 0.05) higher at pH 7.8 and 8.2 than controls. Apparent V _max values (nmol mg protein^?1 min^?1) for cellular uptake of ADMA and l-arginine were ≈11.8 ± 1.2 and 19.5 ± 0.7 for CAT2A, ≈14.3 ± 1.0 and 15.3 ± 0.4 for CAT2B, and 6.3 ± 0.3 and >50 for OCT2, respectively. Apparent K _m values (μmol/l) for cellular uptake of ADMA and l-arginine were ≈3,033 ± 675 and 3,510 ± 419 for CAT2A, ≈4,021 ± 532 and 952 ± 92 for CAT2B, and 967 ± 143 and >10,000 for OCT2, respectively. ADMA and l-arginine are substrates of human CAT2A, CAT2B, OCT2 and MATE1. Transport kinetics of CAT2A, CAT2B, and OCT2 indicate a low affinity, high capacity transport, which may be relevant for renal and hepatic elimination of ADMA or l-arginine.     

The disunity of "Mysidacea" (Crustacea)

New studies on malacostracan relationships have drawn attention to issues concerning monophyly of the order Mysidacea, manifested in recent crustacean classifications that treat the taxon as two separate orders, Lophogastrida and Mysida. We present molecular phylogenies of these orders based on complete sequences of nuclear small-subunit ribosomal DNA (18S rRNA), and morphological evidence is used to revise the classification of the order Mysida to better reflect evolutionary history. A secondary structure model for 18S rRNA was constructed and used to assign putative stem and loop regions to two groups of partitions for phylogenetic analyses. Phylogenies were estimated by maximum-likelihood, Bayesian inference, and maximum-parsimony. The analyses gave strong support for three independently derived lineages, represented by three monophyletic groups, Lophogastrida, Stygiomysida, and Mysida. The family Petalophthalmidae is considered as sister group to the family Mysidae, and Boreomysinae and Rhopalophthalminae are the most early derived of the Mysidae. The tribes contained in the current classification of the subfamily Mysinae are not well-supported by either molecular data or morphology.     

Acute Kidney Injury Urinary Biomarker Time-Courses

Factors which modify the excretion profiles of acute kidney injury biomarkers are difficult to measure. To facilitate biomarker choice and interpretation we modelled key modifying factors: extent of hyperfiltration or reduced glomerular filtration rate, structural damage, and reduced nephron number. The time-courses of pre-formed, induced (upregulated), and filtered biomarker concentrations were modelled in single nephrons, then combined to construct three multiple-nephron models: a healthy kidney with normal nephron number, a non-diabetic hyperfiltering kidney with reduced nephron number but maintained total glomerular filtration rate, and a chronic kidney disease kidney with reduced nephron number and reduced glomerular filtration rate. Time-courses for each model were derived for acute kidney injury scenarios of structural damage and/or reduced nephron number. The model predicted that pre-formed biomarkers would respond quickest to injury with a brief period of elevation, which would be easily missed in clinical scenarios. Induced biomarker time-courses would be influenced by biomarker-specific physiology and the balance between insult severity (which increased single nephron excretion), the number of remaining nephrons (reduced total excretion), and the extent of glomerular filtration rate reduction (increased concentration). Filtered biomarkers have the longest time-course because plasma levels increased following glomerular filtration rate decrease. Peak concentration and profile depended on the extent of damage to the reabsorption mechanism and recovery rate. Rapid recovery may be detected through a rapid reduction in urinary concentration. For all biomarkers, impaired hyperfiltration substantially increased concentration, especially with chronic kidney disease. For clinical validation of these model-derived predictions the clinical biomarker of choice will depend on timing in relation to renal insult and interpretation will require the pre-insult nephron number (renal mass) and detection of hyperfiltration.     

Molecular resistance fingerprint of pemetrexed and platinum in a long-term survivor of mesothelioma

Background

Pemetrexed, a multi-folate inhibitor combined with a platinum compound is the first-line treatment of malignant mesothelioma, but median survival is still one year. Intrinsic and acquired resistance to pemetrexed is common, but its biological basis is obscure. Here we report for the first time a genome-wide profile of acquired resistance in the tumour from an exceptional case with advanced pleural mesothelioma and almost six years survival after 39 cycles of second-line pemetrexed/carboplatin treatment.

Methodology and Principal Findings

Genome-wide analysis with Illumina BeadChip Kit of 25,000 genes was performed on mRNA from pre-treatment and post-resistance biopsies from this individual as well on case and control samples from our previously published study (in total 17 samples). Cell specific expression of proteins encoded by selected genes were analysed by immunohistochemistry. Serial serum levels of CA125, CYFRA21-1 and SMRP levels were examined. TS protein, the main target of pemetrexed was overexpressed. Proteins and genes related to DNA damage response, elongation and telomere extension and repair related directly and indirectly to platinum resistance were overexpressed, as the CHK1 protein and the genes CHEK2, LIG3, POLD1, POLA2, FANCD2, PRPF19, RECQ5 respectively, the last two not previously described in mesothelioma. We observed a down-regulation of leukocyte transendothelial migration and cell adhesion molecules pathways. Silencing of NT5C in two mesothelioma cell lines did not sensitize the cells to Pemetrexed. Proposed resistance markers are TS, KRT7/ CK7, TYMP/ thymidine phosphorylase and down-regulated SPARCL1 and CDKN1B. Moreover, comparison of the primary expression of the sensitive versus a primary resistant case showed multi-fold overexpressed DNA repair, cell cycle, cytokinesis, and spindle formation in the latter. Serum CA125 and SMRP reflected the clinical and radiological course and tumour burden.

Conclusions

Genome-wide microarray of mesothelioma pre- and post-resistance biopsies indicated a novel resistance signature to pemetrexed/carboplatin that deserve validation in a larger cohort.     

A comparative phylogenetic study of genetics and folk music

Computer-aided comparison of folk music from different nations is one of the newest research areas. We were intrigued to have identified some important similarities between phylogenetic studies and modern folk music. First of all, both of them use similar concepts and representation tools such as multidimensional scaling for modelling relationship between populations. This gave us the idea to investigate whether these connections are merely accidental or if they mirror population migrations from the past. We raised the question; does the complex structure of musical connections display a clear picture and can this system be interpreted by the genetic analysis? This study is the first to systematically investigate the incidental genetic background of the folk music context between different populations. Paternal (42 populations) and maternal lineages (56 populations) were compared based on Fst genetic distances of the Y chromosomal and mtDNA haplogroup frequencies. To test this hypothesis, the corresponding musical cultures were also compared using an automatic overlap analysis of parallel melody styles for 31 Eurasian nations. We found that close musical relations of populations indicate close genetic distances (<0.05) with a probability of 82%. It was observed that there is a significant correlation between population genetics and folk music; maternal lineages have a more important role in folk music traditions than paternal lineages. Furthermore, the combination of these disciplines establishing a new interdisciplinary research field of “music-genetics” can be an efficient tool to get a more comprehensive picture on the complex behaviour of populations in prehistoric time.     

HIV-1–cellular interactions analyzed by single virus tracing

Single virus tracing (SVT) allows the direct investigation of the entry pathway of viruses into living cells. Using fluorescently labeled virus-like particles (VLPs) and SVT, we have studied the interaction between human immunodeficiency virus type 1 (HIV-1) and the plasma membrane of living cells. From the trajectories of freely diffusing VLPs in solution, we established that the particle preparation was homogeneous and the particles had a hydrodynamic radius of 86 ± 5 nm, consistent with the size of single HI viruses. The VLPs that come in contact with the cell surface either become immobilized or rapidly dissociate from the cell surface. The fraction of virions that become immobilized on the plasma membrane correlates with the surface heparan sulfate linked proteoglycans (HSPG) concentration of the cell line tested. The particles that are not immobilized make an average of 1.5 contacts with the cell surface before diffusing away. For most cell lines investigated, the contact duration follows an exponential distribution with a lifetime between 20 and 50 ms depending on the cell type.