Replacement names and nomenclatural comments for problematic species-group names in Europe's Neogene freshwater Gastropoda. Part 2

In the course of a new database project on Miocene to Recent freshwater gastropods of Europe, a great many of primary and secondary homonyms were revealed. Such nomenclatural issues need clarification in order to avoid misunderstandings and wrong statements about geographical distributions and temporal ranges. The following 16 new names are introduced to replace existing homonyms: Theodoxus militaris jurisicpolsakae nom. n., Viviparus stevanovici nom. n., Melanopsis haueri ripanjensis nom. n., Melanopsis wolfgangfischeri nom. n., Micromelania ramacanensis nom. n., Pseudamnicola welterschultesi nom. n., Muellerpalia haszprunari nom. n., Muellerpalia pseudovalvatoides nom. n., Lithoglyphus gozhiki nom. n., Valvata heidemariae willmanni nom. n., Radix macaleti nom. n., Gyraulus okrugljakensis nom. n., Gyraulus rasseri nom. n., Gyraulus vrapceanus nom. n., Planorbarius halavatsi nom. n., and Segmentina mosbachensis nom. n. Additionally, six cases of homonyms are discussed that are not replaced by new names, because they are considered junior synonyms.     

Green fluorescent protein – Tagged HCV non-enveloped capsid like particles: Development of a new tool for tracking HCV core uptake

Circulating ‘free’ non-enveloped Hepatitis C virus (HCV) core protein has been demonstrated in HCV-infected patients, and HCV subgenomes with deletions of the envelope proteins have been previously identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell-signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins core₁₇₃-eGFP, eGFP-core₁₉₁ and eGFP-core₁₇₃ can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. By using confocal microscopy and FACS analysis, we provide evidence that the fluorescent nanospheres can not only enter human hepatic cells – the main target of HCV – but also human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. The fluorescent particles might thus be used to trace the intracellular trafficking of naked HCV capsids as showed by live microscopy and to further understand their biological significance.     

Replication and Predictive Value of SNPs Associated with Melanoma and Pigmentation Traits in a Southern European Case-Control Study

Background

Genetic association studies have revealed numerous polymorphisms conferring susceptibility to melanoma. We aimed to replicate previously discovered melanoma-associated single-nucleotide polymorphisms (SNPs) in a Greek case-control population, and examine their predictive value.

Methods

Based on a field synopsis of genetic variants of melanoma (MelGene), we genotyped 284 patients and 284 controls at 34 melanoma-associated SNPs of which 19 derived from GWAS. We tested each one of the 33 SNPs passing quality control for association with melanoma both with and without accounting for the presence of well-established phenotypic risk factors. We compared the risk allele frequencies between the Greek population and the HapMap CEU sample. Finally, we evaluated the predictive ability of the replicated SNPs.

Results

Risk allele frequencies were significantly lower compared to the HapMap CEU for eight SNPs (rs16891982 – SLC45A2, rs12203592 – IRF4, rs258322 – CDK10, rs1805007 – MC1R, rs1805008 - MC1R, rs910873 - PIGU, rs17305573- PIGU, and rs1885120 - MTAP) and higher for one SNP (rs6001027 – PLA2G6) indicating a different profile of genetic susceptibility in the studied population. Previously identified effect estimates modestly correlated with those found in our population (r = 0.72, P<0.0001). The strongest associations were observed for rs401681-T in CLPTM1L (odds ratio [OR] 1.60, 95% CI 1.22–2.10; P = 0.001), rs16891982-C in SCL45A2 (OR 0.51, 95% CI 0.34–0.76; P = 0.001), and rs1805007-T in MC1R (OR 4.38, 95% CI 2.03–9.43; P = 2×10⁻⁵). Nominally statistically significant associations were seen also for another 5 variants (rs258322-T in CDK10, rs1805005-T in MC1R, rs1885120-C in MYH7B, rs2218220-T in MTAP and rs4911442-G in the ASIP region). The addition of all SNPs with nominal significance to a clinical non-genetic model did not substantially improve melanoma risk prediction (AUC for clinical model 83.3% versus 83.9%, p = 0.66).

Conclusion

Overall, our study has validated genetic variants that are likely to contribute to melanoma susceptibility in the Greek population.     

New insights into the chemical and isotopic composition of human-body biominerals. I: Cholesterol gallstones from England and Greece

We have analyzed gallstones from four patients of Europe and particularly from England (including samples from a mother and a daughter) and Greece. According to the XRD, FTIR, NMR and laser micro-Raman results the studied materials correspond to typical cholesterol monohydrate (ChM). The micro-morphology of cholesterol microcrystals was investigated by means of SEM–EDS. The XRF results revealed that Ca is the dominant non-organic metal in all gallstones (up to ～1.95 wt.%) together with Fe, Cu, Pb and Ni (up to ～19 ppm for each metal). Gallstones from England contain additional Mn (up to ～87 ppm) and Zn (up to ～6 ppm) while the sample of the mother contains negligible Zn and Mn, compared to that of her daughter, but significant As (～4.5 ppm). All cholesterol gallstones examined are well enriched in potentially toxic metals (Pb, as well as Ni in one case) and metalloids (As also in one case) as compared to the global average. The position of Zn, which is a characteristic biometal, in the structure of cholesterol, was investigated by molecular simulation using the Accelrys Materials Studio^® software. On the basis of IRMS results, all gallstones examined exhibit a very light δ¹³C signature (average δ¹³C ～?24‰ PDB). Gamma-ray spectrometry measurements indicate the presence of ²¹⁴Pb and ²¹⁴Bi natural radionuclides due to the ²³⁸U series as well as an additional amount of ⁴⁰K.     

KRAS Genotypic Changes of Circulating Tumor Cells during Treatment of Patients with Metastatic Colorectal Cancer

Introduction

Circulating tumor cells (CTCs) could represent a non-invasive source of cancer cells used for longitudinal monitoring of the tumoral mutation status throughout the course of the disease. The aims of the present study were to investigate the detection of KRAS mutations in CTCs from patients with metastatic colorectal cancer (mCRC) and to compare their mutation status during treatment or disease progression with that of the corresponding primary tumors.

Materials and Methods

Identification of the seven most common KRAS mutations on codons 12 and 13 was performed by Peptide Nucleic Acid (PNA)-based qPCR method. The sensitivity of the assay was determined after isolation of KRAS mutant cancer cells spiked into healthy donors'' blood, using the CellSearch Epithelial Cell kit. Consistent detection of KRAS mutations was achieved in samples containing at least 10 tumor cells/7.5 ml of blood.

Results

The clinical utility of the assay was assessed in 48 blood samples drawn from 31 patients with mCRC. All patients had PIK3CA and BRAF wild type primary tumors and 14 KRAS mutant tumors. CTCs were detected in 65% of specimens obtained from 74% of patients. KRAS mutation analysis in CTC-enriched specimens showed that 45% and 16.7% of patients with mutant and wild type primary tumors, respectively, had detectable mutations in their CTCs. Assessing KRAS mutations in serial blood samples revealed that individual patient''s CTCs exhibited different mutational status of KRAS during treatment.

Conclusions

The current findings support the rationale for using the CTCs as a dynamic source of tumor cells which, by re-evaluating their KRAS mutation status, could predict, perhaps more accurately, the response of mCRC patients to targeted therapy.     

Creation of an open-access, mutation-defined fibroblast resource for neurological disease research

Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.     

Initiation of the synthesis of 'stress' ABA by (+)-[(2) H(6) ]ABA infiltrated into leaves of Commelina communis

The 'fettered' fraction of abscisic acid (ABA) that is held within the chloroplasts of unwilted bean and Commelina communis leaves is released when the leaves wilt and it is this 'free' ABA that is now proposed to cause the stomata to close within 2 or 3 min, well before the rise in total ABA can be detected. The large increase in 'stress' ABA begins 2-3 h later. The fettered ABA in a centrifuged homogenate is released by hyperosmotic solutions of mannitol (0.8 M) and NaCl (0.4 M). Dilute solution of halothane (10 mM) and colchicine (1 mM), the detergent sodium dodecyl sulphate (1 mM) the herbicide 2,4-d (0.1 mM) and dithiothreitol (0.01 mM) also caused ABA to be released. Zeatin (0.01 mM), cumene hydroperoxide (0.01 mM) and CaCl(2) (1 mM) had negligible effects. It was postulated that the ABA released from the chloroplasts by wilting could be the signal that initiates the synthesis of the dioxygenase and other enzymes necessary to produce the rise (up to 40-fold) in the amount of stress ABA that is seen 2-3 h later. To test this hypothesis, a solution of (+)-[(2) H(6) ]ABA was vacuum infiltrated into unwilted Commelina leaves to mimic the rise in ABA caused by wilting and gas chromatography/mass spectrometry of the ABA in the extract after 3 h showed that concentrations of (+)-[(2) H(6) ]ABA of up to 0.3 μM stimulated synthesis of endogenous [(1) H]ABA by 15-fold in the unwilted leaves. A 0.5 μM solution blocked the increase in the amount of ABA formed and also reduced the amount of ABA formed in response to a 0.8 M mannitol solution.     

Cysteine-scanning analysis of putative helix XII in the YgfO xanthine permease: ILE-432 and ASN-430 are important

Transmembrane helix XII of UapA, the major fungal homolog of the nucleobase-ascorbate transporter (NAT/NCS2) family, has been proposed to contain an aromatic residue acting as a purine-selectivity filter, distinct from the binding site. To analyze the role of helix XII more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 419ILPASIYVLVENPICAGGLTAILLNIILPGGY450 (the putative helix XII is underlined) was replaced individually with Cys. Of the 32 single-Cys mutants, 25 accumulate xanthine to 80-130% of the steady state observed with C-less YgfO, six (P421C, S423C, I424C, Y425C, L427C, G436C) accumulate to low levels (15-40%), and I432C is inactive. Immunoblot analysis shows that P421C and I432C display low expression in the membrane. Extensive mutagenesis reveals that replacement of Ile-432 with equally or more bulky side chains abolishes active transport without affecting expression, whereas replacement with smaller side chains allows activity but impairs affinity for the analogues 1-methyl and 6-thioxanthine. Only three of the single-Cys mutants of helix XII (V426C, N430C, and N443C) are sensitive to inactivation by N-ethylmaleimide. N430C is highly sensitive, with an IC50 of 10 microm, and is completely protected against inactivation in the presence of 2-thioxanthine, a high affinity substrate analogue. Other xanthine analogues are poorly bound by N430C, whereas replacement of Asn-430 with Thr inactivates the permease. The findings suggest that Ile-432 and Asn-430 of helix XII are crucial for purine uptake and affinity, and Asn-430 is probably at the vicinity of the binding site.