Liquid chromatography-electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.     

Host Factor SAMHD1 Restricts DNA Viruses in Non-Dividing Myeloid Cells

SAMHD1 is a newly identified anti-HIV host factor that has a dNTP triphosphohydrolase activity and depletes intracellular dNTP pools in non-dividing myeloid cells. Since DNA viruses utilize cellular dNTPs, we investigated whether SAMHD1 limits the replication of DNA viruses in non-dividing myeloid target cells. Indeed, two double stranded DNA viruses, vaccinia and herpes simplex virus type 1, are subject to SAMHD1 restriction in non-dividing target cells in a dNTP dependent manner. Using a thymidine kinase deficient strain of vaccinia virus, we demonstrate a greater restriction of viral replication in non-dividing cells expressing SAMHD1. Therefore, this study suggests that SAMHD1 is a potential innate anti-viral player that suppresses the replication of a wide range of DNA viruses, as well as retroviruses, which infect non-dividing myeloid cells.     

Mitochondrial distribution patterns in canine oocytes as related to the reproductive cycle stage

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n = 2), follicular phase (F, n = 4), ovulation (O, n = 2), early luteal (EL, n = 7) and mid/late luteal phase (MLL, n = 2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (I) small aggregates diffused throughout the cytoplasm; (II) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II + III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P < 0.05; O vs. A: P < 0.001), in EL (61%; O vs. EL: P < 0.01), or in MLL (0%; F vs. MLL: P < 0.05; O vs. MLL: P < 0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P < 0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes.     

Utilizing the Dog Genome in the Search for Novel Candidate Genes Involved in Glioma Development—Genome Wide Association Mapping followed by Targeted Massive Parallel Sequencing Identifies a Strongly Associated Locus

Gliomas are the most common form of malignant primary brain tumors in humans and second most common in dogs, occurring with similar frequencies in both species. Dogs are valuable spontaneous models of human complex diseases including cancers and may provide insight into disease susceptibility and oncogenesis. Several brachycephalic breeds such as Boxer, Bulldog and Boston Terrier have an elevated risk of developing glioma, but others, including Pug and Pekingese, are not at higher risk. To identify glioma-associated genetic susceptibility factors, an across-breed genome-wide association study (GWAS) was performed on 39 dog glioma cases and 141 controls from 25 dog breeds, identifying a genome-wide significant locus on canine chromosome (CFA) 26 (p = 2.8 x 10⁻⁸). Targeted re-sequencing of the 3.4 Mb candidate region was performed, followed by genotyping of the 56 SNVs that best fit the association pattern between the re-sequenced cases and controls. We identified three candidate genes that were highly associated with glioma susceptibility: CAMKK2, P2RX7 and DENR. CAMKK2 showed reduced expression in both canine and human brain tumors, and a non-synonymous variant in P2RX7, previously demonstrated to have a 50% decrease in receptor function, was also associated with disease. Thus, one or more of these genes appear to affect glioma susceptibility.     

A new DGGE protocol targeting 2,4-diacetylphloroglucinol biosynthetic gene phlD from phylogenetically contrasted biocontrol pseudomonads for assessment of disease-suppressive soils

In the rhizosphere, biocontrol pseudomonads producing 2,4-diacetylphloroglucinol (Phl) can protect plants from soil-borne pathogens. DGGE of phlD has been proposed to monitor these bacteria, but two distinct protocols were needed for analysis of both the 'Pseudomonas fluorescens' species complex and the strains from rrs restriction group ARDRA-1. Here, a single DGGE protocol performed on 668-bp GC-clamp-containing phlD amplicons was effective with both types of pseudomonads, and 36 reference biocontrol strains from the 'P. fluorescens' complex or group ARDRA-1 gave a total of 11 distinct DGGE bands. phlD amplicons with at least two to seven nucleotidic differences could be discriminated, and the discrimination level was similar to that of phlD restriction analysis with four enzymes. Multiple phlD-DGGE bands were obtained when studying rhizosphere soil containing indigenous phlD+ pseudomonads, and phlD diversity was higher when DGGE was implemented after incubation of tobacco rhizosphere extracts in semi-selective medium (MPN approach) in comparison with approaches based on direct analysis of rhizosphere DNA extracts or assessment of phlD+colonies. phlD-DGGE profiles differed for a soil suppressive and a soil conducive to black root rot of tobacco, and each soil yielded new phlD sequences. In conclusion, this DGGE protocol was useful for monitoring indigenous rhizosphere consortia of phlD+ pseudomonads.     

The dual action of glioma-derived exosomes on neuronal activity: synchronization and disruption of synchrony

Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7–12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca²⁺ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7–12 when the threshold for spike initiation is significantly reduced. At DIV 7–12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.Subject terms: Neuroscience, Preclinical research     

Inhibition of -tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells

It was previously found that -tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM -tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. -Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of -tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high -tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of -tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM -tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from -tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of -tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.