Development of a liquid chromatography-mass spectrometry method for monitoring the angiotensin-converting enzyme inhibitor lisinopril in serum

In this study, a sensitive, specific, precise and accurate method for lisonopril quantitative determination in human serum was developed and validated. The method comprises lisinopril isolation from serum by means of solid-phase extraction followed by its quantification by liquid chromatography-mass spectrometry. Chromatographic separation was performed at 55 degrees C on Kromasil C(18) 5 micrometer 250x3.2 mm HPLC column with mobile phase composed of 50 mM ammonium formate buffer (pH 3)-acetonirile-methanol (72:7:21, v/v/v). A Finnigan AQA benchtop mass spectrometer with a pneumatically assisted electrospray (ES) interface and a single quadrupole mass filter was used to detect and quantify lisinopril in column effluent. Ion signals were acquired by selected ion monitoring of the protonated lisinopril ion m/z=406.5 (M+1). The detector response was linear with r>0.9993 in the investigated concentration range 6-150 ng/ml. The mean recovery of lisinopril from serum samples was 88%. The limit of quantitation for lisinopril was 6 ng/ml with a signal-to-noise ratio at this concentration level S/N=34.75+/-3.9 (n=4).     

Whole blood pro-inflammatory cytokines and adhesion molecules post-lipopolysaccharides exposure in hyperbaric conditions

Hyperbaric oxygen (HBO) is a therapeutic intervention with applications in a large variety of diseases, including traumatic injuries and acute or chronic infections. The presence of pro-inflammatory cytokines regulates certain factors including adhesion molecules, which play a significant role in HBO effects. We have investigated the effect of HBO on pro-inflammatory cytokine release [tumor necrosis factor-alpha (TNF-alpha), interleukin 6 and 8 (IL-6 and IL-8)], and the regulation of adhesion molecules [soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular adhesion molecule (sVCAM)] after lipopolysaccharide (LPS) stimulation in 16 healthy individuals, originating from an urban area. A total number of 64 samples were treated, divided into four groups: Group A: not stimulated with LPS and not exposed to HBO. Group B: stimulated with LPS and not exposed to HBO. Group C: not stimulated with LPS and exposed to HBO. Group D: stimulated with LPS and exposed to HBO. The LPS stimulation dose was 100 pg\ml for 0.1 ml whole blood diluted 1:10. After incubation, samples were exposed to HBO with 100% O2 at 2.4 atmospheres absolute (ATA) for 90 min. TNF-alpha, IL-6, IL-8 and sICAM-1, sVCAM levels were determined in culture supernatant, with ELISA. We observed an enhanced effect of LPS stimulation following exposure to HBO, which caused an increase in cytokine production (TNF-alpha, IL-6, IL-8), a reduction in sICAM, and no change to sVCAM, while their levels without stimulation remained almost invariable. The decrease in sICAM levels could be related to the increased levels of IL-8, as the production of this chemokine is involved in the regulation of adhesion molecules.     

IL-21 activates both innate and adaptive immunity to generate potent antitumor responses that require perforin but are independent of IFN-gamma

IL-21 is a key factor in the transition between innate and adaptive immune responses. We have used the cytokine gene therapy approach to study the antitumor responses mediated by IL-21 in the B16F1 melanoma and MethA fibrosarcoma tumor models in mice. Retrovirally transduced tumor cells secreting biologically functional IL-21 have growth patterns in vitro similar to that of control green fluorescent protein-transduced cells, but are completely rejected in vivo. We show that IL-21 activates NK and CD8(+) T cells in vivo, thus mediating complete rejection of poorly immunogenic tumors. Rejection of IL-21-secreting tumors requires the presence of cognate IL-21R and does not depend on CD4(+) T cell help. Interestingly, perforin, but not IFN-gamma or other major Th1 and Th2 cytokines (IL-12, IL-4, or IL-10), is required for the IL-21-mediated antitumor response. Moreover, IL-21 results in 50% protection and 70% cure of nonimmunogenic tumors when given before and after tumor challenge, respectively, in C57BL/6 mice. We conclude that IL-21 immunotherapy warrants clinical evaluation as a potential treatment for cancer.     

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.     

Intrauterine growth restriction affects bone mineral density of the mandible and the condyle in growing rats

Objectives:To investigate in growing rats the effect of intrauterine growth restriction (IUGR) on the bone mineral density of the mandible and tibia, as well as the quality of the mandibular and condylar bone.Methods:Twelve male rats were born IUGR by mothers sustaining 50% food restriction during pregnancy. Twelve control male rats were born by mothers fed ad libitum. Dual-energy X-ray absorptiometry (DEXA) of the tibia, proximal tibial metaphysis and the mandible, biochemical markers, histology and histomorphometrical analysis on the mandibular and subchondral bone of the condyle were performed.Results:IUGR significantly affected bone mineral density (BMD) of both tibial and mandibular bones. IUGR rats had significantly lower osteocalcin values (p=0.021) and phosphorus (p=0.028), but not 25-OH vitamin D (p=0.352). Bone area percentage in the mandible was significantly lower (51.21±5.54) in IUGR compared to controls (66.00±15.49), and for subchondral bone of the condyle for IUGR (47.01±6.82) compared to controls (68.27±13.37). IUGR had a significant reduction in the fibrous layer, but not the proliferating layer, with the hypertrophic layer significantly increased.Conclusion:Maternal restricted nutrition during gestation can affect BMD of the mandible and the tibia of the offspring animals.     

Presence of Cicadomorpha in olive orchards of Greece with special reference to Xylella fastidiosa vectors

Insects of the infraorder Cicadomorpha cause serious economic impact on agriculture because of pathogens’ transmission. The vector-borne bacterium Xylella fastidiosa, which is associated with olive quick decline syndrome, is exclusively transmitted by insects of this infraorder and more specific by sharpshooters and spittlebugs. Efficient control practices of the insect vectors are necessary for the control of the disease in cases of outbreaks. Therefore, the knowledge of the vectors' species as well as the robust understanding of their biology and ecology is of crucial importance. In the present study, extensive sampling was conducted in 28 olive orchards of Greece during the years 2017 and 2018 aiming to investigate which species of this infraorder are present, with special interest to those which are considered as vectors of the bacterium. During the surveys, 4,350 Cicadomorpha insects were collected. Forty-two, five and one species of the families Cicadellidae, Aphrophoridae and Cercopidae, respectively, were identified. The results of the surveys revealed that the main potential vectors of X. fastidiosa in Greek olive orchards are the spittlebugs Philaenus spumarius and Neophilaenus campestris. Those species have wide distribution in Greece and are present for a long period during the year, and they inhabit both the ground vegetation and the olive trees' canopy. A representative portion of the captured spittlebugs was analysed with molecular methods, and the analysis confirmed the absence of X. fastidiosa in the tested individuals.     

Full genome sequence of bluetongue virus serotype 1 from India

We report the full-genome sequence of an Indian isolate of bluetongue virus serotype 1 (BTV-1), strain IND1992/01. This is the first report of the entire genome sequence (Seg-1 to Seg-10) of an Eastern (e) strain of BTV-1. These sequence data provide a reference for BTV-1e that will help to define the phylogenetic relationships and geographic origins of distinct Indian lineages of BTV-1 as well as their relationships with other BTV strains from around the world. The availability of data for all 10 genome segments of this strain will also help to identify reassortment events involving this and other virus lineages.     

Evaluation of Two Highly-Multiplexed Custom Panels for Massively Parallel Semiconductor Sequencing on Paraffin DNA

Background—Aim

Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA.

Methods

Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible.

Results

Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman’s rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8–99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1–95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3–76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality.

Conclusions

Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.     

Calprotectin: A protein related to cardiovascular risk in adult patients with obstructive sleep apnea

IntroductionIncreased levels of inflammatory mediators, such as hs-CRP, have been detected in patients with obstructive sleep apnea (OSA) and used as cardiovascular risk and disease outcome predictors. Calprotectin is an inflammatory marker regulating atherogenic processes not investigated in adult OSA patients. The aim of the present study as primary objective was to examine the role of calprotectin as an inflammatory molecule, acting through a distinct pathway to the atherogenic process in adult OSA patients and its associations with hs-CRP and the lipidemic profile of the patients. As a secondary objective was the evaluation of the atherogenic markers post-CPAP treatment.Materials and methodsSeventy-four participants underwent full overnight polysomnography. Blood samples were collected for calprotectin, hs-CRP, total cholesterol, triglycerides, LDL, HDL and glucose levels. Thirty-two OSA patients were reexamined 6 months post-CPAP treatment.ResultsOut of 74 participants included in the study, 33 had moderate OSA, 27 had severe OSA and 14 were controls. Calprotectin and hs-CRP were significantly increased in patients with moderate and severe OSA compared to controls (p < 0.0001). Calprotectin and hs-CRP levels were positively correlated with apnea-hypopnea index, BMI and total time of sleep with SaO₂ <90% and inversely correlated with SaO₂ minimum and mean values. Calprotectin and hs-CRP levels were significantly improved post-CPAP treatment (p < 0.0001).DiscussionCalprotectin may serve as a novel and reliable, biomarker of cardiovascular risk severity in OSA patients. The decrease of calprotectin levels post-CPAP treatment combined with hs-CRP amelioration could provide evidence for reduction of cardiovascular risk post CPAP treatment.