Eicosanoid Profiling in an Orthotopic Model of Lung Cancer Progression by Mass Spectrometry Demonstrates Selective Production of Leukotrienes by Inflammatory Cells of the Microenvironment

Eicosanoids are bioactive lipid mediators derived from arachidonic acid¹ (AA), which is released by cytosolic phospholipase A₂ (cPLA₂). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA₂ knockout vs wild-type mice, we demonstrated that prostaglandins (PGE₂, PGD₂ and PGF_2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB₄, LTC₄, LTD₄, LTE₄) production required cPLA₂ expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.     

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent.     

Characterization of the Vaginal Microbiota among Sexual Risk Behavior Groups of Women with Bacterial Vaginosis

Background

The pathogenesis of bacterial vaginosis (BV) remains elusive. BV may be more common among women who have sex with women (WSW). The objective of this study was to use 454 pyrosequencing to investigate the vaginal microbiome of WSW, women who have sex with women and men (WSWM), and women who have sex with men (WSM) with BV to determine if there are differences in organism composition between groups that may inform new hypotheses regarding the pathogenesis of BV.

Methods

Vaginal swab specimens from eligible women with BV at the Mississippi State Department of Health STD Clinic were used. After DNA extraction, 454 pyrosequencing of PCR-amplified 16S rRNA gene sequences was performed. Sequence data was classified using the Ribosomal Database Program classifer. Complete linkage clustering analysis was performed to compare bacterial community composition among samples. Differences in operational taxonomic units with an abundance of ≥2% between risk behavior groups were determined. Alpha and beta diversity were measured using Shannon’s Index implemented in QIIME and Unifrac analysis, respectively.

Results

33 WSW, 35 WSWM, and 44 WSM were included. The vaginal bacterial communities of all women clustered into four taxonomic groups with the dominant taxonomic group in each being Lactobacillus, Lachnospiraceae, Prevotella, and Sneathia. Regarding differences in organism composition between risk behavior groups, the abundance of Atopobium (relative ratio (RR)=0.24; 95%CI 0.11-0.54) and Parvimonas (RR=0.33; 95%CI 0.11-0.93) were significantly lower in WSW than WSM, the abundance of Prevotella was significantly higher in WSW than WSWM (RR=1.77; 95%CI 1.10-2.86), and the abundance of Atopobium (RR=0.41; 95%CI 0.18-0.88) was significantly lower in WSWM than WSM. Overall, WSM had the highest diversity of bacterial taxa.

Conclusion

The microbiology of BV among women in different risk behavior groups is heterogeneous. WSM in this study had the highest diversity of bacterial taxa. Additional studies are needed to better understand these differences.     

High-Throughput Massively Parallel Sequencing for Fetal Aneuploidy Detection from Maternal Plasma

Background

Circulating cell-free (ccf) fetal DNA comprises 3–20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.

Methods

Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.

Results

Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.

Conclusions

These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.     

Solvothermal Preparation of ZnO Nanorods as Anode Material for Improved Cycle Life Zn/AgO Batteries

Nano materials with high surface area increase the kinetics and extent of the redox reactions, thus resulting in high power and energy densities. In this study high surface area zinc oxide nanorods have been synthesized by surfactant free ethylene glycol assisted solvothermal method. The nanorods thus prepared have diameters in the submicron range (300∼500 nm) with high aspect ratio. They have uniform geometry and well aligned direction. These nanorods are characterized by XRD, SEM, Specific Surface Area Analysis, solubility in alkaline medium, EDX analysis and galvanostatic charge/discharge studies in Zn/AgO batteries. The prepared zinc oxide nanorods have low solubility in alkaline medium with higher structural stability, which imparts the improved cycle life stability to Zn/AgO cells.     

Potential evolutionary trade-off between feeding and stability in Cambrian cinctan echinoderms

Reconstructing the function and behaviour of extinct groups of echinoderms is problematic because there are no modern analogues for their aberrant body plans. Cinctans, an enigmatic group of Cambrian echinoderms, exemplify this problem: their asymmetrical body plan differentiates them from all living species. Here, we used computational fluid dynamics to analyse the functional performance of cinctans without assuming an extant comparative model. Three-dimensional models of six species from across cinctan phylogeny were used in computer simulations of water flow. The results demonstrate that cinctans with strongly flattened bodies produced much less drag than species characterized by dorsal protuberances or swellings, suggesting the former were more stable on the seafloor. However, unlike the flattened forms, cinctans with high-relief bodies were able to passively direct flow towards the mouth and associated food grooves, indicating that they were capable of more efficient feeding on particles suspended in the water. This study provides evidence of a previously unknown evolutionary trade-off between feeding and stability in Cambrian cinctan echinoderms.     

Accumulation of Essential and Trace Elements in Guar (Cyamopsis tetragonoloba) and Guar Gum Cultivated in Semi-arid Regions of Sindh,Pakistan

Biological Trace Element Research - For physiological and biochemical studies, it is considered essential to have knowledge about the accumulation of mineral elements in plants and their...     

New insights from old eggs – the shape and thickness of Great Auk Pinguinus impennis eggs

We compared the shape and eggshell thickness of Great Auk Pinguinus impennis eggs with those of its closest relatives, the Razorbill Alca torda, Common Guillemot Uria aalge and Brünnich's Guillemot Uria lomvia, in order to gain additional insights into the breeding biology of the extinct Great Auk. The egg of the Great Auk was most similar in shape to that of Brünnich's Guillemot. The absolute thickness of the Great Auk eggshell was greater than that of the Common Guillemot and Razorbill egg, which is as expected given its greater size, but the relative shell thickness at the equator and pointed end (compared with the blunt end) was more similar to that of the Common Guillemot. On the basis of these and other results we suggest that Great Auk incubated in an upright posture in open habitat with little or no nest, where its pyriform egg shape provided stability and allowed safe manoeuvrability during incubation. On the basis of a recent phylogeny of the Alcidae, we speculate that a single brood patch, a pyriform egg and upright incubation posture, as in the Great Auk and the two Uria guillemots, is the ancestral state, and that the Razorbill – the Great Auk's closest relative – secondarily evolved two brood patches and an elliptical egg as adaptations for horizontal incubation, which provides flexibility in incubation site selection, allowing breeding in enclosed spaces such as crevices, burrows or under boulders, as well as on open ledges.