Focus: A Multifaceted Battle Against Cancer: Extracting Knowledge from Chemical Imaging Data Using Computational Algorithms for Digital Cancer Diagnosis

Fourier transform infrared (FTIR) spectroscopic imaging is an emerging microscopy modality for clinical histopathologic diagnoses as well as for biomedical research. Spectral data recorded in this modality are indicative of the underlying, spatially resolved biochemical composition but need computerized algorithms to digitally recognize and transform this information to a diagnostic tool to identify cancer or other physiologic conditions. Statistical pattern recognition forms the backbone of these recognition protocols and can be used for highly accurate results. Aided by biochemical correlations with normal and diseased states and the power of modern computer-aided pattern recognition, this approach is capable of combating many standing questions of traditional histology-based diagnosis models. For example, a simple diagnostic test can be developed to determine cell types in tissue. As a more advanced application, IR spectral data can be integrated with patient information to predict risk of cancer, providing a potential road to precision medicine and personalized care in cancer treatment. The IR imaging approach can be implemented to complement conventional diagnoses, as the samples remain unperturbed and are not destroyed. Despite high potential and utility of this approach, clinical implementation has not yet been achieved due to practical hurdles like speed of data acquisition and lack of optimized computational procedures for extracting clinically actionable information rapidly. The latter problem has been addressed by developing highly efficient ways to process IR imaging data but remains one that has considerable scope for progress. Here, we summarize the major issues and provide practical considerations in implementing a modified Bayesian classification protocol for digital molecular pathology. We hope to familiarize readers with analysis methods in IR imaging data and enable researchers to develop methods that can lead to the use of this promising technique for digital diagnosis of cancer.     

An electron-capture gas–liquid-chromatographic method for the determination of prostaglandin F2α in biological fluids

A sensitive electron-capture gas-liquid-chromatographic method for the determination of sub-nanogram quantities of prostaglandin F(2alpha) was developed. The method is based on the sub-microgram scale conversion of the prostaglandin into the electron-capturing pentafluorobenzyl ester, and analysis of the latter as the tris-trimethylsilyl ether. The lower limit of detection was 12.5pg of the ester injected ;on-column' as the silylated product. The method was successfully applied to the determination of prostaglandin F(2alpha) in monkey plasma. The specificity of the analytical procedure was increased by incorporating a thin-layer chromatographic fractionation before gas-liquid chromatography. The utility of the analytical methodology developed was demonstrated by its application to the determination of plasma concentrations of intact prostaglandin F(2alpha) in a Rhesus monkey, after subcutaneous administration of a single dose of prostaglandin F(2alpha). The electron-capture gas-liquid-chromatographic assay is compared with the radioimmunoassay and the gas-liquid-chromatographic-mass-spectrometry assay for the determination of prostaglandin F(2alpha).     

Centrosome phosphorylation and the developmental expression of meiotic competence in mouse oocytes.

Previous studies suggested that the transition from an incompetent to a competent meiotic state during the course of oogenesis in the mouse involved a G2/M-like cell cycle transition (Wickramasinghe et al, 1991. Dev. Biol. 143, 162). The present studies tested the hypothesis that centrosome phosphorylation, an event normally induced by MPF, is required for this developmental transition and the expression of meiotic competence in cultured growing mouse oocytes. Multiple fluorescence labeling techniques were used to evaluate centrosome number, phosphorylation status, and microtubule nucleating capacity in competent and incompetent oocytes. Experimental conditions were established for reversibly altering the phosphorylation status of the centrosomes and the effects of these treatments on meiotic resumption were examined. Phosphorylated centrosomes nucleating short microtubules were observed in competent oocytes, whereas nonphosphorylated centrosomes and interphase microtubule arrays were found in incompetent oocytes. Upon recovery from nocodazole-induced microtubule depolymerization, short microtubules formed from centrosomes in competent oocytes, whereas long microtubules reappear in the cytoplasm of incompetent oocytes. Perturbation of the phosphorylation state of oocytes with activators of protein kinase A or protein kinase C resulted in the formation of long interphase microtubules in competent oocytes while centrosome phosphorylation was maintained. Treatment of competent oocytes with the phosphorylation inhibitor 6-dimethylaminopurine also led to formation of long microtubules, although under these conditions centrosomes were dephosphorylated. When competent oocytes were treated simultaneously with puromycin and the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX) for 6 hr, centrosomes became dephosphorylated; centrosomes were rephosphorylated when competent oocytes were further cultured in IBMX without puromycin. Conditions that induced centrosome dephosphorylation in competent oocytes resulted in the loss of the ability to express meiotic competence in culture, whereas maintenance of centrosome phosphorylation in these oocytes was correlated with the ability to resume meiosis. These results suggest that the G2/M transition that occurs when mouse oocytes progress from an incompetent to a competent state in vivo involves the phosphorylation of centrosomes and that the maintenance of centrosome phosphorylation is required for the in vitro expression of meiotic competence.     

Xanthones and 4-phenylcoumarins of Mesua thwaitesii

The constituents of the bark, timber and seeds of Mesua thwaitesii were examined by column chromatography and GC-MS. 1,5-Dihydroxyxanthone, 1,7-dihydroxyxanthone (euxanthone), 1,3-dimethoxy-5-hydroxyxanthone, 1,5,6-trihydroxyxanthone, mammeisin, (4-phenylcoumarin) and sitosterol have been characterized. Nine 4-phenylcoumarins including mammeigin, mesuagin, mammeisin, mesuol, or its isomers, have been identified in the seed extract. One coumarin has not been previously reported.     

Synthetic Antimicrobial Peptide Polybia MP-1 (Mastoparan) Inhibits Growth of Antibiotic Resistant Pseudomonas aeruginosa Isolates From Mastitic Cow Milk

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides (AMPs) offer a potent and effective alternative for treatment of antibiotic resistant microbes. Mastoparans or...     

Substitution of Glutamate Residue by Lysine in the Dimerization Domain Affects DNA Binding Ability of HapR by Inducing Structural Deformity in the DNA Binding Domain

HapR has been given the status of a high cell density master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of the wild type protein. This work aims at investigating the nature of functional inertness of a HapR natural variant harboring a substitution of a conserved glutamate residue at position 117 which participates in forming a salt bridge by lysine (HapR_V2G-E¹¹⁷K). Experimental evidence presented here reveals the inability of this variant to interact with various cognate promoters by in vitro gel shift assay. Furthermore, the elution profiles of HapR_V2G-E¹¹⁷K protein along with the wild type functional HapR_V2G in size-exclusion chromatography as well as circular dichroism spectra did not reflect any significant differences in its structure, thereby indicating the intactness of dimer in the variant protein. To gain further insight into the global shape of the proteins, small angle X-ray scattering analysis (SAXS) was performed. Intriguingly, increased radius of gyration of HapR_V2G-E¹¹⁷K of 27.5 Å in comparison to the wild type protein from SAXS data analyses implied a significant alteration in the global shape of the dimeric HapR_V2G-E¹¹⁷K protein. Structure reconstruction brought forth that the DNA binding domains were substantially “parted away” in this variant. Taken together, our data illustrates that substitution of the conserved glutamate residue by lysine in the dimerization domain induces separation of the two DNA binding domains from their native-like positioning without altering the dimeric status of HapR variant.     

Anion exchange membrane adsorbers for flow‐through polishing steps: Part I. clearance of minute virus of mice

Membrane adsorbers may be a viable alternative to the packed‐bed chromatography for clearance of virus, host cell proteins, DNA, and other trace impurities. However, incorporation of membrane adsorbers into manufacturing processes has been slow due to the significant cost associated with obtaining regulatory approval for changes to a manufacturing process. This study has investigated clearance of minute virus of mice (MVM), an 18–22 nm parvovirus recognized by the FDA as a model viral impurity. Virus clearance was obtained using three commercially available anion exchange membrane adsorbers: Sartobind Q®, Mustang Q®, and ChromaSorb®. Unlike earlier studies that have focused on a single or few operating conditions, the aim here was to determine the level of virus clearance under a range of operating conditions that could be encountered in industry. The effects of varying pH, NaCl concentration, flow rate, and other competing anionic species present in the feed were determined. The removal capacity of the Sartobind Q and Mustang Q products, which contain quaternary ammonium based ligands, is sensitive to feed conductivity and pH. At conductivities above about 20 mS/cm, a significant decrease in capacity is observed. The capacity of the ChromaSorb product, which contains primary amine based ligands, is much less affected by ionic strength. However the capacity for binding MVM is significantly reduced in the presence of phosphate ions. These differences may be explained in terms of secondary hydrogen bonding interactions that could occur with primary amine based ligands. Biotechnol. Bioeng. 2013; 110: 491–499. © 2012 Wiley Periodicals, Inc.