Effect of Processing Route on the Surface Properties of Amorphous Indomethacin Measured by Inverse Gas Chromatography

The aim of this study was to investigate the effect of processing route (i.e., quench cooling and ball milling) on the surface energy heterogeneity and surface chemistry of indomethacin (IMC). Recently developed inverse gas chromatography (IGC) methodology at finite concentrations was employed to determine the surface energy distributions of crystalline, quench cooled and milled IMC samples. Surface properties of crystalline and processed IMC were measurably different as determined by the IGC and other conventional characterization techniques: differential scanning calorimetry and powder X-ray diffraction. Quench cooled IMC was in fully amorphous form. Milled IMC showed no amorphous character by calorimetric or X-ray diffraction studies. It was demonstrated that both processed IMC samples were energetically more active than the crystalline IMC. In particular, milled IMC exhibited a relatively higher dispersive surface energy and higher surface basicity (electron donor capability). This may be attributed to the creation of surface defect sites or exposure of higher energy crystal facets during the milling process. This study confirms that processing route has notable influence on the surface energy distribution and surface acid–base character. IGC was demonstrated as a powerful technique for investigating surface properties of real-world, heterogeneous pharmaceutical materials.Key words: heterogeneity, indomethacin, inverse gas chromatography, surface disorder, surface energy     

Linking engineering and medicine: fostering collaboration skills in interdisciplinary teams

Biomedical engineering embodies the spirit of combining disciplines. The engineer's pragmatic approach to--and appetite for--solving problems is matched by a bounty of technical challenges generated in medical domains. From nanoscale diagnostics to the redesign of systems of health-care delivery, engineers have been connecting advances in basic and applied science with applications that have helped to improve medical care and outcomes. Increasingly, however, integrating these areas of knowledge and application is less individualistic and more of a team sport. Success increasingly relies on a direct focus on practicing and developing collaboration skills in interdisciplinary teams. Such an approach does not fit easily into individual-focused, discipline-based programs. Biomedical engineering has done its fair share of silo busting, but new approaches are needed to inspire interdisciplinary teams to form around challenges in particular areas. Health care offers a wide variety of complex challenges across an array of delivery settings that can call for new interdisciplinary approaches. This was recognized by the deans of the University of Southern California's (USC's) Medical and Engineering Schools when they began the planning process, leading to the creation of the Health, Technology, and Engineering (HTE@USC or HTE for short) program. “Health care and technology are changing rapidly, and future physicians and engineers need intellectual tools to stay ahead of this change,” says Carmen A. Puliafito, dean of the Keck School of Medicine. His goal is to train national leaders in the quest for devices and processes to improve health care.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

From Penicillin-Streptomycin to Amikacin-Vancomycin: Antibiotic Decontamination of Cardiovascular Homografts in Singapore

Background

In February 2012, the National Cardiovascular Homograft Bank (NCHB) became the first tissue bank outside of North America to receive accreditation from the American Association of Tissue Banks. From 2008 to 2009, NCHB had been decontaminating its cardiovascular homografts with penicillin and streptomycin. The antibiotic decontamination protocol was changed in January 2010 as amikacin and vancomycin were recommended, in order to cover bacteria isolated from post-recovery and post- antibiotic incubation tissue cultures.

Aim

The objective of this study is to determine the optimal incubation conditions for decontamination of homografts by evaluating the potencies of amikacin and vancomycin in different incubation conditions. Retrospective reviews of microbiological results were also performed for homografts recovered from 2008 to 2012, to compare the effectiveness of penicillin-streptomycin versus the amikacin-vancomycin regimens.

Methods

Based on microbiological assays stated in United States Pharmacopeia 31, potency of amikacin was evaluated by turbidimetric assay using Staphylococcus aureus, while vancomycin was by diffusion assay using Bacillus subtilis sporulate. Experiments were performed to investigate the potencies of individual antibiotic 6-hours post incubation at 4°C and 37°C and 4°C for 24 hours, after the results suggested that amikacin was more potent at lower temperature.

Findings

Tissue incubation at 4°C for 24 hours is optimal for both antibiotics, especially for amikacin, as its potency falls drastically at 37°C.

Conclusion

The decontamination regimen of amikacin-vancomycin at 4°C for 24 hours is effective. Nevertheless, it is imperative to monitor microbiological trends closely and evaluate the efficacy of current antibiotics regimen against emerging strains of micro-organisms.     

Inhibition of enterovirus 71 (EV-71) infections by a novel antiviral peptide derived from EV-71 capsid protein VP1

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 μM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.     

A comparison study reveals important features of agreement and disagreement between summarized DNA and RNA data obtained from renal cell carcinoma

Cytogenetic abnormalities, such as DNA amplifications and deletions, often lead to significant changes in gene expression levels within a chromosomal region. Instead of generating additional DNA copy number data, one method to identify DNA copy number abnormalities has been to search existing gene expression data for regional perturbations in gene expression. However, it is not clear how well this surrogate method performs in the examination of individual tumors and how we can use both DNA and RNA data to identify candidate genes that may be mutated. Here we report a comparison study using summarized DNA and RNA data to identify chromosomal abnormalities in human samples. Forty-four tissue samples from patients diagnosed as having renal cell carcinoma (RCC) were collected, together with 15 normal kidney samples as controls, and for each sample the genome-wide DNA and RNA data were obtained for comparison using Affymetrix 100K SNP and HGU133plus2 gene expression chips, respectively. The DNA and RNA data was summarized by both chromosome arm and cytogenetic banding patterns and compared. The result of this analysis revealed that the two summarized data sets used to identify cytogenetic changes agreed well. However, some differences between the two were also identified. These differences of large-scale gene expression deregulation without evidence of the comparable DNA copy number alterations may be the result of known mechanisms, such as large-scale methylation or chromosome inactivation, or may be the result of some new mechanism of DNA-RNA translation. The usefulness of the combined data set for identifying regions of mutated genes is also discussed.     

SNP arrays in heterogeneous tissue: highly accurate collection of both germline and somatic genetic information from unpaired single tumor samples

SNP arrays provide reliable genotypes and can detect chromosomal aberrations at a high resolution. However, tissue heterogeneity is currently a major limitation for somatic tissue analysis. We have developed SOMATICs, an original program for accurate analysis of heterogeneous tissue samples. Fifty-four samples (42 tumors and 12 normal tissues) were processed through Illumina Beadarrays and then analyzed with SOMATICs. We demonstrate that tissue heterogeneity-related limitations not only can be overcome but can also be turned into an advantage. First, admixture of normal cells with tumor can be used as an internal reference, thereby enabling highly sensitive detection of somatic deletions without having corresponding normal tissue. Second, the presence of normal cells allows for discrimination of somatic from germline aberrations, and the proportion of cells in the tissue sample that are harboring the somatic events can be assessed. Third, relatively early versus late somatic events can also be distinguished, assuming that late events occur only in subsets of cancer cells. Finally, admixture by normal cells allows inference of germline genotypes from a cancer sample. All this information can be obtained from any cancer sample containing a proportion of 40-75% of cancer cells. SOMATICs is a ready-to-use open-source program that integrates all of these features into a simple format, comprehensively describing each chromosomal event.     

The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan.

The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB(-) mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA(-) and embB(-) mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA(-) and embB(-) mutants. Detailed nuclear magnetic resonance studies coupled with enzyme digestion, chromatography, and mass spectrometry analyses revealed that the disaccharide beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f) extension from the 3-position of the 3,5-linked alpha-d-Ara(f) residue is markedly diminished. As a consequence, a linear terminal beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f) is formed, a motif which is a recognized, nonreducing terminal feature of lipoarabinomannan but not of normal AG. Upon complementation with the embB and embA wild-type genes, the phenotype of the mutants reverted to wild-type, in that normal AG was resynthesized. Our results clearly show that both EmbA and EmbB proteins are involved in the formation of the proper terminal hexaarabinofuranoside motif in AG, thus paving the way for future studies to identify the complete array of arabinosyltransferases involved in the synthesis of mycobacterial cell wall arabinan.