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.     

Biological and cytoselective anticancer properties of copper(II)-polypyridyl complexes modulated by auxiliary methylated glycine ligand

A series of ternary copper(II)-1,10-phenanthroline complexes with glycine and methylated glycine derivatives, [Cu(phen)(aa)(H(2)O)]NO(3)·xH(2)O 1-4 (amino acid (aa): glycine (gly), 1; DL: -alanine (DL: -ala), 2; 2,2-dimethylglycine (C-dmg), 3; sarcosine (sar), 4), were synthesized and characterized by FTIR, elemental analysis, electrospray ionization-mass spectra (ESI-MS), UV-visible spectroscopy and molar conductivity measurement. The determined X-ray crystallographic structures of 2 and 3 show each to consist of distorted square pyramidal [Cu(phen)(aa)(H(2)O)](+) cation, a nitrate counter anion, and with or without lattice water, similar to previously reported structure of [Cu(phen)(gly)(H(2)O)]NO(3)·1?H(2)O. It is found that 1-4 exist as 1:1 electrolytes in aqueous solution, and the cationic copper(II) complexes are at least stable up to 24?h. Positive-ion ESI-MS spectra show existence of only undissociated [Cu(phen)(aa)](+) species. Electron paramagnetic resonance, gel electrophoresis, fluorescence quenching, and restriction enzyme inhibition assay were used to study the binding interaction, binding affinity and selectivity of these complexes for various types of B-form DNA duplexes and G-quadruplex. All complexes can bind selectively to DNA by intercalation and electrostatic forces, and inhibit topoisomerase I. The effect of the methyl substituents of the coordinated amino acid in the above complexes on these biological properties are presented and discussed. The IC(50) values (24?h) of 1-4 for nasopharyngeal cancer cell line HK1 are in the range 2.2-5.2?μM while the corresponding values for normal cell line NP69 are greater than 13.0?μM. All complexes, at 5?μM, induced 41-60?% apoptotic cell death in HK1 cells but no significant cell death in NP69 cells.     

Glycan structures and intrageneric variations of venom acidic phospholipases A(2) from Tropidolaemus pitvipers

Most of the phospholipases A(2) (PLA(2) ; EC3.1.1.4) variants isolated so far from snake venoms are nonglycosylated enzymes. In the present study, we purified an active glycosylated PLA(2) and an inactive nonglycosylated Lys49-like PLA(2) from two geographical venom samples of Tropidolaemus. The PLA(2) variants from the two samples have rather different N-terminal sequences, implying that the samples were probably derived from two species (Tropidolaemus?subannulatus and Tropidolaemus?wagleri). The active PLA(2) s from Sulawesi and Sumatra venoms were designated as Tsu-E6 and Twa-E6, respectively, as a result of the presence of their conserved Glu6 residue. Tsu-E6 inhibited ADP-induced aggregation of mouse and human platelets. Twa-E6 stimulated the aggregation of mouse platelets but inhibited the aggregation of human platelets. Both PLA(2) s were found to be glycosylated at Asn14. Using MALDI-TOF analysis, the released glycans were shown to comprise complex type oligosaccharides without sialylation. This is the first glycan structure of the snake venom PLA(2) to be solved. Furthermore, the enzymatic removal of glycans from both PLA(2) s did not significantly alter their effects on lipid hydrolysis and platelet aggregation. The thermostability of glycosylated Twa-E6 was also found to be as good as that of other homologous PLA(2) s. The presence of these oligosaccharides in PLA(2) s warrants further analyses, which may provide useful insights into the functional regulation of these biomolecules.     

Characterization of a novel prokaryotic GDP dissociation inhibitor domain from the G protein coupled membrane protein FeoB

The FeoB family of membrane embedded G proteins are involved with high affinity Fe(II) uptake in prokaryotes. Here, we report that FeoB harbors a novel GDP dissociation inhibitor-like domain that specifically stabilizes GDP-binding through an interaction with the switch I region of the G protein. We show that the stabilization of GDP binding is conserved between species despite a high degree of sequence variability in their guanine nucleotide dissociation inhibitor (GDI)-like domains, and demonstrate that the presence of the membrane embedded domain increases GDP-binding affinity roughly 150-fold over the level accomplished by action of the GDI-like domain alone. To our knowledge, this is the first example for a prokaryotic GDI, targeting a bacterial G protein-coupled membrane process. Our findings suggest that Fe(II) uptake in bacteria involves a G protein regulatory pathway reminiscent of signaling mechanisms found in higher-order organisms.     

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.