Applications of ATR-FTIR spectroscopic imaging to biomedical samples

FTIR spectroscopic imaging in ATR (Attenuated Total Reflection) mode is a powerful tool for studying biomedical samples. This paper summarises recent advances in the applications of ATR-FTIR imaging to dissolution of pharmaceutical formulations and drug release. The use of two different ATR accessories to obtain chemical images of formulations in contact with water as a function of time is demonstrated. The innovative use of the diamond ATR accessory allowed in situ imaging of tablet compaction and dissolution. ATR-FTIR imaging was also applied to obtain images of the surface of skin and the spatial distribution of protein and lipid rich domains was obtained. Chemical images of cross-section of rabbit aorta were obtained using a diamond ATR accessory and the possibility of in situ imaging of arterial samples in contact with aqueous solution was demonstrated for the first time. This experiment opens an opportunity to image arterial samples in contact with solutions containing drug molecules. This approach may help in understanding the mechanisms of treatment of atherosclerosis.     

Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity

Natural killer (NK) cells and dendritic cells (DCs) are, respectively, central components of innate and adaptive immune responses. We describe here a third DC lineage, termed interferon-producing killer DCs (IKDCs), distinct from conventional DCs and plasmacytoid DCs and with the molecular expression profile of both NK cells and DCs. They produce substantial amounts of type I interferons (IFN) and interleukin (IL)-12 or IFN-gamma, depending on activation stimuli. Upon stimulation with CpG oligodeoxynucleotides, ligands for Toll-like receptor (TLR)-9, IKDCs kill typical NK target cells using NK-activating receptors. Their cytolytic capacity subsequently diminishes, associated with the loss of NKG2D receptor (also known as Klrk1) and its adaptors, Dap10 and Dap12. As cytotoxicity is lost, DC-like antigen-presenting activity is gained, associated with upregulation of surface major histocompatibility complex class II (MHC II) and costimulatory molecules, which formally distinguish them from classical NK cells. In vivo, splenic IKDCs preferentially show NK function and, upon systemic infection, migrate to lymph nodes, where they primarily show antigen-presenting cell activity. By virtue of their capacity to kill target cells, followed by antigen presentation, IKDCs provide a link between innate and adaptive immunity.     

Genomic signatures to guide the use of chemotherapeutics

Using in vitro drug sensitivity data coupled with Affymetrix microarray data, we developed gene expression signatures that predict sensitivity to individual chemotherapeutic drugs. Each signature was validated with response data from an independent set of cell line studies. We further show that many of these signatures can accurately predict clinical response in individuals treated with these drugs. Notably, signatures developed to predict response to individual agents, when combined, could also predict response to multidrug regimens. Finally, we integrated the chemotherapy response signatures with signatures of oncogenic pathway deregulation to identify new therapeutic strategies that make use of all available drugs. The development of gene expression profiles that can predict response to commonly used cytotoxic agents provides opportunities to better use these drugs, including using them in combination with existing targeted therapies.     

Toxicities of DDE on Wheat and Bioremediation of DDE by Fungus Pleurotus pulmonarius

An in vitro study was performed to determine the acute phytotoxicities and genotoxicity of DDE either spiked to soil or added to hydrophonic cultures on wheat Triticum aestivum. A 24-well plate was first used to determine toxicity on individual grains using conventional seed germination/seedling growth toxicity tests whereas a single cell electrophoresis system was applied to measure genotoxicity at single cell level for wheat. Hydrophonic cultures provide a simplified environment to screen for toxicities with high sensitivity. Inverse dose-response relationships were detected between exogenous DDE levels and one of the following parameters: seed germination, seedling growth, and genotoxicity. In contrast, soil reduced the stress on T. aestivum by lowering bioavailability leading to less DDE distributed in radicle and coleoptile, modulated growth, and enhanced tolerance. At all DDE doses spiked to soil including the reference safety level of 0.5 mg/kg, DNA breakage was detected in both radicle and coleoptile but their magnitudes did not correlate with the organ nor the soil DDE contents. Thus, although wheat is highly sensitive to the genotoxic effect of DDE, first demonstrated here, the seed germination test offers a simple quantitative measure of DDE's phytotoxicity in soil and hydrophonic cultures. This study also found that fungus Pleurotus pulmonarius, which secretes extracellular ligninolytic enzymes causing non-specific cleavage of lignin and organopollutants, remediated DDE spiked to soil. In 5 weeks, 78% of 10 mg/kg DDE was biodegraded, and the fungal-treated soil reduced acute toxicity on T. aestivum using the seed germination test.     

Effect of dominant-negative epidermal growth factor receptors on cardiomyocyte hypertrophy

Angiotensin II (AngII) induces heart growth via cardiomyocyte hypertrophy, and central to this is the capacity of the type 1 AngII receptor (AT1R) to "transactivate" epidermal growth factor receptors (EGFRs)--a family with four main subtypes (HER1-4)--although the exact molecular mechanism remains unresolved. In this study, the pharmacological inhibition of AngII-stimulated ERK1/2 activation and cardiomyocyte hypertrophy by increasing concentrations of an EGFR inhibitor, AG1478, indicated that other EGFR subtypes, in addition to HER1, may be involved. We constructed expression vectors and adenoviruses expressing truncated mutant versions of HER1, HER2, and HER4 and determined their capacity to act as dominant-negative inhibitors when co-transfected with full-length EGFRs. It is surprising that adenoviral-mediated expression of these truncated EGFRs in cardiomyocytes led to paradoxical, ligand-independent increases in cardiomyocyte hypertrophy and unusual morphological changes. These results challenge our perception of AT1R-mediated EGFR transactivation and imply that truncated EGFRs may affect cell function through unconventional mechanisms.     

TLR2 and TLR4 differentially regulate B7-1 resulting in distinct cytokine responses to the mycoplasma superantigen MAM as well as to disease induced by Mycoplasma arthritidis

Mycoplasma arthritidis mitogen (MAM) is a superantigen secreted by M. arthritidis, an agent of murine arthritis and toxicity. We previously demonstrated that C3H mouse sub-strains differing in expression of Toll-like receptor 4 (TLR4), differed in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Here we examine the role of B7 co-stimulatory molecules in immune outcome and disease manifestations resulting from these different MAM/TLR2 and MAM/TLR4 interactions. Injections of MAM into C3H/HeJ mice upregulated expression of B7-1 but not B7-2 on peritoneal adherent cells, whereas B7-1 expression was lower on cells from C3H/HeSnJ mice. Anti-B7-1 antibody but not anti-B7-2, injected in vivo, changed the type 1 cytokines in MAM-injected C3H/HeJ mice to a type 2 cytokines and, conversely, the type 2 response in C3H/HeSnJ mice injected with anti-B7-1 shifted to a type 1 pattern. Whereas anti-B7-2 exerted no effect on disease in either mouse strain, anti-B7-1 significantly delayed the lethal toxicity of M. arthritidis in C3H/HeJ mice but enhanced arthritis in C3H/HeSnJ mice. Thus, TLR-mediated regulation of B7-1 results in diverse cytokine profiles in C3H sub-strains, and that the interaction of MAM with different TLR(s) may differentially affect cytokine responses and ultimately, M. arthritidis disease.     

Not so divided: the common basis of plant and animal cell division

Plant cells do not have centrioles and their mitosis is frequently likened to the chromosome-based mechanism seen in acentriolar animal cells. However, this is a false analogy. Although plants can use this mechanism, they generally divide by a method that uses bipolar mitotic caps, which is more similar to the canonical centrosome-based method of animals.     

PCR-based generation of shRNA libraries from cDNAs

Background  

The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries.