Raman Spectroscopic Study of Radioresistant Oral Cancer Sublines Established by Fractionated Ionizing Radiation

Radiotherapy is an important treatment modality for oral cancer. However, development of radioresistance is a major hurdle in the efficacy of radiotherapy in oral cancer patients. Identifying predictors of radioresistance is a challenging task and has met with little success. The aim of the present study was to explore the differential spectral profiles of the established radioresistant sublines and parental oral cancer cell lines by Raman spectroscopy. We have established radioresistant sublines namely, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B from its parental UPCI:SCC029B cell line, by using clinically admissible 2Gy fractionated ionizing radiation (FIR). The developed radioresistant character was validated by clonogenic cell survival assay and known radioresistance-related protein markers like Mcl-1, Bcl-2, Cox-2 and Survivin. Altered cellular morphology with significant increase (p<0.001) in the number of filopodia in radioresistant cells with respect to parental cells was observed. The Raman spectra of parental UPCI:SCC029B, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B cells were acquired and spectral features indicate possible differences in biomolecules like proteins, lipids and nucleic acids. Principal component analysis (PCA) provided three clusters corresponding to radioresistant 50Gy, 70Gy-UPCI:SCC029B sublines and parental UPCI:SCC029B cell line with minor overlap, which suggest altered molecular profile acquired by the radioresistant cells due to multiple doses of irradiation. The findings of this study support the potential of Raman spectroscopy in prediction of radioresistance and possibly contribute to better prognosis of oral cancer.     

Microchips based on three dimensional gel cells: history and perspective

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.     

A Review of Multi-Responsive Membranous Systems for Rate-Modulated Drug Delivery

Membrane technology is broadly applied in the medical field. The ability of membranous systems to effectively control the movement of chemical entities is pivotal to their significant potential for use in both drug delivery and surgical/medical applications. An alteration in the physical properties of a polymer in response to a change in environmental conditions is a behavior that can be utilized to prepare ‘smart’ drug delivery systems. Stimuli-responsive or ‘smart’ polymers are polymers that upon exposure to small changes in the environment undergo rapid changes in their microstructure. A stimulus, such as a change in pH or temperature, thus serves as a trigger for the release of drug from membranous drug delivery systems that are formulated from stimuli-responsive polymers. This article has sought to review the use of stimuli-responsive polymers that have found application in membranous drug delivery systems. Polymers responsive to pH and temperature have been extensively addressed in this review since they are considered the most important stimuli that may be exploited for use in drug delivery, and biomedical applications such as in tissue engineering. In addition, dual-responsive and glucose-responsive membranes have been also addressed as membranes responsive to diverse stimuli.     

Phenotypic and Molecular Characterization of Wheat for Slow Rusting Resistance against Puccinia striiformis Westend. f.sp. tritici

Race‐specific resistance of wheat (Triticum aestivum L.) to yellow rust caused by Puccinia striiformis Westend. f.sp. tritici is often short‐lived. Slow‐rusting resistance has been reported to be a more durable type of resistance. A set of sixteen bread wheat varieties along with a susceptible control Morocco was tested during 2004–05 to 2006–07 in field plots at Peshawar (Pakistan) to identify slow rusting genotypes through epidemiological variables including final rust severity (FRS), apparent infection rate (r), area under disease progress curve (AUDPC), average coefficients of infection (ACI) and leaf tip necrosis (LTN). Epidemiological parameters of resistance were significantly (P < 0.01) different for years/varieties in three seasons, while variety × year interactions remained non‐significant. Sequence tagged site (STS) marker, csLV34 analyses revealed that cultivars Faisalabad‐83, Bahawalpur‐95, Suleman‐96, Punjab‐96, Bakhtawar‐93, Faisalabad‐85, Shahkar‐95 and Kohsar‐95 possessed Yr18 linked allele. Faisalabad‐83, Bahawalpur‐95, Suleman‐96, Punjab‐96, Bakhtawar‐93 and Faisalabad‐85 were relatively more stable over 3‐years where FRS, AUDPC and r values reduced by 80, 84 and 70% respectively compared to control Morocco. These six varieties therefore could be exploited for the deployment of Yr18 in breeding for slow rusting in wheat. Both FRS and ACI are suitable parameters for phenotypic selection.     

SNPdetector: a software tool for sensitive and accurate SNP detection

Identification of single nucleotide polymorphisms (SNPs) and mutations is important for the discovery of genetic predisposition to complex diseases. PCR resequencing is the method of choice for de novo SNP discovery. However, manual curation of putative SNPs has been a major bottleneck in the application of this method to high-throughput screening. Therefore it is critical to develop a more sensitive and accurate computational method for automated SNP detection. We developed a software tool, SNPdetector, for automated identification of SNPs and mutations in fluorescence-based resequencing reads. SNPdetector was designed to model the process of human visual inspection and has a very low false positive and false negative rate. We demonstrate the superior performance of SNPdetector in SNP and mutation analysis by comparing its results with those derived by human inspection, PolyPhred (a popular SNP detection tool), and independent genotype assays in three large-scale investigations. The first study identified and validated inter- and intra-subspecies variations in 4,650 traces of 25 inbred mouse strains that belong to either the Mus musculus species or the M. spretus species. Unexpected heterozygosity in CAST/Ei strain was observed in two out of 1,167 mouse SNPs. The second study identified 11,241 candidate SNPs in five ENCODE regions of the human genome covering 2.5 Mb of genomic sequence. Approximately 50% of the candidate SNPs were selected for experimental genotyping; the validation rate exceeded 95%. The third study detected ENU-induced mutations (at 0.04% allele frequency) in 64,896 traces of 1,236 zebra fish. Our analysis of three large and diverse test datasets demonstrated that SNPdetector is an effective tool for genome-scale research and for large-sample clinical studies. SNPdetector runs on Unix/Linux platform and is available publicly (http://lpg.nci.nih.gov).     

Maintenance of Chloroplast Structure and Function by Overexpression of the Rice MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE Gene Leads to Enhanced Salt Tolerance in Tobacco

In plants, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactodiacylglycerol (DGDG) are major constituents of photosynthetic membranes in chloroplasts. One of the key enzymes for the biosynthesis of these galactolipids is MGDG synthase (MGD). To investigate the role of MGD in the plant’s response to salt stress, we cloned an MGD gene from rice (Oryza sativa) and generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD. The MGD activity in OsMGD transgenic plants was confirmed to be higher than that in the wild-type tobacco cultivar SR1. Immunoblot analysis indicated that OsMGD was enriched in the outer envelope membrane of the tobacco chloroplast. Under salt stress, the transgenic plants exhibited rapid shoot growth and high photosynthetic rate as compared with the wild type. Transmission electron microscopy observation showed that the chloroplasts from salt-stressed transgenic plants had well-developed thylakoid membranes and properly stacked grana lamellae, whereas the chloroplasts from salt-stressed wild-type plants were fairly disorganized and had large membrane-free areas. Under salt stress, the transgenic plants also maintained higher chlorophyll levels. Lipid composition analysis showed that leaves of transgenic plants consistently contained significantly higher MGDG (including 18:3-16:3 and 18:3-18:3 species) and DGDG (including 18:3-16:3, 18:3-16:0, and 18:3-18:3 species) contents and higher DGDG-MGDG ratios than the wild type did under both control and salt stress conditions. These results show that overexpression of OsMGD improves salt tolerance in tobacco and that the galactolipids MGDG and DGDG play an important role in the regulation of chloroplast structure and function in the plant salt stress response.Salt stress is a major environmental factor that poses a serious threat to crop yield and future food production (Møller and Tester, 2007). When plants are exposed to salinity, they suffer two primary obstacles: low external water potentials and high concentrations of toxic ions (Hirayama and Mihara, 1987). These obstacles generally lead to the disruption of various enzymatic processes, changes in membrane lipid composition, alteration in chloroplast structure and function, impairment of photosynthetic capacity, and inhibition of plant growth (Brown and Dupont, 1989; Elkahoui et al., 2004; Munns and Tester, 2008; Sui et al., 2010; Shu et al., 2012).Membranes are the primary matrix for numerous physiological and biochemical activities, and plants easily change their membrane lipid compositions in response to environmental stresses (Harwood, 1996). A number of studies have proved that salt stress can induce changes in plant membrane lipids (Huflejt et al., 1990; Elkahoui et al., 2004; Sui et al., 2010). Sui et al. (2010) found that, in Suaeda salsa, salt stress increased the proportion of phosphatidylglycerol and reduced the proportion of galactolipids. Similar results were observed in Catharanthus roseus cultured cell suspensions, which showed an increase in phospholipid content and a decrease in galactolipid content that were more obvious under 100 mm NaCl than under 50 mm (Elkahoui et al., 2004). Meanwhile, it was shown that salt tolerance in plants is strongly linked with their membrane lipid composition and especially with their galactolipid content, which is positively related to salt tolerance (Hirayama and Mihara, 1987).In plants, galactolipids are major constituents of the photosynthetic membrane, which is the most abundant membrane in nature (Lee, 2000). Two galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are the predominant photosynthetic membrane lipid building blocks, accounting for about 52 mol % and 26 mol % of thylakoid membrane lipids, respectively (Block et al., 1983). These galactolipids are also the major lipids in cyanobacteria, suggesting that MGDG and DGDG are important for all oxygenic photosynthetic organisms (Hölzl and Dörmann, 2007). They are components of PSII and are the indispensable matrix for photosynthesis (Mizusawa and Wada, 2012). Their unique characteristics also give them distinctive packing properties that are important for the organization of thylakoid membranes (Lee, 2000). MGDG has a conical shape due to its smaller head group and a high content of unsaturated fatty acids, giving it nonbilayer-forming characteristics (Webb and Green, 1991; Aronsson et al., 2008); this feature is assumed to play an important structural role in the dense packing of proteins in the membrane (Williams, 1998; Garab et al., 2000). In contrast to MGDG, DGDG has a cylindrical shape that is typical for most plastid lipids and is considered a bilayer-prone lipid (Aronsson et al., 2008); this feature is involved in lipid-mediated contacts between adjacent trimers of light-harvesting complex II (LHCII) when they are packed into two-dimensional crystalline arrays (Lee, 2000). In addition to forming important membrane structures in the thylakoids, MGDG and DGDG are also present in extraplastidic membranes, including the plasma membrane, tonoplasts, endoplasmic reticulum, and Golgi membranes, indicating the crucial role of these galactoglycerolipids in higher plant membrane systems (Yoshida and Uemura, 1986; Brown and Dupont, 1989; Härtel et al., 2000).Two enzymes are involved in the biosynthesis of these galactoglycerolipids: MGDG synthase (MGD), which transfers a galactosyl residue from UDP-Gal to diacylglycerol, and DGDG synthase, which catalyzes the further galactosylation of MGDG to form DGDG (Shimojima et al., 1997; Dörmann et al., 1999; Shimojima and Ohta, 2011). Thus, MGD is the key enzyme in the biosynthesis of both galactolipids and, consequently, also in the formation of photosynthetic membranes (Nakamura et al., 2010). A number of studies have revealed that MGD is vital for plant growth and development. The loss of MGD function in plants leads to a pale-green phenotype, defects in the chloroplast ultrastructure, disruption in the photosynthetic membranes, and complete impairment of photosynthetic ability and photoautotrophic growth, suggesting a unique role for MGD in chlorophyll formation, the structural organization of the plastidic membranes, and photosynthetic growth (Jarvis et al., 2000; Kobayashi et al., 2007; Botté et al., 2011; Myers et al., 2011). The crucial role of MGD under environmental stresses, including phosphorus deficiency and wounding, is also well studied (Kobayashi et al., 2004, 2009a, 2009b; Moellering and Benning, 2011). However, although a number of studies have proved that salt stress can induce changes in plant membrane lipids (Huflejt et al., 1990; Elkahoui et al., 2004; Sui et al., 2010), little is known about the role of MGD and the involvement of galactolipids in response to salt stress.To investigate the function of MGD in plant salt tolerance, we cloned the relevant gene, which is called OsMGD (Qi et al., 2004), from rice (Oryza sativa ‘FR13A’). This gene has a high similarity to Arabidopsis (Arabidopsis thaliana) MGD2 and MGD3, and its expression is induced by several environmental stresses, including salt, drought, and submergence (Qi et al., 2004; Benning and Ohta, 2005). We then generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD and investigated the salt tolerance ability in the transgenic lines and wild-type plants. The results of this study demonstrate that an increase in galactolipid content in leaves is beneficial for maintaining chloroplast structure and function and leads to enhanced salt tolerance in tobacco.     

Preparation and characterization of monoclonal antibodies to the cholera toxin

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.     

Viability and angiogenic activity of mesenchymal stromal cells from adipose tissue and bone marrow under hypoxia and inflammation in vitro

Progenitor stromal cells derived from adipose tissue (ADSC) and bone marrow (BMDSC) hold great promise for use in the cell-based therapy of ischemic diseases. It was demonstrated that these cells secrete a number of angiogenic cytokines that stimulate vascularization. It was demonstrated that ADSC or BMDSC injected intramuscularly or intravenously into the animals with experimental hind-limb ischemia improve vascularization. However, low oxygen levels and inflammation may impair the viability and functional activity of transplanted cells. We have examined ADSC and BMDSC properties in vitro under hypoxic and inflammatory conditions. ADSC and BMDSC derived from Balb/c mice have been cultivated under hypoxia or in the presence of inflammatory cytokines. The viability of cells assessed by annexin V-PE binding and 7AAD storage, as well as by the quantitative TUNEL method, was not changed under hypoxic conditions Cell exposure to inflammatory cytokines induced apoptosis in 70% of cells. Inflammatory cytokines did not stimulate gene expression of angiogenic growth factors. Under hypoxia conditions up-regulation of genes for pro-angiogenic factors and down-regulation of anti-angiogenic genes were more apparent in ADSC. Using angiogenesis models in vitro and in vivo, we demonstrated that stromal cell maintenance under hypoxic conditions increased their ability to stimulate the growth of blood vessels.