Applications of patient-specific induced pluripotent stem cells; focused on disease modeling, drug screening and therapeutic potentials for liver disease

The recent advances in the induced pluripotent stem cell (iPSC) research have significantly changed our perspectives on regenerative medicine by providing researchers with a unique tool to derive disease-specific stem cells for study. In this review, we describe the human iPSC generation from developmentally diverse origins (i.e. endoderm-, mesoderm-, and ectoderm- tissue derived human iPSCs) and multistage hepatic differentiation protocols, and discuss both basic and clinical applications of these cells including disease modeling, drug toxicity screening/drug discovery, gene therapy and cell replacement therapy.     

Effective bioremoval and detoxification of textile dye mixture by Alishewanella sp. KMK6

Alishewanella sp. strain KMK6 was able to degrade mixture of textile dyes (0.5–2.0 g?l^?1) within 8 h. An initial 28 % reduction in COD was observed immediately after decolorization at static anoxic conditions which on further incubation at shaking conditions reduced by 90 %. Partially purified azoreductase was able to utilize different azo dyes as substrates. The HPLC profile of dye degradation showed formation of metabolic products. Further FTIR analysis showed significant changes in the peaks corresponding to functional groups present in dye mixture and its degradation products. The genotoxicity assessment showed that the dye degradation products were non-toxic compared to dye mixture.     

Liposome‐mediated transfection of mature taste cells

The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction‐related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used α‐gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome‐mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common “promiscuous” promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells. © 2005 Wiley Periodicals, Inc. J. Neurobiol, 2005     

Differences in tibial rotation during walking in ACL reconstructed and healthy contralateral knees

This study tested the hypotheses that in patients with a successful anterior cruciate ligament (ACL) reconstruction, the internal–external rotation, varus–valgus, and knee flexion position of reconstructed knees would be different from uninjured contralateral knees during walking. Twenty-six subjects with unilateral ACL reconstructions (avg 31 years, 1.7 m, 68 kg, 15 female, 24 months past reconstruction) and no other history of serious lower limb injury walked at a self-selected speed in the gait laboratory, with the uninjured contralateral knee as a matched control. Kinematic measurements of tibiofemoral motion were made using a previously-described point-cluster technique. Repeated-measures ANOVA (α=0.017) was used to compare ACL-reconstructed knees to their contralateral knees at four distinct points during the stance phase of walking. An offset towards external tibial rotation in ACL-reconstructed knees was maintained over all time points (95%CI 2.3±1.3°). Twenty-two out of twenty-six individuals experienced an average external tibial rotation offset throughout stance phase. Varus–valgus rotation and knee flexion were not significantly different between reconstructed and contralateral knees. These findings show that differences in tibial rotation during walking exist in ACL reconstructed knees compared to healthy contralateral knees, providing a potential explanation why these patients are at higher risk of knee osteoarthritis in the long-term.     

Comparative assessment of bone pose estimation using Point Cluster Technique and OpenSim

Estimating the position of the bones from optical motion capture data is a challenge associated with human movement analysis. Bone pose estimation techniques such as the Point Cluster Technique (PCT) and simulations of movement through software packages such as OpenSim are used to minimize soft tissue artifact and estimate skeletal position; however, using different methods for analysis may produce differing kinematic results which could lead to differences in clinical interpretation such as a misclassification of normal or pathological gait. This study evaluated the differences present in knee joint kinematics as a result of calculating joint angles using various techniques. We calculated knee joint kinematics from experimental gait data using the standard PCT, the least squares approach in OpenSim applied to experimental marker data, and the least squares approach in OpenSim applied to the results of the PCT algorithm. Maximum and resultant RMS differences in knee angles were calculated between all techniques. We observed differences in flexion/extension, varus/valgus, and internal/external rotation angles between all approaches. The largest differences were between the PCT results and all results calculated using OpenSim. The RMS differences averaged nearly 5° for flexion/extension angles with maximum differences exceeding 15°. Average RMS differences were relatively small (< 1.08°) between results calculated within OpenSim, suggesting that the choice of marker weighting is not critical to the results of the least squares inverse kinematics calculations. The largest difference between techniques appeared to be a constant offset between the PCT and all OpenSim results, which may be due to differences in the definition of anatomical reference frames, scaling of musculoskeletal models, and/or placement of virtual markers within OpenSim. Different methods for data analysis can produce largely different kinematic results, which could lead to the misclassification of normal or pathological gait. Improved techniques to allow non-uniform scaling of generic models to more accurately reflect subject-specific bone geometries and anatomical reference frames may reduce differences between bone pose estimation techniques and allow for comparison across gait analysis platforms.     

Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10 g l^?1, pH 8) after 72 h at 30 °C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme was purified to 67-fold with yield of 2.25% having a specific activity of 1670 U mg^?1 and ～36 kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2). The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper and zinc (1 mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3 mM of Fe M^?1 as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about 79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purified keratinase revealed significant degradation (85%) of feather concentrate (20 g l^?1) to 3.9 μM ml^?1 of free amino groups in 24 h at an initial pH of 8.0, 30 °C and 120 rpm shaking. This keratinase activity can be controlled precisely by presence of chemical or metal ions which could be of use in biotechnology industry while the culture can be used in poultry waste management.     

Correlation between substituent constants and hyperpolarizabilities for di-substituted trans-azobenzenes

Nonlinear optical properties of a series of disubstituted trans-azobenzenes were studied. The structures were fully optimized by B3LYP/6-31+G* and both static polarizabilities and hyperpolarizabilities were then calculated by the derivative method. In order to show the relationships between dipole moments, (hyper)polarizabilities and the structures, three kinds of substituent constants were applied to correlate with both ground state dipole moment and hyperpolarizabilities. Both physical properties have a satisfactory correlation with substituent constants Σσ_+/? and bond length alternation. Overall, the electronic excitation contribution to the hyperpolarizabilities is rationalized in terms of the two-level model.     

Infrared and electronic absorption spectra of n-butyronitrile and its ions using Møller Plesset method

We report theoretical infrared and electronic absorption spectra of gauche and anti conformers of n-butyronitrile, their ions and 2-methylpropanenitrile isomer of n-butyronitrile. The coupled cluster theory (CCSD) and second order Møller-Plesset perturbation (MP2) theory with TZVP basis set are used for the study. Vibrational frequencies of gauche and anti conformers of neutral n-butyronitrile at MP2/TZVP and CCSD/TZVP levels are in agreement with the experimental determinations. Rotational and distortion constants are also in good agreement with the available experimental values. Time dependent density functional theory is used to study the electronic absorption spectra of gauche and anti conformers, their ions and an isomer of butyronitrile. All the electronic transitions of gauche and anti conformers of neutral n-butyronitrile and 2-methylpropanenitrile are σ→σ* transitions whereas ions of n-butyronitrile show both σ→σ* as well as π→π* transitions in vacuum UV, far UV and visible regions. This study helps in detection of neutral gauche and anti conformer and their ions in interstellar medium.     

Bioprospecting of thermo- and osmo-tolerant fungi from mango pulp–peel compost for bioethanol production

The persistent edaphic stress on microbial succession due to dynamic changes during composting was explored for selection of multi-stress tolerant microbe(s) desirable for ethanol production. A total of 23 strains were isolated from mango compost using four successive enrichments in YP broth (g l^?1): glucose, 100; 150; 250 with ethanol (40) and cycloheximide (0.4) at 40 °C, pH 6.0. Based on multi-gene ribotyping, 14 yeasts (61 %) of Saccharomycetaceae, 2 filamentous fungi (8.6 %) and 7 bacteria (30.4 %) were obtained. Phenetic and phylogenetic analysis of the 14 yeasts revealed 64.3 % tolerant to 500 g l^?1 glucose, growth at 45 °C and resemblance to Candida sp. (14.3 %), Kluyveromyces marxianus (35.7 %), Pichia kudriavzevii (21.4 %) and Saccharomyces cerevisiae (28.6 %). Assessment of the 14 yeasts in glucose fermentation medium (pH 4.5 at 40 °C) showed ethanol productivity of ≥92 % by 12 yeasts with theoretical yields of 90–97 %. Fermentation of molasses (150 g l^?1 glucose equivalent) by P. kudriavzevii D1C at 40 °C resulted in 73.70 ± 0.02 g l^?1 ethanol and productivity of 4.91 ± 0.01 g l^?1 h^?1. Assessment of P. kudriavzevii D1C revealed multi-stress tolerance towards 5-hydroxymethyl furfural, ethanol (20 %, v/v), high gravity and H₂O₂ (0.3 M) indicating suitability for ethanol production using high gravity molasses and pre-treated lignocellulosic biomass fermentation.