Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol

We present a fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods. The use of enriched genetically modified adult Schwann cells is of interest in the context of autologous cell transplantation within nerve transplants for peripheral nerve repair. Cell preparation from pre-degenerated adult peripheral nerves is described, together with the use of melanocyte growth medium plus forskolin, fibroblast growth factor-2 (FGF-2), pituitary extract and heregulin as a selective, serum-free culture medium and a subsequent cell enrichment step (cold jet). Proliferating adult Schwann cells can be efficiently genetically modified using optimized, non-viral electroporation protocols. The protocol results in Schwann cell cultures that are more than 90-95% pure, and transfection efficiencies vary depending on the initial cell constitution from 20 to 40%. The procedure takes up to 21 d, depending on the length of the pre-degeneration period.     

MUC4 mucin potentiates pancreatic tumor cell proliferation, survival, and invasive properties and interferes with its interaction to extracellular matrix proteins

MUC4, a transmembrane mucin, is aberrantly expressed in pancreatic adenocarcinomas while remaining undetectable in the normal pancreas. Recent studies have shown that the expression of MUC4 is associated with the progression of pancreatic cancer and is inversely correlated with the prognosis of pancreatic cancer patients. In the present study, we have examined the phenotypic and molecular consequences of MUC4 silencing with an aim of establishing the mechanistic basis for its observed role in the pathogenesis of pancreatic cancer. The silencing of MUC4 expression was achieved by stable expression of a MUC4-specific short hairpin RNA in CD18/HPAF, a highly metastatic pancreatic adenocarcinoma cell line. A significant decrease in MUC4 expression was detected in MUC4-knockdown (CD18/HPAF-siMUC4) cells compared with the parental and scrambled short interfering RNA-transfected (CD18/HPAF-Scr) control cells by immunoblot analysis and immunofluorescence confocal microscopy. Consistent with our previous observation, inhibition of MUC4 expression restrained the pancreatic tumor cell growth and metastasis as shown in an orthotopic mouse model. Our in vitro studies revealed that MUC4-associated increase in tumor cell growth resulted from both the enhanced proliferation and reduced cell death. Furthermore, MUC4 expression was also associated with significantly increased invasiveness (P < or = 0.05) and changes in actin organization. The presence of MUC4 on the cell surface was shown to interfere with the tumor cell-extracellular matrix interactions, in part, by inhibiting the integrin-mediated cell adhesion. An altered expression of growth- and metastasis-associated genes (LI-cadherin, CEACAM6, RAC1, AnnexinA1, thrombomodulin, epiregulin, S100A4, TP53, TP53BP, caspase-2, caspase-3, caspase-7, plakoglobin, and neuregulin-2) was also observed as a consequence of the silencing of MUC4. In conclusion, our study provides experimental evidence that supports the functional significance of MUC4 in pancreatic cancer progression and indicates a novel role for MUC4 in cancer cell signaling.     

An efficient algorithm for protein structure comparison using elastic shape analysis

Background

Protein structure comparison play important role in in silico functional prediction of a new protein. It is also used for understanding the evolutionary relationships among proteins. A variety of methods have been proposed in literature for comparing protein structures but they have their own limitations in terms of accuracy and complexity with respect to computational time and space. There is a need to improve the computational complexity in comparison/alignment of proteins through incorporation of important biological and structural properties in the existing techniques.

Results

An efficient algorithm has been developed for comparing protein structures using elastic shape analysis in which the sequence of 3D coordinates atoms of protein structures supplemented by additional auxiliary information from side-chain properties are incorporated. The protein structure is represented by a special function called square-root velocity function. Furthermore, singular value decomposition and dynamic programming have been employed for optimal rotation and optimal matching of the proteins, respectively. Also, geodesic distance has been calculated and used as the dissimilarity score between two protein structures. The performance of the developed algorithm is tested and found to be more efficient, i.e., running time reduced by 80–90 % without compromising accuracy of comparison when compared with the existing methods. Source codes for different functions have been developed in R. Also, user friendly web-based application called ProtSComp has been developed using above algorithm for comparing protein 3D structures and is accessible free.

Conclusions

The methodology and algorithm developed in this study is taking considerably less computational time without loss of accuracy (Table 2). The proposed algorithm is considering different criteria of representing protein structures using 3D coordinates of atoms and inclusion of residue wise molecular properties as auxiliary information.

     

Selection of Optimal Host Strain for Molecular Pathogenesis Studies on <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>

Encapsulated yeast, Cryptococcus gattii (Cg) is a primary and emerging fungal pathogen in North America. It has a predilection for invading the central nervous system of both healthy and immunocompromised humans and animals. Recently, we initiated molecular pathogenesis studies in Cg strain NIH444 (ATCC 32609). In this report, we compared the biology and pathogenic potential of NIH444 to those of WM276, an Australian environmental isolate that is being used for the whole genome-sequencing project. Our data indicated that NIH444 is comparatively more virulent in a mouse model of cryptococcosis than is WM 276. We found robust mating of NIH444, and no mating of WM276, when tested against Cg MATa strain, NIH198. WM276 but not NIH444 was defective in filamentation and sporulation (haploid fruiting). Interestingly, NIH444 has a VGII/AFLP6 genotype similar to that of the genotype of the recent outbreak strains from Vancouver Island, British Columbia, Canada. Additionally, comparisons of nucleotide sequences of various genes also showed differences between NIH444 and WM276. Based on these observations, we conclude that NIH444 should remain the strain of choice for understanding Cg pathogenesis, especially on the North American continent.     

Assessment of Polycyclic Aromatic Hydrocarbons (PAH) and Heavy Metals in the Vicinity of an Oil Refinery in India

Petroleum products are one of the major sources of energy for industry and daily life. Growth of the petroleum industry and shipping of petroleum products has resulted in the pollution. Populations living in the vicinity of oil refinery waste sites may be at greater risk of potential exposure to polycyclic aromatic hydrocarbons (PAH) through inhalation, ingestion, and direct contact with contaminated media. PAH have often been found to coexist with environmental pollutants including heavy metals due to similar pollution sources. The levels and distribution patterns of Σ16 PAH (sum of the 16 PAH) and heavy metals (lead, copper, nickel, cobalt, and chromium) were determined in soil and sediment in the vicinity (5 km radius) of an oil refinery in India. Concentrations of Σ16 PAH in the soils and sediments were found to be 60.36 and 241.23 ppm, respectively. Higher amount of PAH in sediments as compared to soil is due to low water solubility of PAH, settled in the bottom of aquatic bodies. The levels of lead, copper, nickel, cobalt, and chromium (total) in soil were 12.52, 13.52, 18.78, 4.84, and 8.29 ppm, while the concentrations of these metals in sediments were 16.38, 47.88, 50.15, 7.07, and 13.25 ppm, respectively. Molecular diagnostics indices of PAH (Ratio of Phenanthrene/Anthracene, Fluranthene/Pyrene) calculated for soil and sediment samples indicate that the oil refinery environment is contaminated with PAH from petrogenic as well as pyrolytic origin and heavy vehicular traffic on the Agra- Delhi National highway. Sixteen PAH priority pollutants were detected in the United States in entire samples collected near oil refinery areas and concentrations of Σ16 PAH in soil was found to be 1.20 times higher than the threshold value for PAH in soil by ICRCL (Inter-Departmental Committee on the Redevelopment of Contaminated Land). This concentration could lead to disastrous consequences for the biotic and abiotic components of the ecosystem and may affect the soil quality, thus impairing plant growth and its bioaccumulation in food chain.     

Validation of the tetracycline regulatable gene expression system for the study of the pathogenesis of infectious disease

Understanding the pathogenesis of infectious disease requires the examination and successful integration of parameters related to both microbial virulence and host responses. As a practical and powerful method to control microbial gene expression, including in vivo, the tetracycline-regulatable system has recently gained the favor of many investigative groups. However, some immunomodulatory effects of the tetracyclines, including doxycycline, could potentially limit its use to evaluate host responses during infection. Here we have used a well-established murine model of disseminated candidiasis, which is highly dependent on both the virulence displayed by the fungal cells and on the host immune status, to validate the use of this system. We demonstrate that the pathogenesis of the wild type C. albicans CAF2-1 strain, which does not contain any tet-regulatable element, is not affected by the presence of doxycycline. Moreover levels of key cytokines, chemokines and many other biomarkers, as determined by multi-analyte profiling, remain essentially unaltered by the presence of the antibiotic during infection. Our results indicate that the levels of doxycycline needed to control the tetracycline regulatable promoter gene expression system have no detectable effect on global host responses during candidiasis. Because tet-regulatable systems are now being increasingly used in a variety of pathogenic microorganisms, these observations have wide implications in the field of infectious diseases.     

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 μg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 μg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.