Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648‐bp segment near the 5′ terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5′ region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.     

Extent and diversity of human alternative splicing established by complementary database annotation and microarray analysis

Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor, and acceptor sites. In large-scale microarray studies in humans and the mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across six pair of diverse cell lines and validated the database annotation process. Results suggest that splicing in humans is more complex than simple exon-skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts. In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology.     

Colon cancer prediction with genetic profiles using intelligent techniques

Micro array data provides information of expression levels of thousands of genes in a cell in a single experiment. Numerous efforts have been made to use gene expression profiles to improve precision of tumor classification. In our present study we have used the benchmark colon cancer data set for analysis. Feature selection is done using t‐statistic. Comparative study of class prediction accuracy of 3 different classifiers viz., support vector machine (SVM), neural nets and logistic regression was performed using the top 10 genes ranked by the t‐statistic. SVM turned out to be the best classifier for this dataset based on area under the receiver operating characteristic curve (AUC) and total accuracy. Logistic Regression ranks as the next best classifier followed by Multi Layer Perceptron (MLP). The top 10 genes selected by us for classification are all well documented for their variable expression in colon cancer. We conclude that SVM together with t-statistic based feature selection is an efficient and viable alternative to popular techniques.     

Identification, purification, and characterization of a secretory serine protease in an Indian strain of Leishmania donovani

An aprotinin sensitive serine protease was identified in the culture supernatant of the Indian strain of Leishmania donovani (MHOM/IN/1983/AG83). The protease was subsequently purified and characterized. The apparent molecular mass of the enzyme was 115 kDa in SDS-PAGE under non-reducing condition, while on reduction it showed a 56 kDa protein band indicating that the protease is a dimeric protein. The purified enzyme was optimally active at the pH and temperature of 7.5 and 28 degrees C, respectively. Assays of thermal stability indicated that the enzyme preserved 59% of activity even after pretreatment at 42 degrees C for 1 h. The purified protease was not glycosylated and its isoelectric pI was 5.0. N-alpha-p-tosyl-L-arginine methylester (TAME) appeared to be relatively better substrate among the commonly used synthetic substrates. The enzyme was inhibited by Ca(2+) and Mn(2+), but activated by Zn(2+). The protease could play important role(s) in the pathogenesis of visceral leishmaniasis or kala-azar.     

A rapid immunochromatographic assay for the detection of Mycobacterium tuberculosis antigens in pulmonary samples from HIV seropositive patients and its comparison with conventional methods

As tuberculosis generates a highly heterogeneous antibody repertoire, its diagnosis requires tests based on cocktails of antigens. We describe a new, rapid method called rapid immunochromatographic assay (RICA) for cocktail-based diagnosis, which can detect Mycobacterial antigens in sputum specimens. Six antigenic fractions of pathogenic Mycobacterium tuberculosis were used in combination as the capture antigens in the control line of the flow-through assay. Antigen detection of 200 sputum samples from HIV seropositive patients by RICA assay gave a sensitivity of 97.9%, specificity of 99.0%, positive predictive value of 98.9%, negative predictive value of 98.0%, false positive rate of 0.9%, false negative rate of 2.0%, prevalence rate of 49%, likelihood ratio for positive results 97 and likelihood ratio for negative results 0.02. The combination of RICA and AFB staining gave a sensitivity of 100%, specificity of 100%, positive predictive value of 100%, negative predictive value of 100%, false positive rate of 0%, false negative rate of 0%, likelihood ratio for negative results 0. The assay was simple, rapid and economical for the detection of M. tuberculosis infection and suitable for large scale screening of samples in endemic areas without any sophisticated equipment. The results of the assay proved to be superior to conventional methods and combined with clinical data, could form the basis for starting an earlier course of treatment.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.     

Cytokeratin localization in toe pads of the anuran amphibian Philautus annandalii (Boulenger, 1906)

We have examined cytokeratin distribution and their nature in toe pads of the Himalayan tree-frog Philautus annandalii. Toe pads are expanded tips of digits and show modifications of their ventral epidermis for adhesion. The toe pad epidermal cells, being organized into 3–4 rows, possess keratin bundles, especially in surface nanostructures that are involved in adhesion. Immunohistochemical localization using a pan-cytokeratin antibody revealed that cytokeratin immunoreactivity is the strongest in the mid- to basal cell rows of the epidermis, which parallels our previous ultrastructural observation of dense keratin bundles present in this part of the epidermis. The remainder of the epidermis (i.e., the superficial cell layer) showed little immunoreactivity. Immunoblot analysis revealed that toe-pads possessed keratins prominently in the molecular mass of 50 kDa. Possible presence of keratin 5 in toe pad epidermis has been correlated with its usual distribution pattern in mammalian epidermis.     

Activation of proMMP-2 by U46619 occurs via involvement of p38MAPK-NFκB-MT1MMP signaling pathway in pulmonary artery smooth muscle cells

We investigated the mechanism by which TxA₂ mimetic, U46619, activates proMMP-2 in bovine pulmonary artery smooth muscle cells. Our study showed that treatment of the cells with U46619 caused an increase in the expression and subsequently activation of proMMP-2 in the cells. Pretreatment with p³⁸MAPK inhibitor, SB203580; and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by U46619. U46619 also induced increase in MT1-MMP expression, which was inhibited upon pretreatment with SB203580 and Bay11-7082. U46619 treatment to the cells stimulated p³⁸MAPK activity as well as NF-κB activation by IκB-α phosphorylation, translocation of NF-κBp65 subunit from cytosol to nucleus and subsequently, by increasing its DNA-binding activity. Induction of NF-κB activation seems to be mediated through IKK, as transfection of cells with either IKKα or IKKβ siRNA prevented U46619-induced phosphorylation of IκB-α and NF-κBp65 DNA-binding activity. U46619 treatment to the cells also downregulated the TIMP-2 level. Pretreatment of the cells with SB203580 and Bay11-7082 did not show any discernible change in TIMP-2 level by U46619. Overall, U46619-induced activation of proMMP-2 is mediated via involvement of p³⁸MAPK-NFκB-MT1MMP signaling pathway with concomitant downregulation of TIMP-2 expression in bovine pulmonary artery smooth muscle cells.