ECOLOGY AND THE RAPD TECHNIQUES USED TO ASSESS THE GENETIC DIVERSITY IN PTEROLOBIUM HEXAPETALUM, A SCRAMBLING MEDICINAL SHRUB IN MARUTHAMALAI AND CHENNIMALAI HILLS, THE WESTERN GHATS

Objective: To investigate the ecological and genetic diversity, climatic factors, edaphic factors morphological and reproductive characters and RAPD analysis of medicinal plant species Pterolobium hexapetalum in two hills viz., Maruthamalai (arid) and Chennimalai (very arid), which is located in Coimbatore and Erode districts, Tamil Nadu. Methods: The present research was carried out by using a random amplified polymorphic DNA (RAPD) analysis was made to determine the genetic variation between the two populations of the medicinal shrub, Pterolobium hexapetalum in an environmental gradient. Among the five primers tested, the OPN7 (80 %) and OPN17 (71.4 %) produced higher polymorphism was used in RAPD analysis. Results: The results of RAPD analysis showed the presence of 51 individual bands were formed, out of which, 29 were polymorphic bands which showed the existence of genetic variation between populations. A dendrogram was constructed based on Jaccard’s coefficient to determine the degree of genetic relationship among the two populations and analysed. The primers OPN7 and OPN17 were clustered together at a genetic distance level 10. Considering the elevation and proximity, the temperature ranges from 18 °C to 37.6 °C in Maruthamalai hill and 20 °C to 39.4 °C in Chennimalai hill. Conclusion: From the morphoecological studies the results indicated that both arid and very arid climatic conditions showed slight differences in their vegetative and reproductive characters

Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen’s Specific Memory T Cells in Healthy Household Contacts

In vitro mimicking conditions are thought to reflect the environment experienced by M. tuberculosis inside the host granuloma. The majority of the in vitro dormancy experimental models used laboratory adapted strain H37Rv or Erdman strain over the prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from the hypoxic cultures, resolved by two-dimensional electrophoresis (2-DE) and protein spots were characterized by mass spectrometry. Totally 49 spots were characterized as over-expressed or newly appeared between the 3 strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these 2 genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolour flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in circulation of HHC when compared to PTB (p<0.005 for ESAT-6 and p<0.0005 for Lpd). This shows proteins that are predicted to be upregulated during in vitro hypoxia in most prevalent clinical strains would bring the possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also bring the probable true representative antigens that involved during adaption mechanism

Antisense Oligonucleotide-Mediated Splice Switching: Potential Therapeutic Approach for Cancer Mitigation

Splicing is an essential process wherein precursor messenger RNA (pre-mRNA) is reshaped into mature mRNA. In alternative splicing, exons of any pre-mRNA get rearranged to form mRNA variants and subsequently protein isoforms, which are distinct both by structure and function. On the other hand, aberrant splicing is the cause of many disorders, including cancer. In the past few decades, developments in the understanding of the underlying biological basis for cancer progression and therapeutic resistance have identified many oncogenes as well as carcinogenic splice variants of essential genes. These transcripts are involved in various cellular processes, such as apoptosis, cell signaling and proliferation. Strategies to inhibit these carcinogenic isoforms at the mRNA level are promising. Antisense oligonucleotides (AOs) have been developed to inhibit the production of alternatively spliced carcinogenic isoforms through splice modulation or mRNA degradation. AOs can also be used to induce splice switching, where the expression of an oncogenic protein can be inhibited by the induction of a premature stop codon. In general, AOs are modified chemically to increase their stability and binding affinity. One of the major concerns with AOs is efficient delivery. Strategies for the delivery of AOs are constantly being evolved to facilitate the entry of AOs into cells. In this review, the different chemical modifications employed and delivery strategies applied are discussed. In addition to that various AOs in clinical trials and their efficacy are discussed herein with a focus on six distinct studies that use AO-mediated exon skipping as a therapeutic strategy to combat cancer