Sujeet Pratap Singh

Secreted semaphorin 5A suppressed pancreatic tumour burden but increased metastasis and endothelial cell proliferation

Background:Our earlier reports demonstrated that membrane-bound semaphorin 5A (SEMA5A) is expressed in aggressive pancreatic cancer cells and tumours, and promotes tumour growth and metastasis. In this study, we examine whether (1) pancreatic cancer cells secrete SEMA5A and (2) that secreted SEMA5A modulates certain phenotypes associated with tumour progression, angiogenesis and metastasis through various other molecular factors and signalling proteins.Methods and results:In this study, we show that human pancreatic cancer cell lines secrete the extracellular domain (ECD) of SEMA5A (SEMA5A-ECD) and overexpression of mouse Sema5A-ECD in Panc1 cells (not expressing SEMA5A; Panc1-Sema5A-ECD; control cells - Panc1-control) significantly increases their invasion in vitro via enhanced ERK phosphorylation. Interestingly, orthotopic injection of Panc1-Sema5A-ECD cells into athymic nude mice results in a lower primary tumour burden, but enhances the micrometastases to the liver as compared with Panc1-control cells. Furthermore, there is a significant increase in proliferation of endothelial cells treated with conditioned media (CM) from Panc1-Sema5A-ECD cells and a significant increase in microvessel density in Panc1-Sema5A-ECD orthotopic tumours compared with those from Panc1-control cells, suggesting that the increase in liver micrometastases is probably due to increased tumour angiogenesis. In addition, our data demonstrate that this increase in endothelial cell proliferation by Sema5A-ECD is mediated through the angiogenic molecules – interleukin-8 and vascular endothelial growth factor.Conclusion:Taken together, these results suggest that a bioactive, secreted form of Sema5A-ECD has an intriguing and potentially important role in its ability to enhance pancreatic tumour invasiveness, angiogenesis and micrometastases.

Identification of sugarcane microsatellites associated to sugar content in sugarcane and transferability to other cereal genomes

Microsatellites or simple sequence repeats (SSRs) markers are very informative for various applications in genetics and breeding. Information obtained with these markers has contributed to a better understanding of evolution and the complexity of the sugarcane genome. With the objective of identifying a large set of polymorphic microsatellite markers designated as Unigene derived Sugarcane Microsatellite (UGSM) and Sugarcane Enriched Genomic Microsatellite (SEGMS), 351 UGSM and 36 SEGMS were tested to find out informative SSRs marker for sugar content. These markers were screened and validated for their use in genetic diversity, cross transferability and comparative linkage potential in high and low sugar bulk of two segregating progenies and twenty each, cultivated high and low sugar cultivars. 158 (40.83%) of the microsatellite markers (144-UGSM: 14-SEGMS) were found to be highly robust and polymorphic. Cross amplification was estimated among nineteen accessions of six sugarcane cultivars, one inter specific hybrids, five related species, four related genera, and three divergent genera by using 27 UGSM primers. Analysis of 388 alleles, amplified by these markers, indicated the high number of observed allele ranged from 2 to 26, with an average of 14.37 alleles detected per locus. High level of polymorphism detected by these markers among sugarcane species, genera and cultivars was 96.3%, while cross-transferability rate was 98.0% within Saccharum complex and 88.27% to cereals. Wide range of genetic diversity (0.33–0.79 with an average of 0.56) assayed with UGSM markers suggested their importance in various genotypic applications in sugarcane.

Artificial induction of flowering in sugarcane under sub-tropical conditions — A successful approach

Four hours extra dark treatment in artificially made photoperiodic chamber induced early flowering in flowering clones i.e. S. 55/99, S. 187/91,S. 301/86, S. 116/2K.S. 73/89 after 12to 14th weeks. Maximum and minimum temperature ranged from 31.0 and 21.4°C respectively. Control plants of respective genotypes flowered generally after one month of the treated plants. Flowered genotypes in photoperiodic chamber were synchronized with the late flowered clones Saccharum spontaneum and thereby the crosses were made effectively. Flowering behaviour of clones under artificial photoperiodic chamber and natural sunlight (control) were studied and it was observed that there were not adverse effect on pollen fertility and floral organs due to dark treatment.

Genetic diversity of sugarcane hybrid cultivars by RAPD markers

Genetic diversity among sugarcane hybrids (Saccharum spp) is pre-requisite for sugarcane improvement through breeding. Twelve decamer oligonucleotide random-amplified polymorphic DNA (RAPD) markers were utilized to investigate the genetic potential among 24 sugarcane cultivars. A total of 120 fragments were originated by 12 RAPD primers. An average number of fragments were obtained as 11.42 fragments per cultivar, which ranged from 4 to 21 fragments. The genetic similarity among 24 sugarcane cultivars ranged from 0.236 to 0.944 with the mean similarity value of 0.508. On the basis of phylogenetic analysis based on dendrogram, the cultivars were clustered into five groups. Two varieties Co 0118 and CoS 07250 were found as highly diverse sugarcane cultivars. Three most popular cultivars viz, Co 0238, Co 1158, and CoS 08272 were clustered a diverse among particular group. These clusters with their diverse genealogy indicated the influence of parental genome contribution to clustering. Diverse varieties developed for east region were grouped in the separate clusters which indicated the influence of adaptation of varieties to particular agro-climatic condition. Hence, these five diverse hybrid cultivars would be used in further breeding program to get the prominent sugarcane clones which may produced higher cane yield and sugar content.

Prospects of Functional Genomics in Sugarcane

Sugarcane is an important commercial crop cultivated for sugar and energy. However, cultivar improvement may be limited due to the narrow genetic base of desired genes. In this sense, functional genomics is a promising tool to assist in the process of developing improved cultivars. Genetic maps linking DNA markers and traits have been developed, but marker-assisted breeding is in its infancy in sugarcane, and genome sequencing has just recently commenced. Substantial resources are available for the sugarcane transcriptome, and both specific and overlapping gene expression patterns for many traits have been established. Gene silencing and over-expression show promise as tests for gene function in sugarcane, and progress has been made in dissecting sucrose accumulation pathways. To broadly assign functions to unknown genes, different fast and multiple parallel approaches are currently used and developed. Such methods allow analysis of the different constituents of the cell that help to deduce gene function, namely the transcripts, proteins and metabolites. Similarly the phenotypic variations of entire mutant collections can now be analyzed in a much faster and more efficient manner than earlier. The different methodologies have developed to form their own fields within the functional genomics technological platform and are termed transcriptomics, proteomics, metabolomics and phenomics. This chapter focuses on recent technological developments and their impact on the field of sugarcane functional genomics. Understanding the unique biological attributes of sugarcane through functional genomics will provide innovative improvement applications that can underpin future, bio-energy and biomaterial industries.