Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture

Purpose: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.Methods: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction. Results: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.Conclusions: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells

PBEF1/NAmPRTase/Visfatin: a potential malignant astrocytoma/glioblastoma serum marker with prognostic value

Malignant astrocytomas comprise anaplastic astrocytoma (AA; grade III) and Glioblastoma (GBM; grade IV). GBM is the most malignant with a median survival of 10-12 months in patients. Using cDNA microarray based expression profiling of different grades of astrocytomas, we identified several fold increased levels of PBEF1 transcripts in GBM samples. Pre-B-cell colony enhancing factor 1 gene (PBEF1) encodes Nicotinamide phosphoribosyltransferase (NAmPRTase), which catalyses the rate limiting step in the salvage pathway of NAD metabolism in mammalian cells. Further validation using real time RT-qPCR on an independent set of tumor samples (n=91) and normal brain samples (n=9), GBM specific higher expression of PBEF1 was confirmed. Immunohistochemical staining for PBEF1 on a subset of the above samples largely reinforced our finding. We carried out ELISA analysis on serum samples of astrocytoma patients to determine whether this protein levels would correlate with the presence of tumor and tumor grade. PBEF1 serum levels were substantially elevated in many of the AA and GBM patients. Statistical analysis of these data indicates that in patients with astrocytoma, serum PBEF1 levels correlate with tumor grade and is highest in GBM. Immunohistochemical analysis of an independent set of 51 retrospective GBM cases with known survival data revealed that PBEF1 expression in the tumor tissue along with its co-expression with p53 was associated with poor survival. Thus, we have identified PBEF1 as a potential malignant astrocytoma serum marker and prognostic indicator among GBMs

Kloniranje, ekspresija i karakterizacija paraflagelarnog gena Rod 2 bičaša Trypanosoma evansi

Paraflagellar rod is the major structural component of the trypanosomatid flagellum and is identified as a complex lattice of filaments which runs parallel to the axoneme throughout most of the flagellar length. The present study was carried out to investigate the existence of the paraflagellar rod (PFR 2) gene in Trypanosoma evansi infecting Indian cattle. Local isolates of T. evansi collected from naturally infected cow were multiplied in Wistar rats. Complementary DNA (cDNA) was synthesized from the RNA of host cell free T. evansi parasites by reverse transcription. The gel purified PCR product (PFR 2 gene of T. evansi) was cloned into the pTZ57R/T vector system. The nucleotide sequence of the PFR 2 gene of the T. evansi S.V.V.U. isolate (Accession No. KT277497) obtained in the present study revealed 100% homology with T. evansi China isolate and 99% homology with T. evansi Izatnagar and Bikaner isolates. The recombinant protein was sub-cloned into pET 32a and expressed in the BL21 (DE3) pLysS expression system. The PFR 2 gene of T. evansi S.V.V.U. isolate was further characterized by determination of its protein profile with SDS-PAGE and western blotting. Indirect ELISA was optimized for detection of the specific antibody titre against the recombinant protein of the PFR 2 gene of T. evansi. In the kinetoplastid species the PFR 2 gene is highly conserved. Therefore the PFR 2 gene was suggested as a vaccine candidate, as well as a diagnostic antigen.Paraflagelarni štapić glavna je strukturna komponenta tripanosomskog biča i dio je kompleksa filamenaza koji teku paralelno s aksonemom duž biča. Istraživanje je provedeno kako bi se ispitalo postojanje paraflagelarnog gena Rod 2 (PFR2) u bičaša Trypanosoma evansi koji invadira goveda u Indiji. Lokalni izolat T. evansi prikupljen od prirodno invadiranih krava umnožen je u Wistar štakora. Komplementarna DNA (cDNA) sintetizirana je iz RNA obrnutom transkripcijom iz stanica neinvadiranih nositelja T. evansi parazita. Pročišćeni PCR produkt (gen PFR2 bičaša T. evansi) kloniran je u vektorski sustav pTZ57R/T. Nukleotidna sekvencija gena PFR2 bičaša T. evansi, izolat S.V.V.U. (pristupni broj KT277497) dobivena u ovom istraživanju pokazala je 100 %-tnu sličnost s izolatom T. evansi China i 99 %-tnu s izolatom T. evansi Izatnagar i Bikaner. Rekombinantni protein ponovno je kloniran u sustavu pET 32a i prikazan u sustavu BL21 (DE3) pLysS. Gen PFR2 bičaša T. evansi, izolat S.V.V.U. dalje je karakteriziran određivanjem proteinskog profila metodama SDS-PAGE i Western blotting. Indirektni test ELISA optimiziran je za dokaz titra specifičnih protutijela za rekombinantni protein gena PFR2 bičaša T. evansi. U kinetoplastida gen PFR2 izrazito je očuvan. Stoga bi se gen PFR2 mogao upotrijebiti za cjepivo te kao dijagnostički antigen

Developing composites of zinc and hydroxyapatite for degradable orthopedic implant applications

In the present work, Zn-HA composites were developed by powder metallurgy route targeted for bone implant applicaitons. Zn-HA powders with varying HA content (1, 2, and 4 wt.%) were ball milled for 1 hr and sintered to produce composite compacts. X-ray diffraction (XRD) studies were done for all the ball milled powders and sintered compacts. No impurities were observed in the ball milled powders. Microstructural observations revealed the formation of lamellar structure in the composites due to the plastic deformation of the Zn powders during ball milling. Grain size measurements revealed the decreased grain size with increase of addition of HA. Furthermore, aspect ratio (length to thickness ratio) of the grains was measured and found that the aspect ratio was also decreased with the increased HA content. Higher microhardness was measured for all the composites compared with pure Zn. However, composite with 1% has shown higher hardness compared with the remaining composites. Form the preliminary observations, it can be concluded that Zn-HA composites can be successfully produced with lamellar morphology by ball milling followed by sintering for biomedical applications with increased hardness

Identification of potential serum biomarkers of glioblastoma: serum osteopontin levels correlate with poor prognosis

Background: The aim of this study is to identify serum biomarkers with classification and prognosis utility for astrocytoma, in particular glioblastoma (GBM). Methods: Our previous glioma microarray database was mined to identify genes that encode secreted or membrane-localized proteins. Subsequent analysis was done using significant analysis of microarrays, followed by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemical validation in tumor tissues, ELISA and Western blot validation in sera, and correlation with survival of GBM patients. Results: Significant analysis of microarrays identified 31 upregulated and 3 downregulated genes specifically in GBMs. RT-qPCR validation on an independent set of samples confirmed the GBM-specific differential expression of several genes, including three upregulated (CALU, CXCL9, and TIMP1) and two downregulated (GPX3 and TIMP3) novel genes. With respect to osteopontin (OPN), we show the GBM-specific upregulation by RT-qPCR and immunohistochemical staining of tumor tissues. Elevated serum OPN levels in GBM patients were also shown by ELISA and Western blot. GBM patients with high serum OPN levels had poorer survival than those with low serum OPN levels (median survival 9 versus 22 months respectively; P = 0.0001). Further, we also show high serum TIMP1 levels in GBM patients compared with grade II/III patients by ELISA and downregulation of serum GPX3 and TIMP3 proteins in GBMs compared with normal control by Western blot analysis. Conclusions: Several novel potential serum biomarkers of GBM are identified and validated. High serum OPN level is found as a poor prognostic indicator in GBMs. Impact: Identified serum biomarkers may have potential utility in astrocytoma classification and GBM prognosis

Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM

Novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis

Purpose: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. Experimental Design: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II - diffuse astrocytoma, grade III - anaplastic astrocytoma, and grade IV - glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). Results: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage-inducible α (GADD45α) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45α and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45α conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. Conclusions: Our study reveals that GADD45α and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45α overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients

Gene Expression Signature of DMBA-Induced Hamster Buccal Pouch Carcinomas: Modulation by Chlorophyllin and Ellagic Acid

Chlorophyllin (CHL), a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA), a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs) that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy