Role of NGS and SNP genotyping methods in sugarcane improvement programs.

Sugarcane (Saccharum spp.) is one of the most economically significant crops because of its high sucrose content and it is a promising biomass feedstock for biofuel production. Sugarcane genome sequencing and analysis is a difficult task due to its heterozygosity and polyploidy. Long sequence read technologies, PacBio Single-Molecule Real-Time (SMRT) sequencing, the Illumina TruSeq, and the Oxford Nanopore sequencing could solve the problem of genome assembly. On the applications side, next generation sequencing (NGS) technologies played a major role in the discovery of single nucleotide polymorphism (SNP) and the development of low to high throughput genotyping platforms. The two mainstream high throughput genotyping platforms are the SNP microarray and genotyping by sequencing (GBS). This paper reviews the NGS in sugarcane genomics, genotyping methodologies, and the choice of these methods. Array-based SNP genotyping is robust, provides consistent SNPs, and relatively easier downstream data analysis. The GBS method identifies large scale SNPs across the germplasm. A combination of targeted GBS and array-based genotyping methods should be used to increase the accuracy of genomic selection and marker-assisted breeding.

Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells.

BACKGROUND: Several nanoconjugates have been designed to deliver nucleic acids such as small interfering RNA (siRNA) and DNA to cells to study silencing and expression efficacies. In the present study, we prepared novel epithelial cell adhesion molecule (EpCAM) monoclonal antibody conjugated polyethyleneimine (PEI) capped gold nanoparticles (AuNPs) loaded with EpCAM-specific siRNA molecules to knock-down the EpCAM gene in retinoblastoma (RB) cells. We chose EpCAM as a target moiety to deliver siRNA because this molecule is highly expressed in various epithelial cancers and is an ideal target as it is highly expressed in the apical surface of tumor cells while showing basolateral expression in normal cells. METHODS: The EpCAM antibody was conjugated to AuNP-PEI loaded with siRNA molecules to specifically deliver siRNA to EpCAM-expressing RB cells. Conjugation efficiencies were confirmed with ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and agarose and SDS-polyacrylamide gel electrophoresis. The size and zeta potential were measured using a Zeta sizer analyzer. Nanoparticle internalization and uptake were studied using fluorescent microscopy and flow cytometry. Gene silencing efficacy was monitored with western blot analysis and real-time quantitative PCR. RESULTS: Optimal size and neutral zeta potential properties of the AuNP-PEI- EpCAM antibody (EpAb) antibody were achieved for the transfection studies. The AuNP-PEI nanoparticles did not show any cytotoxicity to the cells, which means these nanomaterials are suitable for intracellular delivery of siRNA for therapeutic interventions. With EpCAM antibody conjugation, PEI-capped AuNPs loaded with EpCAM siRNA were significantly internalized in the Y79 cells as observed with fluorescence microscopy and flow cytometry and induced a highly significant reduction in the cell viability of the Y79 cells. Through increased binding of EpCAM antibody-conjugated AuNP-PEI nanoparticles, significant downregulation of EpCAM gene was observed in the Y79 cells when compared to the cells treated with the antibody-unconjugated AuNP-PEI nanoparticles. CONCLUSIONS: Thus, a novel antibody conjugated nanocarrier designed to deliver siRNA holds promise as an effective gene therapy strategy for retinoblastoma in the near future. In addition to serving as an siRNA delivery tool for therapy, gold nanoparticles can also serve as imaging modality in diagnosis.

EpCAM is a putative stem marker in retinoblastoma and an effective target for T-cell-mediated immunotherapy.

PURPOSE: The molecular markers cluster of differentiation (CD)24, CD44, adenosine tri-phosphate (ATP) binding cassette protein G2 (ABCG2), and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of cancer stem cells. In this study we characterized the EpCAM+ retinoblastoma (RB) cells for their cancer stem-like properties in vitro. Additionally, we targeted RB tumor cells via redirecting T cells using bispecific EpCAM×CD3 antibody. METHODS: Flow cytometry was used to study the co-expression of EpCAM with putative cancer stem cell markers, such as CD44, CD24, and ABCG2, in RB primary tumors. In vitro methyl thiazol tetrazolium (MTT) assay, invasion assay, and neurosphere formation assay were performed to characterize EpCAM+ cells for their cancer stem/progenitor cell-like properties. We assessed the in vitro efficacy of bispecific EpCAM×CD3 antibody on RB tumor cell proliferation and validated the results by evaluating effector cytokine production in the culture medium with the ELISA method. RESULTS: EpCAM was co-expressed with all cancer stem cell markers (CD44, CD24, and ABCG2) in primary RB tumors. EpCAM+ cells showed significantly higher proliferative invasive potential and neurosphere formation in vitro compared to EpCAM⁻ Y79 cells. EpCAM+ cells showed higher ß-catenin expression compared to EpCAM- cells. EpCAM×CD3 significantly retarded proliferation of RB primary tumor cells. EpCAM×CD3 effectively induced the secretion of effector cytokines, such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-2, and transforming growth factor (TGF)-ß1, and also perforin levels by pre-activated lymphocytes. CONCLUSIONS: EpCAM might be a novel cancer stem cell marker in RB. EpCAM×CD3 antibody redirecting T cells to attack RB tumor cells may prove effective in RB management. Further preclinical studies are needed to confirm the initial findings of our study.

Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma.

PURPOSE: Previously we showed that epithelial cell adhesion molecule (Ep-CAM), a cell surface molecule, was highly expressed in primary retinoblastoma tumors. In the present study, we studied the genes regulated by Ep-CAM in a retinoblastoma Y79 cell line in vitro using a combination of short interference RNA and microarray technology. METHODS: Flow cytometry, quantitative reverse transcriptase PCR (Q-RT-PCR), and immunohistochemistry were performed to confirm the Ep-CAM re-expression in the Y79 cells treated with 5'-azacytidine (AZC). Ep-CAM expression in AZC-treated Y79 cells was silenced using synthetic anti-Ep-CAM short interference RNA, and whole genome microarray was performed to determine the gene expression changes post Ep-CAM knockdown. Ep-CAM inhibition was confirmed by Q-RT-PCR, western blotting, and immunofluorescence. RESULTS: Ep-CAM expression was significantly restored in Y79 cells on day 5 of AZC treatment. Ep-CAM inhibition significantly affected Y79 cell proliferation. We identified 465 upregulated genes (>or=1.0 fold) and 205 downregulated genes (

Detection of human papillomavirus DNA in retinoblastoma samples: a preliminary study.

Recent studies have shown the presence of human papillomavirus (HPV) genome in retinoblastoma (RB) tumor samples. There is no information on the HPV status in the RB tumors of Indian patients. We studied the presence of HPV genome in RB tumor samples from patients with unilateral tumor. Forty-four fresh RB tumor samples and 30 non-neoplastic donor retinas were analyzed for the presence of HPV 16 and 18 genome by nested and seminested polymerase chain reaction. Tumor tissue sections were also used to assess the expression of the retinoblastoma (Rb) protein. All 30 control tissues were negative for HPV genome. Among the 44 tumor samples, there were 23 tumors with invasion of optic nerve/choroid and 21 tumors with no invasion. HPV DNA was present in 21/44 (47%) RB tumors. Among 21 unilateral RB tumors that were positive for HPV DNA, HPV 16 was detected in 12/21 (57%) tumors. However, HPV 18 was negative in all the tumors. Rb protein was absent in 16 (71%) of 21 tumors that had HPV DNA. However, Rb was also absent in 20 (86%) of 23 tumors that were HPV negative. Children younger than 18 months old were significantly associated with the presence of HPV DNA compared with children above 24 months old (P<0.014). Our study shows the presence of HPV and HPV 16 in a subset of RB tumor samples. However, further studies are in progress to know the role played by HPV in RB.

Expression of high mobility group A2 protein in retinoblastoma and its association with clinicopathologic features.

Retinoblastoma (RB) is the commonest primary intraocular tumor in children. Overexpression of the high mobility group (HMG) A2 protein has been observed in a variety of malignant tumors and often correlates with poor prognosis. We studied the expression of HMGA2 in primary tumor samples and correlated with clinicopathologic features such as invasion, differentiation, and laterality of the tumors. Among 64 tumors, there were 29 tumors with invasion of the optic nerve, choroid, and/or orbit and 35 tumors without invasion. HMGA2 immunoreactivity was evaluated on archival paraffin sections and the results confirmed by Western blotting on 12 fresh tumor samples. Among 29 tumors with invasion, HMGA2 was strongly positive (++) in 10 tumors, moderately positive (+) in 11 tumors. Among 35 tumors without invasion, HMGA2 was strongly positive (++) in 6 tumors, moderately positive (+) in 6 tumors. Tumors with invasion showed significantly higher expression of HMGA2 compared with tumors without invasion (P<0.01). Non-neoplastic retina was negative for HMGA2. There was no correlation between HMGA2 expression and differentiation/laterality. Western blotting revealed that 7 tumors were strongly positive, 2 were moderately positive, and 1 was faintly positive for HMGA2. Our study has demonstrated the HMGA2 expression in a large cohort of primary retinoblastoma tumors and its correlation with invasiveness.

Expression of p63 and p73 in retinoblastoma: a clinicopathological correlation study.

The aim of the study was to explore the expression profile of p63, p73 and their delta isoforms in the retinoblastoma tumor samples and to correlate with clinicopathological parameters. Immunohistochemistry was performed for p63, delta p63, p73 and delta p73 on the archival paraffin sections of retinoblastoma and correlated with clinicopathological features. Western blotting was performed to confirm immunoreactivity results. p63 immunoreactivity was observed in 59% (29/49) of the RB specimens. p63 was expressed in 60% (20/33) low risk tumors and in 56% (9/16) of high risk tumors. p73 was expressed in 77% (38/49) RB specimens. Among the 33 low risk tumors, p73 was expressed in 69% (23/33) tumors and among the 26 high risk tumors, p73 was expressed in 93% (15/16) tumors. High risk tumors showed significantly increased expression of p73 compared to tumors with low risk (P<0.05). This is the first correlation between p63/p73 expression and histopathology in human RB tumors. Our study showed increased expression of p73 in high risk tumors (P<0.05) compared to low risk tumors. Further functional studies are required to explore the role of p63, p73 and their respective isoforms in retinoblastoma.

SRPK1: a cisplatin sensitive protein expressed in retinoblastoma.

Chemotherapy is an essential modality in the treatment of retinoblastoma (RB). Mammalian serine/arginine-rich protein-specific kinase 1 (SRPK1) is a cisplatin-sensitivity-related protein and its downregulation is known to be associated with decreased response to cisplatin and carboplatin. We investigated the expression of SRPK1 in 63 archival RB and correlated its expression with pathologic staging and exposure to chemotherapy. The majority of the RB (62/63) were advanced stage (Groups D and E) with intermediate to high risk of treatment failure according to the new international classification for intraocular RB and SRPK1 was reduced in 32/62 (51%) tumors. SRPK1 protein expression was reduced in (100%) 8/8 RB that had recurred in the orbit or had metastasized. SRPK1 protein expression is reduced in RB with advanced stage of presentation and this may add to drug resistance mechanisms in RB.

Retinoblastoma: expression of HLA-G.

PURPOSE: Human leukocyte antigen (HLA) mediates interactions of tumor cells with cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Retinoblastoma (RB) is the most common intraocular malignant tumor in childhood and is characterized by direct spread to the optic nerve and orbit as well as hematogenous and lymphatic spread. Earlier, we observed that invasive RB showed reduced HLA, which could contribute to its escape from the immune system. Little is known about the role of the nonclassical HLA molecule, HLA-G, in RB and its role in tumor escape mechanisms in RB. METHODS: Forty archival paraffin-embedded RB tumors were analyzed for the expression of HLA-G by immunohistochemistry using a monoclonal antibody; fresh tumor samples were also subjected to Western blot analysis. There were 22 tumors with no invasion and 18 with invasion of the choroid/optic nerve. Immunoanalysis was performed based on the International Histocompatibility Working Group Project Description. RESULTS: HLA-G was negative in the non-neoplastic retina, reduced in 22/22 tumors with no invasion, and positive in 15/18 with invasion. The immunohistochemistry results were confirmed by Western blot analysis. The difference in expression between the two groups was significant ( p < 0.001). There was no correlation of HLA-G expression with differentiation of the tumors. CONCLUSION: Increased expression of HLA-G was observed in invasive RB. This preliminary observation deserves further investigation and may shed more light on the immune escape mechanisms of this tumor and thus enable novel therapeutic strategies.

Reversal of stathmin-mediated microtubule destabilization sensitizes retinoblastoma cells to a low dose of antimicrotubule agents: a novel synergistic therapeutic intervention.

PURPOSE: To explore the possibility of stathmin as an effective therapeutic target and to evaluate the synergistic combination of stathmin RNAi and the antimicrotubule agents paclitaxel and vincristine to retinoblastoma Y79 cells. METHODS: RNAi-mediated specific inhibition of stathmin expression in Y79 cells was shown by real-time quantitative RT-PCR (RT-Q-PCR), its effect on cell proliferation by MTT assay, cell invasion using matrigel, microtubule polymerization by immunohistochemistry, apoptosis, cell cycle analysis by flow cytometry analysis, and the changes in FOXM1 protein expression were studied by Western blot. The effect of combination treatment of stathmin siRNA and paclitaxel/vincristine was studied by assessing cell viability and apoptosis. RESULTS: Short interfering RNA-mediated transient stathmin downregulation resulted in a marked inhibition of retinoblastoma cell proliferation and cell invasion in vitro. Stathmin inhibition promoted Y79 cells to G2/M phase, and ultimately there were increased apoptotic events as evidenced by higher caspase-3 activation and cleaved poly(ADP-ribose) polymerase expression. Cells transfected with stathmin siRNA showed long and bundled microtubule polymers and sensitized the Y79 cells significantly to paclitaxel and vincristine. CONCLUSIONS: Stathmin may be a pivotal determinant for retinoblastoma tumorigenesis and chemosensitivity. Strategies to inhibit stathmin will help to enhance the cytotoxic effect of paclitaxel while reducing toxicity (or side effects) to normal cells caused by high doses.

Molecular pathology of retinoblastoma.

Retinoblastoma (RB) is an embryonic neoplasm of retinal origin. For many years, scientists have sought the fundamental origins of tumorigenesis, with the ultimate hope of discovering a cure. Indeed, these efforts have led to a significant understanding that multiple molecular and genetic aberrations, such as uncontrolled proliferation and the inhibition of apoptosis that contribute to the canonical characteristics of tumor biology. Despite these advances, a thorough understanding, such as the precise cells, which are the targets of neoplastic transformation, especially in solid tumors, is currently lacking. The focus of this review is to emphasize the molecular defects involved in the RB tumor progression and mechanisms associated with inhibition of tumor cell apoptotic processes. This review also discusses the importance of target molecules characterization and their potential therapeutic or prognostic use in RB disease.

Expression of matrix metalloproteinases and their inhibitors in retinoblastoma.

Tumor invasion is the critical step that could lead to metastasis in retinoblastoma (RB), a common childhood cancer. Matrix metalloproteinases (MMPs) degrade extracellular matrix, which is a crucial step involved in various stages of tumor progression, including tumor angiogenesis, tumor growth, and also local invasion and subsequent distant metastasis. We investigated the role of extracellular MMP inducer (EMMPRIN), MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMPs): TIMP-1, TIMP-2 in RB and correlated clinicopathologically. Among 60 tumors, EMMPRIN was expressed in 40 (64%), MMP-2 in 41 (66%), MMP-9 in 38 (61%), TIMP-1 in 35 (56%), and TIMP-2 in 33 (53%) tumors. EMMPRIN was positive (3+) in 13 (39%) out of 33 tumors with invasion and was positive (3+) in only 1 (3%) out of 29 tumors without invasion. MMP-2 (P<0.0001) and MMP-9 (P<0.0001) were significantly positive (3+) in 7 (21%) and 12 (36%) out of 33 tumors with invasion, whereas positive (3+) in 3 (10%) and faint (1+) in 10 (34%) tumors, respectively, out of 29 tumors without invasion. TIMP-1 (P<0.0001) and TIMP-2 (P=0.04) were significantly positive (3+) in 7 (21%) and 10 (30%), respectively out of 33 tumors with invasion, whereas positive (3+) in only 1 (3%) tumor each out of 29 tumors without invasion. Immunoblotting of tumors confirmed the presence of EMMPRIN, MMPs, and TIMPs. In conclusion, both MMPs and TIMPs may be involved RB invasion and EMMPRIN could play a role in up-regulation of MMP-2 in invasive RB.

Expressions of Rac1, Tiam1 and Cdc42 in retinoblastoma.

The Rho GTPases are the molecular regulators of the cell motility processes and are involved in cell cycle progression and gene transcription. We studied the expression of Rho-like GTPases molecules, particularly Rac, Tiam1 and cdc42, in retinoblastoma and correlated these with clinicopathological parameters of the tumors. Sixty-seven tumors were included which were divided in to two groups; group A: tumors with optic nerve/choroidal/orbital invasion (n=35) and group B: tumors with no invasion (n=32). Immunohistochemistry was done on paraffin sections for all the proteins and were confirmed by Western blot on fresh tumor samples. In group A tumors, Rac was positive in 10/35 (28%), cdc42 was positive in 12/35 (34%) and Tiam1 was positive in 30/35 (85%) tumors. In group 2 tumors, Rac was positive in 5/32 (15%), cdc42 was positive in 4/32 (12%) and Tiam1 was positive in 30/32 (93%) tumors. Two groups (both invasive and non-invasive tumors) showed decreased expression of Rac1 and cdc42 whereas Tiam1 was significantly expressed in invasive tumors compared to non-invasive tumors (P<0.0001). We observed a 70K cleavage product of Tiam1 along with an 110K product by blotting in RB samples. Caspase-3 was also demonstrated in RB samples, which showed Tiam1 cleavage products. This is the first study that showed the expression patterns of Rac, cdc42 and Tiam1 in retinoblastoma tumors. Thus, further studies are required to prove the involvement of caspase-3 in the cleavage of Tiam1 in vitro in RB cells and to trace out alternative pathways involved in tumor progression.

Expression of Fas ligand in retinoblastoma.

BACKGROUND: The importance of the Fas-Fas ligand (FasL) mechanism for the immune evasion by tumors provided a strong rationale for the examination of FasL expression in retinoblastoma. In an earlier publication, the authors reported that invasive retinoblastomas decreased Fas expression. Because to the authors' knowledge there is not much information regarding the effect of FasL expression on retinoblastoma, the authors studied the expression of FasL in retinoblastoma and correlated it with invasiveness. METHODS: Thirty-six archival retinoblastoma specimens were divided into 2 groups. Group A (n = 17) was comprised of specimens from tumors with no invasion and Group B (n = 19) was comprised of specimens from tumors with invasion of the choroid (focal, diffuse), optic nerve (laminar, postlaminar, surgical end), and orbit. Sections were immunostained with a monoclonal antibody to FasL and the immunoreactivity was assessed. RESULTS: In Group A, FasL was negative in 100% (17 of 17) of the tumor specimens. In Group B, FasL was expressed in 79% (15 of 19) of the tumor specimens (positive in 9 tumors and heterogeneous in 6 tumors). The difference in FasL expression between the two groups was significant (P < 0.001) CONCLUSIONS: Increased expression of FasL was observed in specimens taken from patients with aggressive tumors. Thus, Loss of Fas and gain of aberrant FasL expression were common features of malignant transformation. The data suggested that the Fas/FasL pathway is potentially immunosuppressive and may be involved in the escape of retinoblastoma cells from immune destruction.