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Matrix stiffness regulates the physiology of the cells and plays an important role in maintaining its homeostasis. It has been reported to regulate cell division, proliferation, migration, extracellular uptake and various other physiological processes. The alteration in matrix stiffness has also been well reported in various disease pathologies. However, in ocular system, Keratoconus (KC) is an ideal model to study the effect of matrix stiffness on endocytosis since the progression of the disease is controlled by increasing the stromal elasticity. Our study using corneal epithelial and retinal pigment epithelial cell lines showed that ocular cells do respond to matrix stiffness by altering their morphology and endocytic uptake of FITC-Dextran 20 kDa. Further, by using KC epithelium as a clinical model, we hypothesize that change in stromal elasticity may also affect the endocytosis of KC epithelium. Our results clearly showed alteration in the expression of actin binding proteins such as Phosphorylated Cofilin, Profilin, Focal adhesion kinase, and Vinculin. Apart from cytoskeletal rearrangement proteins, we also observed endocytic proteins such as Clathrin, Caveolin1 and Rab 11 to be affected by matrix stiffness. Our study thus establishes connecting role between endocytosis and matrix stiffness which could be used to understand the pathophysiology of keratoconus that it is influenced by both mechanical and biochemical factors.

Inactivation of RB is one of the hallmarks of cancer, however gaps remain in our understanding of how RB-loss changes human cells. Here we show that pRB-depletion results in cellular reprogramming, we quantitatively measured how RB-depletion altered the transcriptional, proteomic and metabolic output of non-tumorigenic RPE1 human cells. These profiles identified widespread changes in metabolic and cell stress response factors previously linked to E2F function. In addition, we find a number of additional pathways that are sensitive to RB-depletion that are not E2F-regulated that may represent compensatory mechanisms to support the growth of RB-depleted cells. To determine whether these molecular changes are also present in RB1

Keratoconus (KC) is non-inflammatory, bilateral progressive corneal ectasia, and a disease of established biomechanical instability. The etiology of KC is believed to be multifactorial. Although previous studies gained insight into the understanding of the disease, little is known thus far on global protein phosphorylation changes in keratoconus. We performed phosphoproteome analysis of corneal epithelium from control (N = 5) and KC patients. Tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography (IMAC) were used for the phosphopeptides enrichment and quantitation. Enriched peptides were analyzed on Orbitrap Fusion Tribrid mass spectrometer. This leads to the identification of 2939 unique phosphopeptides derived from 1270 proteins. We observed significant differential phosphorylation of 591 phosphopeptides corresponding to 375 proteins. Our results provide first phosphoproteomic signature of the keratoconus disease and identified dysregulated signaling pathways that can be targeted for therapy in future studies.

Inherited retinal diseases (IRDs) are a group of phenotypically diverse disorders with varied genetic mutations, which result in retinal degeneration leading to visual impairment. When a patient presents to a clinician who is not an IRD expert, establishing a correct diagnosis can be challenging. The patient and the family members are often anxious about further vision loss. They are eager to know the prognosis and chance of further worsening of the vision. It is important for every eye specialist to educate himself/herself about the basics of IRD. It would help to familiarize oneself about how to approach a patient with an IRD. An early and accurate diagnosis can help predict the vision loss and also help the patient plan his/her education and choose appropriate career choices. An updated knowledge about the genetic mutations, mode of inheritance, and possible therapies would empower the eye specialist to help his/her patients. This article gives a broad plan of how to approach a patient with IRD with regards to characterization and diagnosis of the disorder, visual rehabilitation, and possible therapy.

Retinoblastoma is a malignant tumour of the retina which most often occurs in children. Earlier studies on retinoblastoma have concentrated on the identification of key players in the disease and have not provided information on activated/inhibited signalling pathways. The dysregulation of protein phosphorylation in cancer provides clues about the affected signalling cascades in cancer. Phosphoproteomics is an ideal tool for the study of phosphorylation changes in proteins. Hence, global phosphoproteomics of retinoblastoma (RB) was carried out to identify signalling events associated with this cancer. Over 350 proteins showed differential phosphorylation in RB compared to control retina. Our study identified stress response proteins to be hyperphosphorylated in RB which included H2A histone family member X (H2AFX) and sirtuin 1. In particular, Ser140 of H2AFX also known as gamma-H2AX was found to be hyperphosphorylated in retinoblastoma, which indicated the activation of DNA damage response pathways. We also observed the activation of anti-apoptosis in retinoblastoma compared to control. These observations showed the activation of survival pathways in retinoblastoma. The identification of hyperphosphorylated protein kinases including Bromodomain containing 4 (BRD4), Lysine deficient protein kinase 1 (WNK1), and Cyclin-dependent kinase 1 (CDK1) in RB opens new avenues for the treatment of RB. These kinases can be considered as probable therapeutic targets for RB, as small-molecule inhibitors for some of these kinases are already in clinical trials for the treatment other cancers.

Retinoblastoma (RB) is a childhood eye cancer. Currently, chemotherapy, local therapy, and enucleation are the main ways in which these tumors are managed. The present work is the first study that uses constraint-based reconstruction and analysis approaches to identify and explain RB-specific survival strategies, which are RB tumor specific. Importantly, our model-specific secretion profile is also found in RB1-depleted human retinal cells in vitro and suggests that novel biomarkers involved in lipid metabolism may be important. Finally, RB-specific synthetic lethals have been predicted as lipid and nucleoside transport proteins that can aid in novel drug target development.

We present here the life and research of V. S. Rama Das, a distinguished Indian botanist who specialized in photosynthesis. He was the first to purify chloroplasts that were free of mitochondrial contamination. He then studied C4, C3-C4 intermediate and CAM pathways, as well as their taxonomic distribution in tropical climates. His most valuable legacy is that he, as a philosopher, inspired and guided many students to pursue their research career in India. Also see Narayana and Pullaiah (Eminent Indian Botanists: Past and present: Biographies and contributions, pp 394-401, 2010) and Raghavendra and Reddy (Curr Sci 101:798-799, 2011) for further information on Rama Das.

Purpose Retinoblastoma (RB) is a pediatric ocular cancer which is caused due to the aberrations in the RB1 gene. The changes in the membrane proteomics would help in understanding the development of the retinoblastoma and could identify candidates for biomarkers and therapy. Experimental design Quantitative proteomics is performed on the enriched membrane fractions from pooled normal retina (n = 5) and pooled retinoblastoma tissues (n = 5). The proteins are tryptic-digested and tagged with iTRAQ labels. Orbitrap mass spectrometry is used to analyze and quantify the deregulated membrane proteins involved in the RB tumor progression. Immunohistochemistry (IHC) is used to further validate few of the differentially expressed proteins. Results A total of 3122 proteins are identified of which, 663 proteins are found to be deregulated with ≥two fold change in the RB tumor compared to the retina. 282 proteins are upregulated and 381 are downregulated with ≥2 peptide identifications. Bioinformatic analysis revealed that, most of the proteins are involved in the transport, cellular communication, and growth. Overexpression of lamin B1 (LMNB1) and transferrin receptor (TFRC) are observed in RB tumors using IHC. Conclusion and clinical relevance The present study, is the first comprehensive quantitative membrane proteomic atlas of the differentially regulated proteins in RB compared to the retina. LMNB1 and TFRC could be potential biomarkers for this childhood cancer.

In this paper, we report a simple, rapid, and robust method to synthesize surface-enhanced Raman-scattered gold nanoparticles (GNPs) based on green chemistry. Vitis vinifera L. extract was used to synthesize noncytotoxic Raman-active GNPs. These GNPs were characterized by ultraviolet-visible spectroscopy, dynamic light-scattering, Fourier-transform infrared (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Raman spectroscopy. The characteristic surface plasmon-resonance band at ~ 528 nm is indicative of spherical particles, and this was confirmed by TEM. The N–H and C–O stretches in FTIR spectroscopy indicated the presence of protein molecules. The predominant XRD plane at (111) and (200) indicated the crystalline nature and purity of GNPs. GNPs were stable in the buffers used for biological studies, and exhibited no cytotoxicity in noncancerous MIO-M1 (Müller glial) and MDA-MB-453 (breast cancer) cell lines. The GNPs exhibited Raman spectral peaks at 570, 788, and 1,102 cm−1. These new GNPs have potential applications in cancer diagnosis, therapy, and ultrasensitive biomarker detection., Video abstract

The importance of the polo-like kinase 1 (PLK1) gene is increasing substantially both as a biomarker and as a target for highly specific cancer therapy. This is due to its involvement in multiple points of cell progression and carcinogenesis. PLK1 inhibitors' efficacy in treating human cancers has been limited due to the lack of a specific targeting strategy. Here, we describe a method of targeted downregulation of PLK1 in cancer cells and the concomitant rapid detection of surface lipidomic perturbations using desorption electrospray ionization mass spectrometry (DESI MS). The efficient delivery of siRNA targeting PLK1 gene selectively to the cancer cells is achieved by targeting overexpressed cell surface epithelial cell adhesion molecule (EpCAM) by the EpDT3 aptamer. The chimeric aptamer (EpDT3-siPLK1) showed the knockdown of PLK1 gene expression and PLK1 protein levels by quantitative PCR and western blotting, respectively. The abundant surface lipids, phosphatidylcholines (PCs), such as PC(32:1) (m/z 754.6), PC(34:1) (m/z 782.6), and PC(36:2) (m/z 808.6), were highly expressed in MCF-7 and WERI-RB1 cancer cells compared to normal MIO-M1 cells and they were observed using DESI MS. These overexpressed cell surface lipids in the cancer cells were downregulated upon the treatment of EpDT3-siPLK1 chimera indicating a novel role of PLK1 to regulate surface lipid expression in addition to the efficient selective cancer targeting ability. Our results indicate that DESI MS has a potential ability to rapidly monitor aptamer-mediated cancer therapy and accelerate the drug discovery process. Graphical abstract Binding of aptamer chimera to the cells and changes in lipid profile.

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

Circulating serum microRNAs (miRNAs) are promising biomarkers for disease diagnosis. The quantification of the serum miRNA copy number is a challenge due to the presence of low levels in the serum. Here, we report on a direct measurement of the miRNA copy number from human serum using a locked nucleic acid (LNA) modified beacon probe with a single step using fluorescence spectroscopy and microscopy. We had used a minimum volume of 0.1 μL healthy human serum and retinoblastoma serum to show the biological variation of the miRNA copy number.

Various disease conditions, particularly tumours, can be understood easily by studying changes in the lipid profile of cells. While lipid profiles of tissues have been recorded by desorption electrospray ionization mass spectrometric (DESI-MS) imaging, there is paucity in standardized protocols for sample preparation involving cell cultures to generate reliable results. In this study, we report a method for the direct analysis of lipids from cultured cells by incorporating them onto Whatman 42 filter paper as a substrate for reliable DESI-MS analysis.The WERI-RB1 cell line was spotted on commonly used substrates for DESI-MS analysis, such as glass slides, Teflon coated glass slides, thin layer chromatography (TLC) plates, and Whatman 42 filter paper. A comparison of mass spectrometric images with two different lipids was made to understand the behaviour of different surfaces when the same sample was spotted on them. Relative intensities of different lipid peaks in the WERI-RB1 cell line were compared and relative lipid abundances were also compared across two different human retinoblastoma cell lines; WERI-RB1 and Y79.The study demonstrates that good lipid signals can be obtained by DESI-MS when the cells are spotted on Whatman 42 filter paper. Tandem mass spectrometry was performed to identify the lipids as glycerophosphocholines (PC). Better lipid images from assembly of cells were obtained with distinct boundary when they were spotted on Whatman 42 filter paper than other surfaces.We demonstrate the use of a simple substrate for reliable DESI-MS analysis of cultured cells. This method has the potential to understand various interactions of cells with other external agents. The current method would help in the application of DESI-MS for biology in general and medical sciences in particular.

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

Background Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. Methodology/Principal Findings To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using “Composite Organic-Inorganic Nanoparticles” (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. Conclusions/Significance Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.

Statins are a class of drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMGcoA) reductase, a critical enzyme in the mevalonate pathway. Several reports document that statins may prevent different human cancers. However, whether or not statins can prevent cancer is controversial due to discordant results. One possible explanation for these conflicting conclusions is that only some tumors or specific statins may be effective. Here, we demonstrate in an in vivo transgenic model in which atorvastatin reverses and prevents the onset of MYC-induced lymphomagenesis, but fails to reverse or prevent tumorigenesis in the presence of constitutively activated K-Ras (G12D). Using phosphoprotein fluorescence-activated cell sorter (FACS) analysis, atorvastatin treatment was found to result in the inactivation of the Ras and ERK1/2 signaling pathways associated with the dephosphorylation and inactivation of MYC. Correspondingly, tumors with a constitutively activated K-Ras (G12D) did not exhibit dephosphorylation of ERK1/2 and MYC. Atorvastatin's effects on MYC were specific to the inhibition of HMGcoA reductase, as treatment with mevalonate, the product of HMG-CoA reductase activity, abrogated these effects and inhibited the ability of atorvastatin to reverse or suppress tumorigenesis. Also, RNAi directed at HMGcoA reductase was sufficient to abrogate the neoplastic properties of MYC-induced tumors. Thus, atorvastatin, by inhibiting HMGcoA reductase, induces changes in phosphoprotein signaling that in turn prevent MYC-induced lymphomagenesis.