Identification of dysregulation of sphingolipids in retinoblastoma using liquid chromatography-mass spectrometry.

Retinoblastoma (RB) is a rare ocular cancer seen in children that counts for approximately 3% of all childhood cancers. It is found that mutation in RB1, a tumour Suppressor Gene on chromosome 13 as the cause of malignancy. Retinoblastoma protein is the target for ceramide to cause apoptosis. We studied lipidomics of two RB cell lines, one aggressive cell line (NCC-RbC-51) derived from a metastatic site and one non aggressive cell line (WERI-Rb1) in comparison with a control cell line (MIO-M1). Lipid profiles of all the cell lines were studied using high resolution mass spectrometer coupled to high performance liquid chromatography. Data acquired from all the three cell lines in positive mode were analyzed to identify differentially expressed metabolites. Several phospholipids and lysophospholipids were found to be dysregulated. We observed upregulation of hexosyl ceramides, and down regulation of dihydroceramides and higher order sphingoglycolipids hinting at a hindered sphingolipid biosynthesis. The results obtained from liquid chromatography-mass spectrometry are validated by using qPCR and it was observed that genes involved in ceramide biosynthesis pathway are getting down regulated.

Removal and identification of a subconjunctival parasite by light and scanning electron microscopy.

Background: Ophthalmic dirofilariasis is an uncommon zoonotic parasitic infection caused by species of Dirofilaria, a dog tapeworm that is transmitted to human by mosquitoes. Man is a dead-end host for the parasite. Ophthalmic involvement is rare and includes periorbital, subconjunctival, subtenon, and intra-ocular involvement. We report the removal of a subconjunctival worm and identification by light microscopy (LM) and scanning electron microscopy (SEM). Purpose: : A 62-year-old female presented with complaints of redness, discharge, and foreign body sensation with difficulty in opening eyes in the left eye for the last 3 days. The patient is a non-vegetarian. On examination, her best corrected visual acuity in both eyes was 20/20. On slit lamp examination, there was a long, thin, round, coiled white subconjunctival live worm in the left eye superiorly. The rest of anterior segment evaluation, intra-ocular pressure, and fundus was normal in both eyes. The parasite was removed under local anesthesia from subconjunctival space [Video]. External surface morphology under LM revealed fine transverse cuticular striations with tapered cephalic and caudal ends. Uterus was long and coiled with indistinguishable masses inside. The finding was also confirmed by SEM. Synopsis: A subconjuctival parasite was removed and identified as Dirofilaria repens by characteristic LM and SEM findings. Highlight: Dirofilaria species may lodge in many tissues of human bodies including eye and adnexa. Dirofilaria is a natural parasite of carnivorous animals, mostly dogs, cats, and foxes.[1] The most common mode of transmission to human is usually by bite of mosquitoes like Culex and Aedes, which are considered as vectors, and it is often thought that parasitemia is because of accidental conduction.[1] Simple surgical removal of the worm is curative. After removal, the worm should be visualized directly under LM. All the internal structures of the transparent worm could be seen and compared with those under SEM.

Synthesis and Characterization of a Novel Peptide Targeting Human Tenon Fibroblast Cells To Modulate Fibrosis: An Integrated Empirical Approach.

Fibrosis is the primary factor influencing the prognosis of glaucoma post-trabeculectomy surgery, an eye condition characterized by increased intraocular pressure (IOP). Despite advancements in surgical procedures and aftercare, it continues to be a serious impediment. During the clinical intervention of scarring, fibrosis is managed by using topical application of combined antifibrotic drugs (mitomycin C). But still, scarring remains a key problem due to minimal drug penetration and nonbioavailability. In this study, we synthesized a cell-specific peptide for modulating scarring in human tenon fibroblasts undergoing epithelial-mesenchymal transition (EMT). The peptide was also conjugated with mitomycin C in order to investigate the effect of the drug conjugation on human tenon fibroblasts from the nanofiber composite system and to evaluate the fibrosis process. Peptide VRF2019 was identified using a subtractive proteomics approach, including solubility, cell penetration, and amphipathic properties. The peptide structure was determined using circular dichroism spectroscopy. The peptide and drug was conjugated using N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) chemistry, and the conjugation efficiency was evaluated using high-pressure liquid chromatography. The conjugated product and polycaprolactone (PCL) were electrospun to form a composite nanofiber, which was tested for cytotoxicity and drug release on human tenon fibroblast cells. The modeled VRF2019 peptide structure formed an α-helical structure with all residues spanning the allowed regions of the Ramachandran plot. Subsequent molecular dynamics simulations also demonstrated its membrane penetration potential. The peptide uptake was also studied in human tenon fibroblast cells. High-pressure liquid chromatography (HPLC) and mass spectrometry measurements confirmed peptide-drug conjugation and stability. Furthermore, scanning electron microscopy (SEM) investigation revealed the structure and size of the PCL composite nanofiber. We infer from early research that the PCL composite nanofiber matrix can greatly increase drug delivery and bioavailability.

Understanding the cellular response of human tenon fibroblast on polycaprolactone-Aloe vera blend fiber.

The signalling response is determined by the cell's reaction to different biochemical and biophysical inputs such as stiffness, topological, and structural alignment. The surface patterns at the nano-scale can be an influential factor in cell signalling behaviour. It is important to understand the cellular response to the biophysical cues for biomedical applications. Biomaterials have an important role in regenerative tissue engineering. In this study, we have fabricated electrospun polycaprolactone (PCL) and PCL-Aloe vera (PCL-AV) nanofibrous matrix and studied its effect on the human tenon fibroblast (HTF) cellular and morphological changes. The electrospun fibers were characterized using Scanning Electron Microscope (SEM), Fourier Transform Infrared spectroscopy (FTIR), Atomic Force Microscopy (AFM) and Brunaur, Emette and Teller (BET) analysis for their morphology, composition, topography, surface area and porosity. The results revealed fiber size, roughness and porosity has been altered by addition of AV. The HTF cell viability, proliferation and expression of focal adhesion proteins, such as FAK, Ezrin, Vasp and Cofilin on the PCL-AV fiber matrix were examined. The results showed a change in cellular morphology and a significant change in the cofilin phosphorylation on PCL-AV nanofiber. The influence of Aloe vera composition on the nano-dimension of the PCL has made a significant impact on the cellular morphology at both gene and protein levels. This observation suggests that AV composition in the nanofiber can significantly influence the HTF cellular adhesions.

Graphitic Carbon Nitride Causes Widespread Global Molecular Changes in Epithelial and Fibroblast Cells.

For scaffold and imaging applications, nanomaterials such as graphene and its derivatives have been widely used. Graphitic carbon nitride (g-C3N4) is among one such derivative of graphenes, which draws strong consideration due to its physicochemical properties and photocatalytic activity. To use g-C3N4 for biological applications, such as molecular imaging or drug delivery, it must interact with the epithelium, cross the epithelial barrier, and then come in contact with the extracellular matrix of the fibroblast cells. Thus, it becomes essential to understand its molecular mechanism of action. Hence, in this study, to understand the molecular reprogramming associated with g-C3N4, global gene expression using DNA microarrays and proteomics using tandem mass tag (TMT) labeling and mass spectrometry were performed in epithelial and fibroblast cells, respectively. Our results showed that g-C3N4 can cross the epithelial barrier by regulating the adherens junction proteins. Further, using g-C3N4-PDMS scaffolds as a mimic of the extracellular matrix for fibroblast cells, the common signaling pathways were identified between the epithelium and fibroblast cells. These pathways include Wnt signaling, integrin signaling, TGF-ß signaling, cadherin signaling, oxidative stress response, ubiquitin proteasome pathway, and EGF receptor signaling pathways. These altered signature pathways identified could play a prominent role in g-C3N4-mediated cellular interactions in both epithelial and fibroblast cells. Additionally, ß catenin, EGFR, and MAP2K2 protein-protein interaction networks could play a prominent role in fibroblast cell proliferation. The findings could further our knowledge on g-C3N4-mediated alterations in cellular molecular signatures, enabling the potential use of these materials for biological applications such as molecular imaging and drug delivery.

Proteomics-based approach for differentiation of age-related macular degeneration sub-types.

Purpose: Age-related macular degeneration (AMD) is one of the leading causes of irreversible central vision loss in the elderly population. The current study aims to find non-invasive prognostic biomarkers in the urine specimens of the AMD patients. Methods: Peripheral blood and urine samples were collected from 23 controls and 61 AMD patients. Genomic DNA was extracted from the buffy coat of peripheral blood. Allele specific PCR was used to assay SNPs in complement factor H (CFH), complement component 3 (C3). Comparative proteomic analysis of urine samples from early AMD, choroidal neovascular membrane (CNVM), geographic atrophy (GA), and healthy controls was performed using isobaric labelling followed by mass spectrometry. Validation was performed using enzyme-linked immunosorbent assay (ELISA). Results: Comparative proteomic analysis of urine samples identified 751 proteins, of which 383 proteins were found to be differentially expressed in various groups of AMD patients. Gene ontology classification of differentially expressed proteins revealed the majority of them were involved in catalytic functions and binding activities. Pathway analysis showed cell adhesion molecule pathways (CAMs), Complement and coagulation cascades, to be significantly deregulated in AMD. Upon validation by ELISA, SERPINA-1 (Alpha1 antitrypsin), TIMP-1 (Tissue inhibitor of matrix metaloprotease-1), APOA-1 (Apolipoprotein A-1) were significantly over-expressed in AMD (n = 61) patients compared to controls (n = 23). A logistic model of APOA-1 in combination with CFH and C3 polymorphisms predicted the risk of developing AMD with 82% accuracy. Conclusion: This study gives us a preliminary data on non-invasive predictive biomarkers for AMD, which can be further validated in a large cohort and translated for diagnostic use.

Correction to: EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex.

An amendment to this paper has been published and can be accessed via the original article.

Understanding the Uptake Mechanism and Interaction Potential of the Designed Peptide and Preparation of Composite Fiber Matrix for Fungal Keratitis.

The conventional use of antibiotics for the treatment of infectious keratitis currently faces two major challenges: poor drug penetration and the emergence of antibiotic resistance in microbial strains. Cell-penetrating peptides (CPPs) with antimicrobial properties have the potential to address these challenges. However, their mode of action, mechanism of uptake, and interaction potential have not been explored in detail. In this study, we probed the mechanism of uptake and interaction potential of our previously designed peptides (VRF005 and VRF007). Our results showed that VRF005 undergoes direct translocation and induces a rough membrane surface, whereas VRF007 undergoes clathrin-mediated endocytic uptake. The gel shift assay showed that VRF005 is bound to genomic DNA, whereas VRF007 is bound to chitin and ß-d-glucan. Gene expression studies revealed the effect of peptide VRF005 on Candida albicans transcription. Molecular docking and simulations showed that VRF005 forms noncovalent interactions (such as H-bonding and water bridges) with natamycin. It exhibited synergistic antifungal activity in the colony-forming assay. VRF005, functionalized in the polycaprolactone fiber matrix, showed sustained delivery and antifungal activity.

Role of Topical Ethanol in the Treatment of Pythium insidiosum Keratitis-A Proof of Concept.

PURPOSE: To study the efficacy and safety profile of topical absolute ethanol in the treatment of Pythium insidiosum keratitis. METHOD: Microbiological, clinical, and histopathological assessments were performed to study the effects of absolute ethanol on P. insidiosum keratitis. In addition, infrared spectroscopy was performed to assess the corneal penetration of ethanol. RESULTS: Microbiological tests revealed that ethanol inhibited the growth of P. insidiosum at concentrations even as low as 20% as compared to Candida albicans and Aspergillus flavus, where minimal growth was noted. However, at 40%, 60%, 80%, and 99.9% of ethanol, complete inhibition of growth was noted for all organisms. Histopathology of the absolute ethanol-treated cadaveric cornea showed the compaction of collagen and no stromal necrosis. Infrared spectroscopy revealed secondary structural changes in collagen in the ethanol-treated cadaveric corneas as compared to controls. Clinically, 1 case with a recurrence of P. insidiosum after therapeutic penetrating keratoplasty resolved with the topical application of absolute ethanol, and the other case, where corneal scraping had grown Pythium within 24 hours, failed to grow the organism from the corneal button which was treated with absolute alcohol preoperatively. After therapeutic penetrating keratoplasty, there was no recurrence, and the graft epithelized well. CONCLUSIONS: Ethanol can be considered an option for treating P. insidiosum keratitis; however, the exact dose and strength of ethanol which will be most effective needs further work.

Monitoring of changes in lipid profiles during PLK1 knockdown in cancer cells using DESI MS.

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.

Ethanol Extracts of Selected Cyathea Species Decreased Cell Viability and Inhibited Growth in MCF 7 Cell Line Cultures.

Cancer is the cause of more than 6 million deaths worldwide every year. For centuries, medicinal plants have been used in the treatment of cancer. Chemotherapy, radiotherapy, surgery and acupuncture point stimulation are also used to treat cancer. The present study was intended to reveal the cytotoxic and anticancer potential of selected Cyathea species and to highlight their importance in the pharmaceutical industry for the development of cost-effective drugs. Cytotoxic studies using brine shrimp lethality bioassays and MCF 7 cell line cultures were carried out. Compared to petroleum ether, chloroform and acetone extracts, the ethanol extracts of selected Cyathea species were found to be more effective against brine shrimps. The ethanol extracts were further subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assays. A decrease in cell viability and an increase in growth inhibition were observed for the MCF 7 cell line. The maximum percentage of cell inhibition was observed in Cyathea crinit, followed by Cyathea nilgirensis and Cyathea gigantea. The results of the present study suggest that Cyathea species are an effective source of cytotoxic compounds.

EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation. RESULTS: Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was -30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells. CONCLUSIONS: The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo.

Resonance Energy Transfer between protein and rhamnolipid capped ZnS quantum dots: application in in-gel staining of proteins.

The interaction of proteins with quantum dots is an interesting field of research. These interactions occur at the nanoscale. We have probed the interaction of Bovine Serum Albumin (BSA) and Candida rugosa lipase (CRL) with rhamnolipid capped ZnS (RhlZnSQDs) using absorption and fluorescence spectroscopy. Optical studies on mixtures of RhlZnSQDs and proteins resulted in Förster's Resonance Energy Transfer (FRET) from proteins to QDs. This phenomenon has been exploited to detect proteins in agarose gel electrophoresis. The activity of the CRL was unaffected on the addition of QDs as revealed by zymography.