Retinoblastoma: A review of the molecular basis of tumor development and its clinical correlation in shaping future targeted treatment strategies.

Retinoblastoma is a retinal cancer that affects children and is the most prevalent intraocular tumor worldwide. Despite tremendous breakthroughs in our understanding of the fundamental mechanisms that regulate progression of retinoblastoma, the development of targeted therapeutics for retinoblastoma has lagged. Our review highlights the current developments in the genetic, epigenetic, transcriptomic, and proteomic landscapes of retinoblastoma. We also discuss their clinical relevance and potential implications for future therapeutic development, with the aim to create a frontline multimodal therapy for retinoblastoma.

Secreted Ly-6/uPAR-related protein-1 (SLURP1) is a pro-differentiation factor that stalls G1-S transition during corneal epithelial cell cycle progression.

PURPOSE: Previously we demonstrated that the secreted Ly-6/uPAR related protein-1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an anti-inflammatory and anti-angiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal phenotype for the first time. METHODS: We compared the 10-week-old wild type (WT) and Slurp1X-/- mouse corneal (i) histology by hematoxylin-eosin and periodic acid-Schiff's reagent staining, (ii) cell proliferation by immunostaining for Ki67, (iii) cell adhesion molecules by immunostaining for desmosomal and tight junction proteins, (iv) barrier function by fluorescein staining and (v) wound-healing by epithelial debridement. Effect of SLURP1 on cell cycle was quantified in human corneal limbal epithelial (HCLE) cells engineered to express SLURP1 (HCLE-SLURP1). RESULTS: WT and Slurp1X-/- corneal histology was largely comparable, other than a few loosely attached superficial cells in Slurp1X-/- corneas. Compared with the WT, Slurp1X-/- corneas displayed (i) increase in Ki67+ cells, (ii) altered expression and/or localization of tight junction proteins Tjp1 and Pard3, and desmosomal Dsp, (iii) increased superficial fragility and (iv) slower CE wound healing. HCLE-SLURP1 cells displayed (i) decrease in Ki67+ cells, (ii) increased cell number doubling time, (iii) stalling in G1-S phase transition during cell cycle, and (iv) downregulation of cyclins CCNE and CCND1/D2, cyclin-dependent kinases CDK4 and CDK6, and upregulation of CDK inhibitor p15/CDKN2B. CONCLUSIONS: Collectively, these results elucidate that Slurp1X-/- CE cell homeostasis is altered and suggest that SLURP1 is a pro-differentiation factor that stalls G1-S transition during cell cycle progression by downregulating cyclins and upregulating p15/CDKN2B.

Infectious Keratitis: An Update on Role of Epigenetics.

Epigenetic mechanisms modulate gene expression and function without altering the base sequence of DNA. These reversible, heritable, and environment-influenced mechanisms generate various cell types during development and orchestrate the cellular responses to external stimuli by regulating the expression of genome. Also, the epigenetic modifications influence common pathological and physiological responses including inflammation, ischemia, neoplasia, aging and neurodegeneration etc. In recent past, the field of epigenetics has gained momentum and become an increasingly important area of biomedical research As far as eye is concerned, epigenetic mechanisms may play an important role in many complex diseases such as corneal dystrophy, cataract, glaucoma, diabetic retinopathy, ocular neoplasia, uveitis, and age-related macular degeneration. Focusing on the epigenetic mechanisms in ocular diseases may provide new understanding and insights into the pathogenesis of complex eye diseases and thus can aid in the development of novel treatments for these diseases. In the present review, we summarize the clinical perspective of infectious keratitis, role of epigenetics in infectious keratitis, therapeutic potential of epigenetic modifiers and the future perspective.

Mini Review: Current Trends and Understanding of Exosome Therapeutic Potential in Corneal Diseases.

Exosomes are a subset of extracellular vesicles (EVs) that are secreted by most cell types. They are nanosized EVs ranging from 30 to 150 nm. The membrane-enclosed bodies originate by the process of endocytosis and mainly comprise DNA, RNA, protein, and lipids. Exosomes not only act as cell-to-cell communication signaling mediators but also have the potential to act as biomarkers for clinical application and as a promising carrier for drug delivery. Unfortunately, the purification methods for exosomes remain an obstacle. While most of the exosome researches are mainly focused on cancer, there are limited studies highlighting the importance of exosomes in ocular biology, specifically cornea-associated pathologies. Here, we summarize a brief description of exosome biogenesis, roles of exosomes and exosome-based therapies in corneal pathologies, and exosome bioengineering for tissue-specific therapy.

Current perspectives of limbal-derived stem cells and its application in ocular surface regeneration and limbal stem cell transplantation.

Limbal stem cells are involved in replenishing and maintaining the epithelium of the cornea. Damage to the limbus due to chemical/physical injury, infections, or genetic disorders leads to limbal stem cell deficiency (LSCD) with partial or total vision loss. Presently, LSCD is treated by transplanting limbal stem cells from the healthy eye of the recipient, living-related, or cadaveric donors. This review discusses limbal-derived stem cells, the importance of extracellular matrix in stem cell niche maintenance, the historical perspective of treating LSCD, including related advantages and limitations, and our experience of limbal stem cell transplantation over the decades.

Evaluation and Comparison of the Effect of Elastomeric Chain and Stainless Steel Ligature Wire on Maxillary Orthodontic Miniscrew Failure.

CONTEXT: Orthodontic miniscrews are used for the purpose of conservation of anchorage. AIMS: The aim of the study was to evaluate the orthodontic miniscrew failure between the elastomeric chain-supported retraction and stainless steel (SS) ligature-aided retraction. SETTINGS AND DESIGN: This was a cross-sectional split mouth randomized controlled trial. MATERIALS AND METHODS: The sample (30) was divided equally among the control group and the experimental group (15 each). Miniscrews were placed between second premolar and the first molar of maxilla. The experimental group was based on the split mouth technique wherein right or left side of the maxillary arch was treated using either an elastomeric power chain (EPC) engaged to the miniscrews directly (Group 1) or an EPC engaged indirectly to miniscrews with the help of SS ligature wire (Group 2). In control group, implants were placed in maxilla without any retraction force. Clinical signs of inflammation was assessed at the following interval; 7th day, 14th day, 1st month, 2nd month, and at the time of removal of implant. STATISTICAL ANALYSIS USED: Kruskal-Wallis ANOVA test was used. RESULTS: Mean rank of gingival inflammation was 28.33 at the 1st-month interval in Group 1 and inflammation remained high in the this group for all time intervals in comparison to Group 2. Group 2 showed highest mean rank of inflammation of 26.10 at 7th day. In control group, the inflammation remained low at all the time intervals. Moreover, the difference noted was statistically significant. CONCLUSIONS: The gingival inflammation around the peri-implant tissue with the application of EPC at various interval remained high in comparison to the EPC with SS group. The gingival inflammation in the control group was very less, and it remained less throughout the different time periods.

KLF4 Coordinates Corneal Epithelial Apical-Basal Polarity and Plane of Cell Division and Is Downregulated in Ocular Surface Squamous Neoplasia.

Purpose: Previously, we demonstrated that Krüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) homeostasis by suppressing epithelial-mesenchymal transition (EMT) and TGF-ß signaling. As TGF-ß affects epithelial apicobasal polarity (ABP) and plane of division, we investigated the role of KLF4 in these processes. Methods: Klf4 was ablated in adult ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mouse CE using doxycycline chow. ABP and plane of division markers' expression in Klf4Δ/ΔCE and human ocular surface squamous neoplasia (OSSN) tissues relative to controls was evaluated by quantitative PCR, immunoblots, and/or immunofluorescent staining. Results: Klf4Δ/ΔCE CE cells displayed downregulation of apical Pals1 and Crumbs1, apicolateral Par3, and basolateral Scribble, as well as upregulation of Rho family GTPase Cdc42, suggesting disruption of ABP. Phalloidin staining revealed that the Klf4Δ/ΔCE CE actin cytoskeleton is disrupted. Klf4Δ/ΔCE cells favored vertical plane of division within 67.5° to 90° of the CE basement membrane (39% and 47% of the dividing cells relative to 23% and 26% in the control based on phospho-histone-H3 and survivin, respectively), resulting in more dividing cells within the Klf4Δ/ΔCE CE as reported previously. KLF4 was downregulated in human OSSN tissues that displayed EMT and downregulation of PAR3, PALS1, and SCRIB, consistent with a protective role for KLF4. Conclusions: By demonstrating that Klf4 ablation affects CE expression of ABP markers and Cdc42, cytoskeletal actin organization, and the plane of cell division and that KLF4 is downregulated in OSSN tissues that display EMT and lack ABP, these results elucidate the key integrative role of KLF4 in coordinating CE cell polarity and plane of division, loss of which results in OSSN.

The secreted Ly-6/uPAR related protein-1 (SLURP1) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production.

The secreted Ly-6/uPAR related protein-1 (SLURP1) is an anti-angiogenic and anti-inflammatory peptide highly expressed by the mucosal epithelial cells. SLURP1 is abundantly expressed by the corneal epithelial cells and is significantly downregulated when these cells are transformed and adapted for culture in vitro. Here we studied the effect of overexpressing SLURP1 in Human Corneal Limbal Epithelial (HCLE) cells cultured in vitro. The expression of DSP1, DSG1, TJP1 and E-Cadherin was significantly upregulated in two different SLURP1-overexpressing HCLE cell (HCLE-SLURP1) clones. HCLE-SLURP1 cells also displayed a significant decrease in tumor necrosis factor-α (TNF-α)-induced upregulation of (i) IL-8 from 7.4- to 2.9- and 2.1-fold, (ii) IL-1ß from 4.9- to 3.9- and 2.9-fold, (iii) CXCL1 from 9- to 3.3- and 5.5-fold, and (iv) CXCL2 from 4.8- to 2.1- and 2.8-fold. ELISAs revealed a concomitant decrease in IL-8 levels in cell culture supernatants from 789 pg/ml in the control, to 503 and 352 pg/ml in HCLE-SLURP1 cells. Consistently, cytosolic IκB expression was elevated in HCLE-SLURP1 cells with a concurrent suppression of TNF-α-activated nuclear translocation of NF-κB. Collectively, these results elucidate the beneficial effects of SLURP1 in stabilizing the HCLE intercellular junctions and suppressing the TNF-α-induced upregulation of inflammatory cytokines by suppressing NF-κB nuclear translocation.

The secreted Ly6/uPAR-related protein-1 suppresses neutrophil binding, chemotaxis, and transmigration through human umbilical vein endothelial cells.

The secreted Ly-6/uPAR Related Protein-1 (SLURP1) is an immunomodulatory protein that promotes corneal immune- and angiogenic-privilege. Here, we have examined the influence of SLURP1 on neutrophil-vascular endothelial cell interactions using human umbilical vein endothelial cells (HUVEC) and differentiated neutrophil-like HL-60 (dHL-60) cells, or primary human neutrophils. SLURP1 blocked the tumor necrosis factor-alpha (TNF-α)-activated dHL-60 cells (i) binding to TNF-α-activated HUVEC with a concurrent reduction in endothelial cell adhesion molecule E-selectin, (ii) transmigration through TNF-α-activated confluent HUVEC monolayer by stabilizing VE-cadherin and ß-catenin on endothelial cell cytoplasmic membranes, (iii) chemotaxis towards chemoattractant formyl Met-Leu-Phe (fMLP) coupled with their decreased polarization, and (iv) TNF-α-stimulated matrix metalloproteinase-9 (MMP9) expression and activity. SLURP1 also suppressed the primary human neutrophil chemotaxis, and interaction with HUVEC. Furthermore, SLURP1 suppressed fMLP-induced phosphorylation of protein kinase-B (AKT) in dHL-60 cells. Collectively, these results provide evidence that SLURP1 suppresses neutrophil (i) docking on HUVEC cells by decreasing endothelial cell adhesion molecule E-Selectin production, (ii) transmigration through HUVEC monolayer by stabilizing endothelial cell membrane localization of VE-cadherin and ß-catenin complex and promoting their barrier function, and (iii) chemotaxis by modulating their polarization and TNF-α-stimulated MMP9 production.

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-ß Signaling and Upregulating CDK Inhibitors P16 and P27.

Purpose: Krüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) cell fate while suppressing mesenchymal properties. TGF-ß plays a crucial role in cell differentiation and development, and if dysregulated, it induces epithelial-mesenchymal transition (EMT). As KLF4 and TGF-ß regulate each other in a context-dependent manner, we evaluated the role of the crosstalk between KLF4 and TGF-ß-signaling in CE homeostasis. Methods: We used spatiotemporally regulated ablation of Klf4 within the adult mouse CE in ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/ Krt12rtTA/rtTA/ Tet-O-Cre) mice and short hairpin RNA (shRNA)-mediated knockdown or lentiviral vector-mediated overexpression of KLF4 in human corneal limbal epithelial (HCLE) cells to evaluate the crosstalk between KLF4 and TGF-ß-signaling components. Expression of TGF-ß signaling components and cyclin-dependent kinase (CDK) inhibitors was quantified by quantitative PCR, immunoblots, and/or immunofluorescent staining. Results: CE-specific ablation of Klf4 resulted in (1) upregulation of TGF-ß1, -ß2, -ßR1, and -ßR2; (2) downregulation of inhibitory Smad7; (3) hyperphosphorylation of Smad2/3; (4) elevated nuclear localization of phospho-Smad2/3 and Smad4; and (5) downregulation of CDK inhibitors p16 and p27. Consistently, shRNA-mediated knockdown of KLF4 in HCLE cells resulted in upregulation of TGF-ß1 and -ß2, hyperphosphorylation and nuclear localization of SMAD2/3, downregulation of SMAD7, and elevated SMAD4 nuclear localization. Furthermore, overexpression of KLF4 in HCLE cells resulted in downregulation of TGF-ß1, -ßR1, and -ßR2 and upregulation of SMAD7, p16, and p27. Conclusions: Collectively, these results demonstrate that KLF4 regulates CE cell cycle progression by suppressing canonical TGF-ß signaling and overcomes the undesirable concomitant decrease in TGF-ß-dependent CDK inhibitors p16 and p27 expression by directly upregulating them.

Cancer medicine: a direction.

OBJECTIVE: The medicine of cancer is directed in this paper. The pie theory is applied for the proposed medicine. The improbability and un-constancy are the major theories, which are used to design this anti-cancer medicine.

Amelioration of collagen antibody induced arthritis in mice by an antibody directed against the fibronectin type III repeats of tenascin-C: Targeting fibronectin type III repeats of tenascin-C in rheumatoid arthritis.

Tenascin-C (TN-C) levels are elevated in the synovial tissue and fluid, as well as cartilage of rheumatoid arthritis (RA) patients. In addition, the presence of TN-C fragments has also been documented in arthritic cartilage. We have previously shown that a single chain variable fragment antibody (TN64), directed against the fibronectin type III repeats 1-5 (TNfnIII 1-5) of TN-C, effectively inhibits fibrotic pathology. Given that fibrosis results from chronic inflammation, and the fact that increased levels of TN-C in the synovial fluid of patients with RA contributes to synovial inflammation and joint destruction, we aimed to investigate the role of TNfnIII 1-5 region of TN-C in RA pathogenesis. Using either the wild type or variants of the two integrin-binding motifs (RGD and AEIDGIEL) present within the TNfnIII 1-5 polypeptide, we demonstrate that the adhesion and migration of synovial fibroblasts is RGD-dependent. The antibody TN64 is effective in inhibiting migration of cells in response to TnfnIII 1-5, and prevents fibroblast-mediated destruction of cartilage. The TN64 antibody was further tested in collagen antibody induced arthritic (CAIA) mice. Our data shows the efficacy of TN64 in preventing induction of arthritis, with significant downregulation of RA-associated cytokines. This suggests that components of the extracellular matrix such as the TNfnIII 1-5 region of TN-C could be exploited to develop therapies to suppress inflammation seen in RA. The TN64 antibody is one such promising candidate in the development of novel treatments for RA.

Spatiotemporally Controlled Ablation of Klf5 Results in Dysregulated Epithelial Homeostasis in Adult Mouse Corneas.

Purpose: Corneal epithelial (CE) homeostasis requires coordination between proliferation and differentiation. Here we examine the role of cell proliferation regulator Krüppel-like factor 5 (Klf5) in adult mouse CE homeostasis. Methods: Klf5 was ablated in a spatiotemporally restricted manner by inducing Cre expression in 8-week-old ternary transgenic Klf5LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre (Klf5Δ/ΔCE) mouse CE by administering doxycycline via chow. Normal chow-fed ternary transgenic siblings served as controls. The control and Klf5Δ/ΔCE corneal (1) histology, (2) cell proliferation, and (3) Klf5-target gene expression were examined using (1) periodic acid Schiff reagent-stained sections, (2) Ki67 expression, and (3) quantitative PCR and immunostaining, respectively. The effect of KLF4, KLF5, and OCT1 on gastrokine-1 (GKN1) promoter activity was determined by transient transfection in human skin keratinocyte NCTC-2544 cells. Results: Klf5 expression was decreased to 23% of the controls in Klf5Δ/ΔCE corneas, which displayed increased fluorescein uptake, downregulation of tight junction proteins Tjp1 and Gkn1, desmosomal Dsg1a, and basement membrane Lama3 and Lamb1, suggesting defective permeability barrier. In transient transfection assays, KLF5 and OCT1 synergistically stimulated GKN1 promoter activity. Klf5Δ/ΔCE CE displayed significantly fewer cell layers and Ki67+ proliferative cells coupled with significantly decreased cyclin-D1, and elevated phospho(Ser-10) p27/Kip1 expression. Expression of Krt12, E-cadherin, and ß-catenin remained unaltered in Klf5Δ/ΔCE corneas. Conclusions: Klf5 contributes to adult mouse CE homeostasis by promoting (1) permeability barrier function through upregulation of Tjp1, Gkn1, Dsg1a, Lama3, and Lamb1, and (2) basal cell proliferation through upregulation of cyclin-D1 and suppression of phospho(Ser-10) p27/Kip1, without significantly affecting the expression of epithelial markers Krt12, E-cadherin, and ß-catenin.

Assessment of Changes in Nickel and Chromium Levels in the Gingival Crevicular Fluid during Fixed Orthodontic Treatment.

BACKGROUND: Various components of appliances used in fixed orthodontic treatment are fabricated from materials that are highly resistant in nature and have high strength and biocompatibility. Corrosion of materials occurs inside the oral cavity due to numerous environmental or oral factors that act on them. These factors include temperature, pH variation, salivary conditions, mechanical loads, microbiological and enzymatic activity, and various food components. Gingival crevicular fluid (GCF) is the material obtained from the gingival sulcus and might act as a potential source for various biomarkers in the orthodontic setup because inflammatory-induced response is directly related to orthodontic forces in GCF. In the light of above-mentioned data, we planned this study to assess and evaluate the changes occurring in nickel and chromium levels in the GCF during fixed orthodontic treatment. MATERIALS AND METHODS: This study included assessment of 30 patients who underwent fixed orthodontic treatment. Three samples were taken from the GCF of the patients giving a total of 90 samples. The samples were collected at the following time intervals: At baseline (pretreatment time), 1 month after the start of orthodontic treatment, and at 6 months after the commencement of orthodontic treatment. Cellulose strips were used for isolation of the tooth region. For GCF collection, a standardized cellulose acetate absorbent strip was used. Placement of the strips was done in the sulcus for 60 seconds for the collection of the samples. Refrigeration of the specimen bottles was done for a minimum of 7 days and was then sent to a laboratory where specimens were transferred for atomic absorption spectrophotometry. All the results were analyzed by Statistical Package for the Social Sciences software. RESULTS: At 1 month, the mean value of nickel and chromium in GCF was found to be 4.5 and 4.9 ug/gm of GCF respectively. While comparing the mean nickel levels between 1 and 6 months and between baseline and 6 months, significant results were obtained. Significant results were also obtained while comparing the mean values of chromium in GCF between baseline and 6 months and between 1 and 6 months. Gingival health index of the patients was found to be associated with increased inflammation with the progression of time of orthodontic treatment. CONCLUSION: Levels of nickel and chromium might show considerable elevation in the GCF with time along with an increase in the severity of inflammation in the gingival health in patients undergoing fixed orthodontic treatment. CLINICAL SIGNIFICANCE: Regular oral prophylaxis of the patients undergoing orthodontic treatment should be done to avoid tox-icities caused by the release of nickel and chromium and for maintenance of good oral hygiene and oral health.

KLF4 Plays an Essential Role in Corneal Epithelial Homeostasis by Promoting Epithelial Cell Fate and Suppressing Epithelial-Mesenchymal Transition.

Purpose: The purpose of this study was to test the hypothesis that KLF4 promotes corneal epithelial (CE) cell fate by suppressing the epithelial-mesenchymal transition (EMT), using spatiotemporally regulated CE-specific ablation of Klf4 in Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mice. Methods: CE-specific ablation of Klf4 was achieved by feeding Klf4Δ/ΔCE mice with doxycycline chow. The wild-type (WT; normal chow-fed littermates) and the Klf4Δ/ΔCE histology was compared by hematoxylin and eosin-stained sections; EMT marker expression was quantified by quantitative PCR, immunoblots, and immunofluorescent staining; and wound healing rate was measured by CE debridement using Algerbrush. KLF4 and EMT markers were quantified in human corneal limbal epithelial (HCLE) cells undergoing TGF-ß1-induced EMT by quantitative PCR, immunoblots, and immunofluorescent staining. Results: The epithelial markers E-cadherin, Krt12, claudin-3, and claudin-4 were down-regulated, whereas the mesenchymal markers vimentin, ß-catenin, survivin, and cyclin-D1 and the EMT transcription factors Snail, Slug, Twist1, Twist2, Zeb1, and Zeb2 were up-regulated in the Klf4Δ/ΔCE corneas. The Klf4Δ/ΔCE cells migrated faster, filling 93% of the debrided area within 16 hours compared with 61% in the WT. After 7 days of wounding, the Klf4Δ/ΔCE cells that filled the gap failed to regain epithelial characteristics, as they displayed abnormal stratification; down-regulation of E-cadherin and Krt12; up-regulation of ß-catenin, survivin, and cyclin-D1; and a 2.5-fold increase in the number of proliferative Ki67+ cells. WT CE cells at the migrating edge and the HCLE cells undergoing TGF-ß1-induced EMT displayed significant down-regulation of KLF4. Conclusions: Collectively, these results reveal that KLF4 plays an essential role in CE homeostasis by promoting epithelial cell fate and suppressing EMT.

Control of fibrotic changes through the synergistic effects of anti-fibronectin antibody and an RGDS-tagged form of the same antibody.

TGF-ß and myofibroblasts play a key role in fibrosis, characterized by aberrant synthesis and deposition of extracellular matrix (ECM) proteins, such as fibronectin (Fn) and collagen type I. There are two major roles played by integrins in the fibrotic pathology: (i) Fn-integrin interaction, coupled with cytokines like TGF-ß, facilitates the self-polymerization of Fn and regulates cell-matrix fibrillar adhesions, thereby promoting fibrillogenesis; (ii) Integrin interaction with an RGD (arginine-glycine-aspartic) consensus sequence in the latent TGF-ß, resulting in its activation. This study describes an anti-fibrotic strategy using a combination of two antibodies: Fn52 (targeted against the N-terminal 30 kDa region of fibronectin, a major site for Fn self-association), and its engineered form, Fn52RGDS (which binds to integrins). Interestingly, a synergistic effect of the cocktail in causing a decline in fibrotic features was confirmed in the context of fibrotic posterior capsular opacification (PCO), mediated by the lens epithelial cells (left behind after cataract surgery). Inclusion of Fn52RGDS to Fn52 aids in better diffusion of the antibodies; such combination therapies could be useful in the context of pathologies involving extensive remodeling of the fibronectin matrix, where the thick ECM offers a major challenge for efficient drug delivery.

Isothiocyanates: a class of bioactive metabolites with chemopreventive potential.

In recent years, growing interest has been focused on the field of chemoprevention using natural therapies. The reason to turn toward "natural" remedies is associated with diverse beneficial pharmacological properties of natural compounds. Isothiocyanates (ITCs), the major pharmacological active constituents of cruciferous vegetables, are derived from the enzymatic hydrolysis of glucosinolates (GSLs). ITCs govern many intracellular targets including cytochrome P 450 (CYP) enzymes, proteins involved in antioxidant response, tumorigenesis, apoptosis, cell cycle, and metastasis. Investigation of the mechanisms of anti-cancer drugs has given important information regarding the use of natural chemopreventive compounds. This extensive review covers various molecular aspects of the interactions of ITCs with their recognized cellular targets involved in cancer treatment in order to enhance anti-tumor outcome with decreased toxicity to patients.

Targeting the fibronectin type III repeats in tenascin-C inhibits epithelial-mesenchymal transition in the context of posterior capsular opacification.

PURPOSE: Posterior capsular opacification (PCO) is a common complication following extracapsular surgery, associated with fibrosis, opacification, and contraction of the posterior lens capsule. It is characterized by increased expression of extracellular matrix proteins such as tenascin-C, fibronectin, collagens, and proteoglycans. Tenascin-C is known to be critical for injury-induced epithelial-mesenchymal transition (EMT) in the lens epithelium. We aimed to target fibronectin type III repeats 1-5 within tenascin-C (TNfnIII 1-5) using an scFv (single-chain variable fragment) antibody, and to evaluate its effectiveness in the context of lens epithelial cells. METHODS: Phage display library screening was used to generate an antibody against TNfnIII 1-5. Lens epithelial cells were cultured in the presence of the scFv antibodies to evaluate the effects on cell proliferation, migration, fibronectin polymerization and deposition, matrix metalloprotease (MMP) regulation, actin stress fiber distribution, and expression of EMT markers. The effect on SMAD-dependent and SMAD-independent pathways was also examined. RESULTS: The scFv TN64 was found to be effective in regulating the proliferation, migration, and expression of MMP-2 and MMP-9, fibronectin polymerization and deposition, and expression of EMT markers. TN64 did not interfere with SMAD3 phosphorylation. Altered localization of ß-catenin, as well as downregulation of phosphorylation of mitogen-activated protein (MAP) kinases and focal adhesion kinase (FAK), was involved. CONCLUSIONS: Our data suggest that the TNfnIII 1-5 repeats play an important role in PCO pathology. The inhibition of EMT by TN64 is mediated by SMAD-independent, integrin-ß-catenin-FAK signaling pathway, and is therefore proposed as a novel antifibrotic therapeutic candidate.

Expression of granulocyte colony stimulating factor and its receptor by retinal pigment epithelial cells: a role in maintaining differentiation-competent state.

PURPOSE: Granulocyte colony stimulating factor (GCSF) is a potent hematopoietic factor that stimulates the growth of neutrophil granulocyte precursors, and also regulates the differentiation and survival of neutrophils by inhibiting apoptosis. Incidentally, GCSF is also known to act as an endogenous ligand for brain cells, counteracting acute neuronal degeneration and contributing to long-term plasticity of progenitor cells after cerebral ischemia. Since GCSF was recently reported to be present in retinal ganglions, we examined its expression in retinal pigment epithelial (RPE) cells, which, together with retinal neurons, arise from the same underlying precursor cells. METHODS: We used reverse transcriptase polymerase chain reaction (PCR) to assay expression of GCSF and GCSF receptor (GCSFR) genes; immunostaining and flow cytometry to assay the presence of GCSFR on cell surfaces; bromodeoxyuridine (BrdU) incorporation measurement to monitor DNA synthesis; and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to monitor cell proliferation. The effect of GCSF on differentiation of RPE cells was assessed by immunocytochemistry to detect the presence of various marker proteins. RESULTS: The D407 RPE cells, as well as RPE derived from cadaver eyes, were found to express both GCSF and GCSFR. Despite the presence of the GCSF receptor, exogenously added GCSF did not result in any proliferation of these cells. We found that GCSF acts like a de-differentiating factor, maintaining RPE cells in the rounded form, and in a transdifferentiation-competent state. CONCLUSIONS: The expression of GCSF and GCSFR by D407 RPE may be an important factor in RPE cell maintenance.

Multivitamin and micronutrient treatment improves semen parameters of azoospermic patients with maturation arrest.

The study was undertaken to evaluate the efficacy of multivitamin and micronutrient supplementation in azoospermic patients with maturation arrest. A total of 35 azoospermic patients showing maturation arrest on testicular biopsy were recruited in this study. The patients were divided into two groups. Untreated group (n=11) without any treatment and treated group (n=24) who received multivitamins, micronutrients and co-enzyme Q10. The sperm concentration, motility and morphology were evaluated at monthly interval. The results showed reduction in liquefaction time and relative viscosity of the semen in the treated group. Further, in treated group there was appearance of spermatozoa (4.0 million/ml) exhibiting progressive motility (7%) and normal morphology (6%), even in the first follow up visit. The sperm count, motility and normal morphology increased significantly on subsequent visits. Within 3 months (3 visits) 2 pregnancies were reported. These observations indicate that multivitamin and micronutrient supplementation improve the qualitative and quantitative parameters of seminogram in patients with azoospermia of maturation arrest.