A Global Comprehensive Study of the Distribution of Type I-E and Type I-E* CRISPR-Cas Systems in Klebsiella pneumoniae.

Background: The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems are the short DNA sequences and RNA-dependent nuclease involved in the adaptive immunity in bacteria and archaea. The type of CRISPR-Cas system influences antibiotic susceptibility in Klebsiella pneumoniae. Here, our objective was to study the diversity of CRISPR-Cas system in the genome of K. pneumoniae from the available whole genome sequencing (WGS) data. Material and Methods: We identified the CRISPR-Cas systems of K. pneumoniae using the CRISPR-CasFinder database. The complete genome sequence and its submission details were obtained from the National Center for Biotechnology Information (NCBI) database. Results: A total of 1607 K. pneumoniae whole genome sequences were analyzed. The major contributors of WGS data of K. pneumoniae were China (26.6%), United States (21.5%), Australia (10%), South Korea (8%), India (5.5%), and United Kingdom (4.9%). Out of 1607 genomes analyzed, almost one-fourth were CRISPR-Cas positive (403/1607) and three-fourth were CRISPR-Cas negative (1204/1607). Among CRISPR-Cas positive strains, 220 belonged to type I-E* and 183 were type I-E. Furthermore, type I-E* CRISPR-Cas systems were significantly higher in Asia (P < 0.001), whereas type I-E were significantly higher in Europe (P < 0.01). Among countries, typically, type I-E* strains were found to be higher in China (P < 0.01) and India (P < 0.01), whereas type I-E strains were higher in Germany (P < 0.01). Conclusion: Hence, it is important to know the type of CRISPR-Cas systems in K. pneumoniae strains across the countries and it can help to understand the diversity of CRISPR-Cas systems worldwide.

Nitric oxide mitigates thalidomide-induced abnormalities during germination and development of fennel seeds.

Thalidomide causes teratogenic effects in several animal species and in humans. Accordingly, the World Health Organization banned thalidomide when mothers who took thalidomide during pregnancy delivered abnormal fetuses. After four decades, thalidomide underwent drug "re-purposing" since its antiangiogenic and immunomodulatory effects were therapeutic for multiple myeloma. There are no reports of thalidomide's effects on prokaryotes, but it showed teratogenic effects in Arabidopsis thaliana, an ancestor of the plant kingdom. This proof of concept study clearly shows that thalidomide caused a significant and reproducible decrease in germination rate, nitric oxide (NO) production, and chlorophyll content of fennel plantlets. Thalidomide also induced the formation of abnormal fennel plantlets with stunting, wrinkling, and curling of fennel shoots and leaves. Notably, quantitative analysis showed that thalidomide caused a 50% increase in the formation of abnormal fennel plantlets and that these negative effects of thalidomide showed a 2.50- to 4-fold decrease when fennel seeds were co-incubated with an NO donor (Spermine NoNoate) or a stable cGMP analog 8-bromo Guanosine 3',5'-cyclic monophosphate (8-Bromo-cGMP). This study is important because it confirms that thalidomide's negative effects on fennel seed germination and growth are mediated by attenuation of NO and disruption of NO signaling. This reproducible model of thalidomide-induced, NO-dependent damage in a plant system can be used to further investigate the molecular mechanisms of thalidomide action in plants. Importantly, this study establishes a link between the evolution of development of higher plants and mammals.

Intussusceptive angiogenesis as a key therapeutic target for cancer therapy.

Deregulation of angiogenesis is a key reason for tumor growth and progression. Several anti-angiogenic drugs in clinical practice attempt to normalize abnormal tumor vasculature. Unfortunately, these drugs are ineffective due to the development of resistance in patients after drug holidays. A sizable literature suggests that resistance to these anti-angiogenic drugs occurs due to various compensatory mechanisms of tumor angiogenesis. Therefore, we describe different compensatory mechanisms of tumor angiogenesis, and explain why intussusceptive angiogenesis (IA), is a crucial mechanism of compensatory angiogenesis in tumors which resist anti-VEGF (vascular endothelial growth factor) therapies. IA is often overlooked due to the scarcity of experimental models. Therefore, we examine data from existing experimental models and our novel ex-ovo model of angiogenesis in chick embryos, and explain the important genes and signaling pathways driving IA. Using bio-informatic analyses of major genes regulating conventional sprouting angiogenesis (SA) and intussusceptive angiogenesis, we provide fresh insights on the 'angiogenic switch' which regulates the transition from SA to IA. Finally, we examine the interplay between molecules regulating SA, IA, and molecules known to promote tumor progression. Based on these analyses, we conclude that intussusceptive angiogenesis (IA) is a promising therapeutic target for developing effective anti-cancer treatment regimes.

Can the vagus nerve serve as biomarker for vata dosha activity?

This 'discussion paper' raises 'provocative questions' to identify physiological systems underlying vata dosha and candidate biomarkers for vata activity. We explained the strong correlations between survival and homeostatic functions of the parasympathetic vagus nerve, and functions governed by the five major sub-types of vata dosha (Praana, Udana, Vyaana, Samaana, and Apana). Four reasons were provided to hypothesize that vagal activity is a reliable candidate biomarker of important vata dosha functions. First, normal vata dosha and the vagus maintain neural, respiratory, and digestive homeostasis, and dysfunctions in both entities cause very similar diseases. Second, vata dosha regulates higher neural functions such as mental health and behaviour, and the 'polyvagal theory' proposes similar functions for the vagus. Third, the similar roles of vata dosha and vagus in maintaining gut homeostasis, suggest that vagal activity in the 'gut-brain' link is a candidate biomarker of pakwashaya (lower gut), a primary regulatory site for vata dosha. Fourth, the vagus is the only vital nerve whose activity can be reliably measured and manipulated. Indeed, vagal nerve stimulation is a USA-FDA approved therapy for certain ailments attributed to impaired vata dosha. No other nerve or dosha, has such multi-functional and life-sustaining properties. These arguments position vagal activity as a suitable candidate biomarker for certain functions of vata dosha.

Microarray and pattern miner analysis of AXL and VIM gene networks in MDA­MB­231 cells.

MDA­MB­231 cells represent malignant triple­negative breast cancer, which overexpress epidermal growth factor receptor (EGFR) and two genes (AXL and VIM) associated with poor prognosis. The present study aimed to identify novel therapeutic targets and elucidate the functional networks for the AXL and VIM genes in MDA­MB­231 cells. We identified 71 genes upregulated in MDA­MB­231 vs. MCF7 cells using BRB­Array tool to re­analyse microarray data from six GEO datasets. Gene ontology and STRING analysis showed that 43/71 genes upregulated in MDA­MB­231 compared with MCF7 cells, regulate cell survival and migration. Another 19 novel genes regulate migration, metastases, senescence, autophagy and chemoresistance. The Pattern Miner systems biology tool uses specific genes as inputs or 'baits' to identify outputs from the NCI­60 database. Using five genes regulating cancer cell migration (AXL, VIM, EGFR, CAPN2, and COL4A1) as input 'baits', we used pattern miner to identify statistically significant, co­expressed genes from the list of 71 genes upregulated in MDA­MB­231 compared with MCF7 cells. Outputs were subsets of the 71 genes, which showed significant co­expression with one or more of the five input genes. These outputs were used to develop functional networks for AXL and VIM. Analysis of these networks verified known properties of AXL and VIM, and suggested novel functions for these two genes. Thus, genes in the AXL network promote migration, metastasis and chemoresistance, whereas the VIM gene network regulates novel tumorigenic processes, such as lipogenesis, senescence and autophagy. Notably, these two networks contain 12 genes not reported for TNBC.

MicroRNAs: Impaired vasculogenesis in metal induced teratogenicity.

Certain metals have been known for their toxic effects on embryos and fetal development. The vasculature in early pregnancy is extremely dynamic and plays an important role in organogenesis. Nascent blood vessels in early embryonic life are considered to be a primary and delicate target for many teratogens since the nascent blood islands follow a tightly controlled program to form vascular plexus around and inside the embryo for resourcing optimal ingredients for its development. The state of the distribution of toxic metals, their transport mechanisms and the molecular events by which they notch extra-embryonic and embryonic vasculatures are illustrated. In addition, pharmacological aspects of toxic metal induced teratogenicity have also been portrayed. The work reviewed state of the current knowledge of specific role of microRNAs (miRNAs) that are differentially expressed in response to toxic metals, and how they interfere with the vasculogenesis that manifests into embryonic anomalies.

Epigallocatechin gallate & curcumin prevent transforming growth factor beta 1-induced epithelial to mesenchymal transition in ARPE-19 cells.

Background & objectives: Proliferative vitreoretinopathy (PVR) is characterized by the presence of epiretinal membrane (ERM), which exerts traction and detaches the retina. Epithelial to mesenchymal transition (EMT) of the retinal pigment epithelial (RPE) cells underlies ERM formation. Adjuvant therapies aimed at preventing recurrence of PVR after surgery mostly failed in clinical trials. This study was aimed to evaluate the anti-EMT properties of bio-active compounds epigallocatechin gallate (EGCG), curcumin and lycopene as inhibitors of EMT induced by transforming growth factor beta 1 (TGF-ß1) in cultured ARPE-19 cells. Methods: ARPE-19 cells were treated with TGF-ß1 alone or co-treated with EGCG (1-50 µM), lycopene (1-10 µM) and curcumin (1-10 µM). The mRNA and protein expression of EMT markers, alpha-smooth muscle actin, vimentin, zonula occludens-1 and matrix metalloproteinase-2 (MMP-2), were assessed by reverse transcription polymerase chain reaction/quantitative polymerase chain reaction and immunofluorescence/enzyme linked immunosorbent assay. Activity of MMP-2 was assessed by zymography. Functional implications of EMT were assessed by proliferation assay (MTT assay) and migration assay (scratch assay). Western-blot for phosphorylated Smad-3 and total Smad-3 was done to delineate the mechanism. Results: EGCG and curcumin at 10 µM concentration reversed EMT, inhibited proliferation and migration through Smad-3 phosphorylation, when induced by TGF-ß1 in ARPE-19 cells. Lycopene did not prevent EMT in ARPE-19 cells. Interpretation & conclusions: EGCG and curcumin are potent in preventing EMT induced by TGF-ß1 in ARPE-19 cells and therefore, proposed as potential molecules for further pre-clinical evaluation in PVR management.

Mechanical perturbations trigger endothelial nitric oxide synthase activity in human red blood cells.

Nitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the intravascular NO pool and regulates physiological functions. However the regulatory mechanisms and clinical implications of RBC-eNOS are unknown. The present study investigated regulation and functions of RBC-eNOS under mechanical stimulation. This study shows that mechanical stimuli perturb RBC membrane, which triggers a signaling cascade to activate the eNOS. Extracellular NO level, estimated by the 4-Amino-5-Methylamino-2', 7'-Difluorofluorescein Diacetate probe, was significantly increased under mechanical stimuli. Immunostaining and western blot studies confirmed that the mechanical stimuli phosphorylate the serine 1177 moiety of RBC-eNOS, and activates the enzyme. The NO produced by activation of RBC-eNOS in vortexed RBCs promoted important endothelial functions such as migration and vascular sprouting. We also show that mechanical perturbation facilitates nitrosylation of RBC proteins via eNOS activation. The results of the study confirm that mechanical perturbations sensitize RBC-eNOS to produce NO, which ultimately defines physiological boundaries of RBC structure and functions. Therefore, we propose that mild physical perturbations before, after, or during storage can improve viability of RBCs in blood banks.

Microarray Analysis of Differentially-Expressed Genes Encoding CYP450 and Phase II Drug Metabolizing Enzymes in Psoriasis and Melanoma.

Cytochrome P450 drug metabolizing enzymes are implicated in personalized medicine for two main reasons. First, inter-individual variability in CYP3A4 expression is a confounding factor during cancer treatment. Second, inhibition or induction of CYP3A4 can trigger adverse drug-drug interactions. However, inflammation can downregulate CYP3A4 and other drug metabolizing enzymes and lead to altered metabolism of drugs and essential vitamins and lipids. Little is known about effects of inflammation on expression of CYP450 genes controlling drug metabolism in the skin. Therefore, we analyzed seven published microarray datasets, and identified differentially-expressed genes in two inflammatory skin diseases (melanoma and psoriasis). We observed opposite patterns of expression of genes regulating metabolism of specific vitamins and lipids in psoriasis and melanoma samples. Thus, genes controlling the turnover of vitamin D (CYP27B1, CYP24A1), vitamin A (ALDH1A3, AKR1B10), and cholesterol (CYP7B1), were up-regulated in psoriasis, whereas melanomas showed downregulation of genes regulating turnover of vitamin A (AKR1C3), and cholesterol (CYP39A1). Genes controlling abnormal keratinocyte differentiation and epidermal barrier function (CYP4F22, SULT2B1) were up-regulated in psoriasis. The up-regulated CYP24A1, CYP4F22, SULT2B1, and CYP7B1 genes are potential drug targets in psoriatic skin. Both disease samples showed diminished drug metabolizing capacity due to downregulation of the CYP1B1 and CYP3A5 genes. However, melanomas showed greater loss of drug metabolizing capacity due to downregulation of the CYP3A4 gene.

Microarray analysis of differentially expressed genes regulating lipid metabolism during melanoma progression.

A new hallmark of cancer involves acquisition of a lipogenic phenotype which promotes tumorigenesis. Little is known about lipid metabolism in melanomas. Therefore, we used BRB (Biometrics Research Branch) class comparison tool with multivariate analysis to identify differentially expressed genes in human cutaneous melanomas, compared with benign nevi and normal skin derived from the microarray dataset (GDS1375). The methods were validated by identifying known melanoma biomarkers (CITED1, FGFR2, PTPRF, LICAM, SPP1 and PHACTR1) in our results. Eighteen genes regulating metabolism of fatty acids, lipid second messengers and gangliosides were 2-9 fold upregulated in melanomas of GDS-1375. Out of the 18 genes, 13 were confirmed by KEGG pathway analysis and 10 were also significantly upregulated in human melanoma cell lines of NCI-60 Cell Miner database. Results showed that melanomas upregulated PPARGC1A transcription factor and its target genes regulating synthesis of fatty acids (SCD) and complex lipids (FABP3 and ACSL3). Melanoma also upregulated genes which prevented lipotoxicity (CPT2 and ACOT7) and regulated lipid second messengers, such as phosphatidic acid (AGPAT-4, PLD3) and inositol triphosphate (ITPKB, ITPR3). Genes for synthesis of pro-tumorigenic GM3 and GD3 gangliosides (UGCG, HEXA, ST3GAL5 and ST8SIA1) were also upregulated in melanoma. Overall, the microarray analysis of GDS-1375 dataset indicated that melanomas can become lipogenic by upregulating genes, leading to increase in fatty acid metabolism, metabolism of specific lipid second messengers, and ganglioside synthesis.

Tolerance of ARPE 19 cells to organophosphorus pesticide chlorpyrifos is limited to concentration and time of exposure.

Age related macular degeneration is a blinding disease common in elder adults. The prevalence of age related macular degeneration has been found to be 1.8% in the Indian population. Organophosphates are widely used insecticides with well documented neurological effects, and the persistent nature of these compounds in the body results in long term health effects. Farmers exposed to organophosphorus pesticides in USA had an earlier onset of age related macular degeneration when compared to unexposed controls. A recent study found significant levels of an organophosphate, termed chlorpyrifos, in the blood samples of Indian farmers. Therefore, in understanding the link between age related macular degeneration and chlorpyrifos, the need for investigation is important. Our data show that ARPE-19 (retinal pigment epithelial cells) exhibit a cytoprotective response to chlorpyrifos as measured by viability, mitochondrial membrane potential, superoxide dismutase activity, and increased levels of glutathione peroxidase and reduced glutathione, after 24 h exposure to chlorpyrifos. However, this cytoprotective response was absent in ARPE-19 cells exposed to the same range of concentrations of chlorpyrifos for 48 h. These results have physiological significance, since HPLC analysis showed that effects of chlorpyrifos were mediated through its entry into ARPE-19 cells. HPLC analysis also showed that chlorpyrifos remained stable, as we recovered up to 80% of the chlorpyrifos added to 6 different ocular tissues.

Augmented sensitivity to methotrexate by curcumin induced overexpression of folate receptor in KG-1 cells.

Folate receptors are targets of various strategies aimed at efficient delivery of anti-cancer drugs. Folate receptors also play a role in the uptake of antifolate drugs which are used for therapeutic intervention in leukemia. Therefore, it is important to identify compounds which regulate expression of folate receptors in leukemic cells. The present study examined if curcumin could modulate the uptake and cytotoxicity of the antifolate drug methotrexate, in KG-1 leukemic cells. This is the first report to show that curcumin (10-50 µM) causes a significant, dose-dependent, 2-3 fold increase in uptake of radiolabelled folic acid and methotrexate into KG-1 cells both at 24 h and 48 h of treatment. Interestingly, pre-treatment of KG-1 leukemic cells with curcumin (10 µM and 25 µM) also caused a statistically significant enhancement in the cytotoxicity of methotrexate. We performed Real Time Quantitative RT-PCR to confirm the upregulation of FRß mRNA in curcumin treated cells. Immunocytochemistry and Western blotting showed that curcumin caused increased expression of folate receptor ßin KG-1 cells. Our data show that the mechanism of curcumin action involves up-regulation of folate receptor ß mRNA and protein in KG-1 cells. Therefore, combination of non-toxic concentrations of curcumin and methotrexate, may be a viable strategy for therapeutic intervention for leukemias using a folate receptor-targeted drug delivery system.

Ayurvedic medicine offers a good alternative to glucosamine and celecoxib in the treatment of symptomatic knee osteoarthritis: a randomized, double-blind, controlled equivalence drug trial.

OBJECTIVE: To demonstrate clinical equivalence between two standardized Ayurveda (India) formulations (SGCG and SGC), glucosamine and celecoxib (NSAID). METHODS: Ayurvedic formulations (extracts of Tinospora cordifolia, Zingiber officinale, Emblica officinalis, Boswellia serrata), glucosamine sulphate (2 g daily) and celecoxib (200 mg daily) were evaluated in a randomized, double-blind, parallel-efficacy, four-arm, multicentre equivalence drug trial of 24 weeks duration. A total of 440 eligible patients suffering from symptomatic knee OA were enrolled and monitored as per protocol. Primary efficacy variables were active body weight-bearing pain (visual analogue scale) and modified WOMAC pain and functional difficulty Likert score (for knee and hip); the corresponding a priori equivalence ranges were ±1.5 cm, ±2.5 and ±8.5. RESULTS: Differences between the intervention arms for mean changes in primary efficacy variables were within the equivalence range by intent-to-treat and per protocol analysis. Twenty-six patients showed asymptomatic increased serum glutamic pyruvic transaminase (SGPT) with otherwise normal liver function; seven patients (Ayurvedic intervention) were withdrawn and SGPT normalized after stopping the drug. Other adverse events were mild and did not differ by intervention. Overall, 28% of patients withdrew from the study. CONCLUSION: In this 6-month controlled study of knee OA, Ayurvedic formulations (especially SGCG) significantly reduced knee pain and improved knee function and were equivalent to glucosamine and celecoxib. The unexpected SGPT rise requires further safety assessment. TRIAL REGISTRATION: Clinical Drug Trial Registry-India, www.ctri.nic.in, CTRI/2008/091/000063.

Insights on personalized medicine from Ayurveda.

The "omics" era of research has provided vital information on the genetic and biochemical diversity of individuals. This has lead to the emergence of "personalized medicine," wherein one aims to design specific drugs for individual patients or subtypes of patients. Indeed, the ongoing patent wars on this matter, suggest that personalized medicine represents a major goal for today's pharmaceutical industries. Although the concept of personalized medicine is new to modern medicine, it is a well-established concept in Ayurveda, the traditional system of Indian medicine that is still being practiced. Therefore, this article discusses topics that are crucial for the advancement of modern personalized medicine. These topics include disease susceptibility, disease subtypes, and Ayurvedic therapeutics. First, we explain how Ayurveda, Traditional Chinese Medicine, and Traditional Korean medicine or Sasang Constitutional medicine; conceptualize disease susceptibility and disease subtypes. Next, we focus on conceptual similarities between molecular medicine and Ayurvedic concepts of disease susceptibility and disease subtypes. For each topic, we explain the relevant experimental evidence reported in the literature. We also propose new hypotheses and suggest experimental approaches for their testing and validation.

Identification of natural compounds which inhibit biofilm formation in clinical isolates of Klebsiella pneumoniae.

Klebsiella pneumoniae, an important opportunistic pathogen, exists as a biofilm in persistent infections and in-dwelling medical devices. With the objective of identifying natural compounds inhibiting biofilm formation in K. pneumoniae, 35 clinical isolates were screened,out of which 7 strong biofilm producers were identified. Six natural compounds were tested for their inhibitory effects on bacterial growth and biofilm formation by determining the minimum inhibitory concentration and minimum concentration for biofilm inhibition (MBIC) for each compound. The results show that reserpine followed by linoleic acid, were the most potent biofilm inhibitors. Reserpine, an efflux pump inhibitor was effective at biofilm inhibition at a concentration of 0.0156 mg/mL, 64-fold lower concentration than its MIC. Linoleic acid, an essential fatty acid was effective as a biofilm inhibitor at 0.0312 mg/mL, which is 32-fold lower than its MIC. Berberine, another plant derived antimicrobial, chitosan and eugenol had an MBIC value of 0.0635 mg/mL. Curcumin, a natural phenolic compound was effective at biofilm inhibition at a concentration of 0.25 mg/mL, which is 50 fold less than its MIC. Notably, the MIC and MBIC data on these 6 natural compounds was reproducible in all seven high biofilm forming isolates of K. pneumoniae. The present report is a comprehensive comparative analysis of the dose dependent inhibition of various natural compounds on biofilm formation in K. pneumoniae.

Cancer, inflammation, and insights from ayurveda.

A recent, exciting discovery relates to the concept of "shared pathology" between cancer and metabolic syndrome. One major pathway common to cancer and metabolic syndrome is chronic inflammation, which is a major driving force in carcinogenesis. Indeed, chronic inflammation precedes most cancers and is considered a "hallmark" of the neoplastic process. We discuss molecular and biochemical evidence which links diet, obesity, abnormal lipid metabolism, and type 2 diabetes mellitus with chronic inflammation. We also explain how each of these factors is linked with biochemical aberrations of carcinogenesis and the prevalence and risk of cancer. While there are reliable biomarkers for chronic inflammation, there are few markers for a mechanistic link between early inflammation and digestive disorders. Discovery of such a marker could lead to identification of a new subtype of patients with digestive disorders that predispose them to cancer and/or metabolic syndrome. In this context, we discuss the ayurvedic concept of "Ama" which is thought to be a toxic, proinflammatory waste-product of improper digestion. We then develop hypotheses and outline preclinical and clinical experiments designed to prove whether "Ama" can serve as a novel and reliable biomarker that links abnormal digestive status, with the onset of chronic inflammation.

Cellular chemosensitivity assays: an overview.

Data on cell viability have long been obtained from in vitro cytotoxicity assays. Today, there is a focus on markers of cell death, and the MTT cell survival assay is widely used for measuring cytotoxic potential of a compound. However, a comprehensive evaluation of cytotoxicity requires additional assays which -measure short and long-term cytotoxicity. Assays which measure the cytostatic effects of compounds are not less important, particularly for newer anticancer agents. This overview discusses the advantages and disadvantages of different non-clonogenic assays for measuring short and medium-term cytotoxicity. It also discusses clonogenic assays, which accurately measure long-term cytostatic effects of drugs and toxic agents. For certain compounds and cell types, the advent of high throughput, multiparameter, cytotoxicity assays, and gene expression assays have made it possible to predict cytotoxic potency in vivo.

GLUT-1 expression in bovine retinal capillary endothelial cells and pericytes exposed to advanced glycation end products.

PURPOSE: Glucose uptake and glucose transporter GLUT-1 expressions are characteristic of retinal capillary endothelial cells and pericytes in response to high glucose. In this study, the effects of advanced glycation end product (AGE-BSA) exposure on these parameters were tested. METHODS: Primary cultures of bovine retinal capillary endothelial cells (BRECs) and bovine retinal capillary pericytes (BRPs) were exposed to AGE-BSA (100 µg/mL) for 6 days. Glucose uptake was measured using U (14)C-glucose and the GLUT-1 mRNA expression by RT-PCR. GLUT-1 protein was detected by immunofluorescence and subjected to FACS analysis. RESULTS: The authors observed that there was no significant decrease in the GLUT-1 protein expression, and this was confirmed by glucose uptake by (14)C-labeled glucose in both BRECs and BRPs. Even though there was a slight decrease in the mRNA expression of GLUT-1 in AGE-BSA-treated cells compared with both untreated control and BSA treated, the decrease was not significant. CONCLUSIONS: This is the first report to show that there is no difference in glucose uptake in BRECs and BRPs on exposure to AGE-BSA.

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.

Chondroprotective Potential of Fruit Extracts of Phyllanthus emblica in Osteoarthritis.

There is a need for effective nutraceuticals for osteoarthritis care. The fruit of Phyllanthus emblica is used as a powerful rejuvenator in Ayurvedic medicine. This study measured the chondroprotective potential of P. emblica ('Amalaki') fruits in vitro. We used aqueous extracts of unprocessed P. emblica fruit powder (powder A), and the powder obtained after hot water extraction and drying of powder A (powder B). Chondroprotection was measured in three different assay systems. First, we tested the effects of both fruit powders on the activities of the enzymes hyaluronidase and collagenase type 2. Second, an in vitro model of cartilage degradation was set-up with explant cultures of articular knee cartilage from osteoarthritis patients. Cartilage damage was assayed by measuring glycosaminoglycan release from explants treated with/without P. emblica fruit powders. Aqueous extracts of both fruit powders significantly inhibited the activities of hyaluronidase and collagenase type 2 in vitro. Third, in the explant model of cartilage matrix damage, extracts of glucosamine sulphate and powder B (0.05 mg/ml) exhibited statistically significant, long-term chondroprotective activity in cartilage explants from 50% of the patients tested. This result is important since glucosamine sulphate is the leading nutraceutical for osteoarthritis. Powder A induced a statistically significant, short-term chondroprotective activity in cartilage explants from all of the patients tested. This is the first study to identify and quantitate new chondroprotective activities of P. emblica fruits. These data provide pilot pre-clinical evidence for the use of P. emblica fruits as a chondroprotective agent in osteoarthritis therapy.