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MicroRNAs of the miR-16 and miR-34 families have been reported to inhibit cell cycle progression, and their loss has been linked to oncogenic transformation. Utilizing a high-throughput, genome-wide screen for miRNAs and mRNAs that are differentially regulated in osteosarcoma (OS) cell lines, we report that miR-449a and miR-424, belonging to the miR-34 and miR-16 families, respectively, target the major S/G(2) phase cyclin, cyclin A2 (CCNA2), in a bipartite manner. We found that the 3′-UTR of CCNA2 is recognized by miR-449a, whereas the CCNA2 coding region is targeted by miR-424. Of note, we observed loss of both miR-449a and miR-424 in OS, resulting in derepression of CCNA2 and appearance of aggressive cancer phenotypes. Ectopic expression of miR-449a and miR-424 significantly decreased cyclin A2 levels and inhibited proliferation rate, migratory potential, and colony-forming ability of OS cells. To further probe the roles of miR-449a and miR-424 in OS, we developed an OS mouse model by intraosseous injection of U2OS cells into the tibia bone of NOD-scid mice, which indicated that miR-449a and miR-424 co-expression suppresses tumor growth. On the basis of this discovery, we analyzed the gene expression of human OS biopsy samples, revealing that miR-449a and miR-424 are both down-regulated, whereas cyclin A2 is significantly up-regulated in these OS samples. In summary, the findings in our study highlight that cyclin A2 repression by miRNAs of the miR-16 and miR-34 families is lost in aggressive OS.

Purpose The aim of this work was to study the association of serum ionized calcium with retinal photoreceptor apoptosis on spectral domain optical coherence tomography (SD-OCT) in diabetic retinopathy (DR). Methods Sixty consecutive cases with Type 2 diabetes mellitus were categorized into three groups: no diabetic retinopathy; non-proliferative DR; proliferative DR. The eye with more severe form of the disease was considered. Twenty healthy controls were also included. Best corrected visual acuity (BCVA) was measured on logMAR scale. Retinal photoreceptor apoptosis was defined as disruption of retinal photoreceptor ellipsoid zone (EZ). Ellipsoid zone disruption was assessed using SD-OCT. Serum levels of total and ionized calcium were measured using standard protocol. Results EZ disruption was found to be positively associated with serum total calcium and ionized calcium. Also, EZ disruption was found to be positively associated with logMAR BCVA. Conclusion Increased serum ionized calcium induces retinal photoreceptor apoptosis resulting in increased EZ disruption in DR.

The tumor microenvironment is characterized by nutrient-deprived conditions in which the cancer cells have to adapt for survival. Serum starvation resembles the growth factor deprivation characteristic of the poorly vascularized tumor microenvironment and has aided in the discovery of key growth regulatory genes and microRNAs (miRNAs) that have a role in the oncogenic transformation. We report here that miR-874 down-regulates the major G1/S phase cyclin, cyclin E1 (CCNE1), during serum starvation. Because the adaptation of cancer cells to the tumor microenvironment is vital for subsequent oncogenesis, we tested for miR-874 and CCNE1 interdependence in osteosarcoma cells. We observed that miR-874 inhibits CCNE1 expression in primary osteoblasts, but in aggressive osteosarcomas, miR-874 is down-regulated, leading to elevated CCNE1 expression and appearance of cancer-associated phenotypes. We established that loss of miR-874–mediated control of cyclin E1 is a general feature of osteosarcomas. The down-regulation of CCNE1 by miR-874 is independent of E2F transcription factors. Restoration of miR-874 expression impeded S phase progression, suppressing aggressive growth phenotypes, such as cell invasion, migration, and xenograft tumors, in nude mice. In summary, we report that miR-874 inhibits CCNE1 expression during growth factor deprivation and that miR-874 down-regulation in osteosarcomas leads to CCNE1 up-regulation and more aggressive growth phenotypes.

Elevated lipid levels increase complications of diabetic retinopathy (DR). Uncontrolled diabetes increases these complications and causes unintentional weight loss, indicating an apparently normal body mass index (BMI). Thus, it is easy to assume that patients with DR and a normal BMI have optimal lipid status. Apolipoprotein (Apo) A-I and Apo B levels differentially indicate serum lipid status in DR. Subjective Global Assessment (SGA) scores are associated with DR status. If SGA scores and serum Apo A-I and B levels are found to be interrelated, their relationship can reflect lipid defects in patients with DR despite apparently normal BMI. The aim of the present study was to investigate the possible relationship between serum Apo A-I and B levels and SGA scores of patients with DR.This was a case-control study conducted from November 2011 to April 2014. Serum Apo A-I and B levels and SGA scores were calculated for 40 healthy controls, 48 individuals without DR, 49 nonproliferative DR cases, and 48 proliferative DR cases. Pearson's correlation analysis was applied between Apo A-I, Apo B, Apo B/Apo A-I ratio, and SGA scores.Negative correlation was observed between serum Apo A-I level (r = -0.567, P 0.001) and positive correlation between serum Apo B level (r = 0.451, P 0.001) and Apo B/Apo A-I ratio (r = 0.597, P 0.001) with escalating SGA scores.To our knowledge, this is the first study to report a novel correlation between serum Apo A-I, Apo B and Apo B/Apo A-I ratio and SGA scores. SGA scores can help predict lipid abnormalities in patients with DR even when they have an apparently normal BMI.

Cloud computing offers computational, storage, and applications capabilities as subscription oriented services. It offers a platform explore computational assets and use them based on "pay per use arrangement". Thus it opens ways to access boundless assets with negligible equipment and programming at the customers' end. This paper focuses on the advancement of a cloud administration's provisioning structure by building up a dynamic load-balancer for the cloud. It proposes a framework and protocol for the resource environment in the cloud. Distributed Hash Table (DHT) protocol has been utilized for a service query to perform a job agreed by the user. For load balancing, gossip protocol has been used for inter/intra-cluster gossip. For inter-cluster gossip, the load is balanced among the leaders of every cluster. The proposed protocol uses the inter-cloud resource management, where a leader is selected from the cloud that interacts to other cloud and decides on virtual machine (VM) migration. The decision about job allocation is not acknowledged by a single machine, which generates the scalable architecture of the proposed protocol. The protocol considers the current load situation and decides at the time of request submission. This protocol is adaptable, reliable and scalable and supports green computing by utilizing server solidification.

To correlate serum levels of N-epsilon-carboxy methyl lysine (N(ε)-CML) with severity of retinopathy, in vivo macular edema and disruption of external limiting membrane (ELM) and photoreceptor ellipsoid zone in type 2 diabetes mellitus (DM).Consecutive cases of type 2 DM [diabetes mellitus with no retinopathy (No DR) (n=20); non- proliferative diabetic retinopathy (NPDR) with diabetic macular edema (n=20); proliferative diabetic retinopathy with diabetic macular edema (PDR) (n=20)] and healthy controls (n=20) between the ages of 40 and 65 years were included (power of study=93.8%). In vivo histology of retinal layers was assessed using spectral domain optical coherence tomography. Every study subject underwent macular thickness analysis using the macular cube 512×128 feature. Disruption of ELM and photoreceptor ellipsoid zone was graded: grade 0, no disruption of ELM and ellipsoid zone; grade 1, ELM disrupted and ellipsoid zone intact; grade 2, both ELM and ellipsoid zone disrupted. Data were statistically analyzed.The mean levels of N(ε)-CML were 31.34±21.23 ng/ml, 73.88±35.01 ng/ml, 91.21±66.65 ng/ml, and 132.08±84.07 ng/ml in control, No DR, NPDR and PDR respectively. N(ε)-CML level was significantly different between the study groups (control, No DR, NPDR and PDR) (p0.001). Mean logMAR visual acuity decreased with increased levels of N(ε)-CML (p0.001). The association of N(Ɛ)CML with the grades of disruption was found to be statistically significant (F value=18.48, p0.001). Univariate analysis was done with N(Ɛ)-CML as a dependent variable. The values of N(Ɛ)-CML were normalized (log10) and were subjected to univariate analysis with fasting blood glucose level, glycosylated hemoglobin, central subfield macular thickness and cube average thickness among the diseased groups (NPDR and PDR) that act as confounders. It was found that none of the variables had significant effect on N(Ɛ)-CML (fasting blood glucose p=0.12, HBA1c p=0.65, central subfield macular thickness p=0.13, cube average thickness p=0.19). N(Ɛ)-CML tends to be a significant and important predictor of grade of ELM and ellipsoid zone disruption in diabetic retinopathy.Increased N(ε)-CML levels are associated with increased severity of diabetic retinopathy, macular edema and structural changes in macula that is ELM and ellipsoid zone disruption, which serves as a prognosticator of visual outcome.

Caveolin‐1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine‐14 (Y14), contains the highly‐conserved membrane‐proximal caveolin scaffolding domain (CSD; amino acids 82‐101). Here we show, using CSD mutants (F92A/V94A) and membrane‐permeable CSD competing peptides, that Src kinase‐dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension and cancer cell migration is CSD‐dependent. Quantitative proteomic analysis of Cav1‐GST (1‐101 amino acids) pulldowns showed 6‐fold increased binding of vinculin and, to a lesser extent, α‐actinin, talin and filamin, to phosphomimetic Cav1Y14D relative to non‐phosphorylatable Cav1Y14F. Consistently, pY14Cav1 enhanced CSD‐dependent vinculin tension in focal adhesions, dampening force fluctuation and synchronously stabilizing cellular focal adhesions in a high‐tension mode, paralleling effects of actin stabilization. By promoting focal adhesion traction, functional interaction between Cav1 Y14 phosphorylation and the CSD shifts the molecular clutch linking adhesions and the actin cytoskeleton to high gear, and thereby drives cancer cell migration.

Hyperglycemia in diabetes causes endogenous formation of Advanced Glycation End-products (AGEs) which accumulate in various body parts including retina causing diabetic retinopathy. AGEs also originate from exogenous dietary sources contributing to the body's AGE pool. Currently, curing of diabetic retinopathy is mainly focused on medication, surgical or laser interventions and not much emphasis is given on preventing or halting its occurrence or advancement to more severe stages, nutritionally. Planning a 'low glycemic index-low AGE' diet therapy for diabetic subjects can reduce endogenous and exogenous origin AGEs in the body and help in controlling retinopathy. Sound and accurate assessment of nutritional status is a crucial step for planning a therapeutic diet for this condition. As this aspect has not gained sufficient attention till now we are assessing the association of serum Advanced Glycation End-product (AGE) levels with the severity of diabetic retinopathy and for the first time estimating the nutritional status of subjects with this eye disorder for long term patient care.This was a tertiary care centre-based, case-control study involving sixty three consecutive cases with diabetes divided as 21 cases with diabetes but no retinopathy, 21 cases with non proliferative diabetic retinopathy (NPDR), 21 cases with proliferative diabetic retinopathy (PDR) along with 21 healthy controls. Serum AGE levels of all the cases and controls were evaluated by Enzyme Linked Immuno Sorbent Assay (ELISA) and nutritional status was assessed by anthropometric measurements and SGA scores.Serum AGE levels were found significantly elevated in PDR group when compared with no retinopathy (p 0.05) and control (p 0.001) group. Control group was also significantly different from (p 0.05) from NPDR group. Increase in SGA scores was statistically significant amongst the four study groups though other indices of nutritional status showed no definite trend with the increasing severity of retinopathy.Our study shows that serum AGE levels are potential risk markers of diabetic retinopathy and SGA can be used as a regular tool for the assessment of nutritional status of diabetic retinopathy subjects which will help planning a 'low glycemic index-low AGE' therapeutic diet for halting this morbidity.

Impairment of vision is a devastating complication of tuberculous meningitis which may occur as a result of increased intracranial pressure, compression over the visual pathways or vasculitis. We herein present occurrence of neuroretinitis in a 35-year-old lady presenting with low grade fever and headache for one month, and associated with diminution of vision from 3 weeks. She was diagnosed as a case of definite tuberculous meningitis and initiated on anti-tuberculous treatment as per WHO guidelines with supplemental corticosteroids. Marked improvement in vision was observed and at 3 months of follow-up the patient was asymptomatic. Direct ophthalmoscopy, visual field analysis, fluorescein angiography, optical coherence tomography and magnetic resonance imaging of the brain were done to document the ophthalmological findings. Neuroretinitis, being an unusual cause of visual impairment in tuberculous meningitis, must be considered in patients without any evidence of raised intracranial pressure or compression, and with normal fluorescein angiography. We suggest that neuroretinitis may be added to list of causes of visual impairment in patients with tuberculous meningitis.

The most imperative organ, kidney has been widely studied in zebrafish for its simplified structures and development. Understanding the proteomic component of kidney might lead to a better insight for understanding the structural and functional complexity of kidney. In this study we have analyzed the proteome profile of the zebrafish kidney based on gel based proteome mapping techniques involving single dimension gel electrophoresis nanoflow liquid chromatography mass spectrophotometer, single dimension gel electrophoresis microflow ESI liquid chromatography mass spectrophotometer and two dimensional gel electrophoresis matrix assisted laser desorption/ionization assay mass spectrophotometer analysis. A total of 385 proteins were identified consensually from the analysis as zebrafish kidney specific protein which includes 313, 55, and 87 proteins identified based on 1-DE FTMS/ITMSMS, 1-DE ESI-LCMS/MS and 2-DE MALDI MS/MS approaches respectively. The identified kidney proteome dataset was found to be representatives of diverse pI, mass, localization, process and functions. The kidney proteome dataset was found to be significantly associated with various metabolic, catabolic, cytoskeleton remodeling and rectal disease pathways. The engendered kidney protein catalog will serve as a template for understanding kidney functions and biomarker identification related to different kidney disorders.

Geminin is a cellular protein that associates with Cdt1 and inhibits Mcm2-7 loading during S phase. It prevents multiple cycles of replication per cell cycle and prevents episome replication. It also directly inhibits the HoxA11 transcription factor. Here we report that geminin forms a parallel coiled-coil homodimer with atypical residues in the dimer interface. Point mutations that disrupt the dimerization abolish interaction with Cdt1 and inhibition of replication. An array of glutamic acid residues on the coiled-coil domain surface interacts with positive charges in the middle of Cdt1. An adjoining region interacts independently with the N-terminal 100 residues of Cdt1. Both interactions are essential for replication inhibition. The negative residues on the coiled-coil domain and a different part of geminin are also required for interaction with HoxA11. Therefore a rigid cylinder with negative surface charges is a critical component of a bipartite interaction interface between geminin and its cellular targets.

Uveitis is an inflammation of the uveal tract and is one of the major causes of visual impairment. Several lines of evidence suggest an important role for activated T lymphocytes in the perpetuation of posterior uveitis. In sequel to our preliminary observations with human S-antigen, we have further investigated the proliferative response of peripheral blood lymphocytes of posterior uveitis patients against 20 linear and 9 overlapping peptides of retinal S-antigen. The expression of surface markers CD4, CD8, CD29, CD45RA in peripheral blood was detected by flow cytometry. We have also assessed the pattern of cytokines present in peripheral blood mononuclear cells (PBMCs) using ribonuclease protection assay (RPA). Nineteen out of 32 patients' lymphocytes showed proliferative response to S-antigen, one or more of its 20 linear and nine overlapping synthetic peptides. Six patients showed significant lymphoproliferative response against various peptides. The maximum response was found to peptides from the 231–270 amino acid region of human S-antigen sequence. The percentage of CD29 + (memory cells) and CD45RA + (naive cells) T-lymphocytes was higher in patients compared to healthy volunteers. There was a demonstrable difference in the percentage of CD4 + and CD8 + lymphocytes in the patients ( P ≤ 0.05) as compared to controls. Higher message for interleukin (IL)-5, IL-10, IL-15, IL-9, IL-2, IL-13, and interferon (IFN)-γ was observed in uveitis patients than in healthy individuals. In brief, our study suggests that a particular region of S-antigen plays an important role in idiopathic uveitis.

A 21-base pair RNA duplex that perfectly matches an endogenous target mRNA selectively degrades the mRNA and suppresses gene expression in mammalian tissue culture cells. A single base mismatch with the target is believed to protect the mRNA from degradation, making this type of interference highly specific to the targeted gene. A short RNA with mismatches to a target sequence present in multiple copies in the 3′-untranslated region of an exogenously expressed gene can, however, silence it by translational repression. Here we report that a mismatched RNA, targeted to a single site in the coding sequence of an endogenous gene, can efficiently silence gene expression by repressing translation. The antisense strand of such a mismatched RNA requires a 5′-phosphate but not a 3′-hydroxyl group. G·U wobble base pairing is tolerated as a match for both RNA degradation and translation repression. Together, these findings suggest that a small inhibitory RNA duplex can suppress expression of off-target cellular proteins by RNA degradation or translation repression. Proper design of experimental small inhibitory RNAs or a search for targets of endogenous micro-RNAs must therefore take into account that these short RNAs can affect expression of cellular genes with as many as 3–4 base mismatches and additional G·U mismatches.

Uveitis, an intraocular inflammatory disease which affects the uveal tract and the retina of the eye in humans, is one of the major causes of visual impairment. Posterior uveitis is often associated with inflammation of the retina and vitreous. Unfortunately, etiological diagnosis of the disease is not possible in the majority of patients. It is generally felt that an autoimmune mechanism may be involved in so-called idiopathic cases. The role of retinal S-antigen, its 20 linear peptides spanning the entire sequence, and 2 additional peptides, known to be uveitopathogenic in experimental animals, was studied in 26 patients with uveitis. Lymphocyte proliferative response was tested in vitro to identify the epitopes of S-antigen involved and to establish their role in the pathogenesis of uveitis. Of 26 uveitis patients tested, 11 showed a significant T cell proliferative response in vitro to at least 1 antigen used. None among the controls showed any response to the peptides or native S-antigen used in this study. We have found that uveitis patients respond most frequently to peptide 4 (61–80), peptide 5 (81–100), peptide 8 (141–160), peptide 9 (161–180), peptide 12 (221–240), and peptide 13 (241–260) of the human S-antigen. These results further confirm that autoimmunity to retinal S-antigen may play a role in the etiopathogenesis of a subset of patients with idiopathic uveitis.

Arh1p is an essential mitochondrial protein of yeast with reductase activity. Here we show that this protein is involved in iron metabolism. A yeast strain was constructed in which the open reading frame was placed under the control of a galactose-regulated promoter. Protein expression was induced by galactose and repressed to undetectable levels in the absence of galactose, although cells grew quite well in the absence of inducer. Under noninducing conditions, cellular iron uptake was dysregulated, exhibiting a failure to repress in response to medium iron. Iron trafficking within the cell was also disturbed. Exposure of Arh1p-depleted cells to increasing iron concentrations during growth led to drastic increases in mitochondrial iron, indicating a loss of homeostatic control. Activity of aconitase, a prototype Fe-S protein, was deficient at all concentrations of mitochondrial iron, although the protein level was unaltered. Heme protein deficiencies were exacerbated in the iron-loaded mitochondria, suggesting a toxic side effect of accumulated iron. Finally, a time course correlated the cellular depletion of Arh1p with the coordinated appearance of various mutant phenotypes including dysregulated cellular iron uptake, deficiency of Fe-S protein activities in mitochondria and cytoplasm, and deficiency of hemoproteins. Thus, Arh1p is required for control of cellular and mitochondrial iron levels and for the activities of Fe-S cluster proteins.

Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane. These translocases reversibly cooperate during protein import. We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Δ1-pyrroline-5-carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes. Consequently, the two membranes of mitochondria become progressively “zippered” together, forming long stretches in which they are in close contact (Schulke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci. U. S. A.94, 7314–7319). We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70. Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery.

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2–4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.