Potential of Capric Acid in Neurological Disorders: An Overview.

To solve the restrictions of a classical ketogenic diet, a modified medium-chain triglyceride diet was introduced which required only around 60% of dietary energy. Capric acid (CA), a small molecule, is one of the main components because its metabolic profile offers itself as an alternate source of energy to the brain in the form of ketone bodies. This is possible with the combined capability of CA to cross the blood-brain barrier and achieve a concentration of 50% concentration in the brain more than any other fatty acid in plasma. Natural sources of CA include vegetable oils such as palm oil and coconut oil, mammalian milk and some seeds. Several studies have shown that CA has varied action on targets that include AMPA receptors, PPAR-γ, inflammatory/oxidative stress pathways and gut dysbiosis. Based on these lines of evidence, CA has proved to be effective in the amelioration of neurological diseases such as epilepsy, affective disorders and Alzheimer's disease. But these studies still warrant more pre-clinical and clinical studies that would further prove its efficacy. Hence, to understand the potential of CA in brain disease and associated comorbid conditions, an advance and rigorous molecular mechanistic study, apart from the reported in-vitro/in-vivo studies, is urgently required for the development of this compound through clinical setups.

Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL.

BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.

Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications.

Mesenchymal stem (stromal) cells (MSCs) are multipotent cells with the ability to differentiate into several cell types, thus serving as a cell reservoir for regenerative medicine. Much of the current interest in therapeutic application of MSCs to various disease settings can be linked to their immunosuppressive and anti-inflammatory properties. One of the key mechanisms of MSC anti-inflammatory effects is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA, and proteins from their cells of origin. Recent animal model-based studies suggest that EVs have significant potential as a novel alternative to whole cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function. In this article, we review current knowledge related to the potential use of MSC-derived EVs in various diseases and discuss the promising future for EVs as an alternative, cell-free therapy.

miR-630 targets IGF1R to regulate response to HER-targeting drugs and overall cancer cell progression in HER2 over-expressing breast cancer.

BACKGROUND: While the treatment of HER2 over-expressing breast cancer with recent HER-targeted drugs has been highly effective for some patients, primary (also known as innate) or acquired resistance limits the success of these drugs. microRNAs have potential as diagnostic, prognostic and predictive biomarkers, as well as replacement therapies. Here we investigated the role of microRNA-630 (miR-630) in breast cancer progression and as a predictive biomarker for response to HER-targeting drugs, ultimately yielding potential as a therapeutic approach to add value to these drugs. METHODS: We investigated the levels of intra- and extracellular miR-630 in cells and conditioned media from breast cancer cell lines with either innate- or acquired- resistance to HER-targeting lapatinib and neratinib, compared to their corresponding drug sensitive cell lines, using qPCR. To support the role of miR-630 in breast cancer, we examined the clinical relevance of this miRNA in breast cancer tumours versus matched peritumours. Transfection of miR-630 mimics and inhibitors was used to manipulate the expression of miR-630 to assess effects on response to HER-targeting drugs (lapatinib, neratinib and afatinib). Other phenotypic changes associated with cellular aggressiveness were evaluated by motility, invasion and anoikis assays. TargetScan prediction software, qPCR, immunoblotting and ELISAs, were used to assess miR-630's regulation of mRNA, proteins and their phosphorylated forms. RESULTS: We established that introducing miR-630 into cells with innate- or acquired- resistance to HER-drugs significantly restored the efficacy of lapatinib, neratinib and afatinib; through a mechanism which we have determined to, at least partly, involve miR-630's regulation of IGF1R. Conversely, we demonstrated that blocking miR-630 induced resistance/insensitivity to these drugs. Cellular motility, invasion, and anoikis were also observed as significantly altered by miR-630 manipulation, whereby introducing miR-630 into cells reduced cellular aggression while inhibition of miR-630 induced a more aggressive cellular phenotype. CONCLUSIONS: Taken together, our findings suggest miR-630 as a key regulator of cancer cell progression in HER2 over-expressing breast cancer, through targeting of IGF1R. This study supports miR-630 as a diagnostic and a predictive biomarker for response to HER-targeted drugs and indicates that the therapeutic addition of miR-630 may enhance and improve patients' response to HER-targeting drugs.

miR-34a is an intracellular and exosomal predictive biomarker for response to docetaxel with clinical relevance to prostate cancer progression.

BACKGROUND: Docetaxel-resistance limits successful treatment of castration resistant prostate cancer. We previously demonstrated that extracellular vesicles (exosomes) may play a role in regulating docetaxel resistance. Here, we investigated intracellular and extracellular (exosomal) miRNAs related to docetaxel resistance. METHODS: Following global miRNA profiling of cell line models of docetaxel-resistance and their corresponding exosomes, we investigated the clinical relevance of four selected miRNAs (miR-598, miR-34a, miR-146a, miR-148a) in four publically available clinical cohorts representing both primary and advanced disease in tissue and urine specimens. One of these miRNAs, miR-34a was selected for functional evaluation by miRNA inhibition and over-expression in vitro. We further assessed the panel of miRNAs for their combined clinical relevance as a biomarker signature by examining their common predicted targets. RESULTS: A strong correlation was found between the detection of miRNAs in exosomes and their corresponding cells of origin. Of the miRNAs chosen for further validation and clinical assessment, decreased miR-34a levels showed substantial clinical relevance and so was chosen for further analysis. Manipulating miR-34a in prostate cancer cells confirms that this miRNA regulates BCL-2 and may, in part, regulate response to docetaxel. When combined, these miRNAs are predicted to regulate a range of common mRNA targets, two of which (e.g., SNCA, SCL7A5) demonstrate a strong relationship with prostate cancer progression and poor prognosis. CONCLUSIONS: This study supports the extracellular environment as an important source of minimally invasive predictive biomarkers representing their cellular origin. Using miR-34a as example, we showed that biomarkers identified in this manner may also hold functional relevance.

miRNAs as biomarkers of therapeutic response to HER2-targeted treatment in breast cancer: A systematic review.

Breast cancer is the most common type of lethal cancer in women globally. Women have a 1 in 8 chance of developing breast cancer in their lifetime. Among the four primary molecular subtypes (luminal A, luminal B, HER2+, and triple-negative), HER2+ accounts for 20-25 % of all breast cancer and is rather aggressive. Although the treatment outcome of HER2+ breast cancer patients has been significantly improved with anti-HER2 agents, primary and acquired drug resistance present substantial clinical issues, limiting the benefits of HER2-targeted treatment. MicroRNAs (miRNAs) play a central role in regulating acquired drug resistance. miRNA are single-stranded, non-coding RNAs of around 20-25 nucleotides, known for essential roles in regulating gene expression at the post-transcriptional level. Increasing evidence has demonstrated that miRNA-mediated alteration of gene expression is associated with tumorigenesis, metastasis, and tumor response to treatment. Comprehensive knowledge of miRNAs as potential markers of drug response can help provide valuable guidance for treatment prognosis and personalized medicine for breast cancer patients.

miRNA Biogenesis and Regulation of Diseases: An Updated Overview.

MicroRNAs (miRNAs) are small RNA molecules, with their role in gene silencing and translational repression by binding to the target mRNAs. Since it was discovered in 1993, miRNA is found in all eukaryotic cells conserved across the species. miRNA-size molecules are also known to be found in prokaryotes. Regulation of miRNAs is extensively studied for their role in biological processes as well as in development and progression of various human diseases including neurodegenerative diseases, cardiovascular disease, and cancer. miRNA-based therapy has a promising application, and with a good delivery system, miRNA therapeutics can potentially be a success. miRNAs and EVs have potential therapeutic and prognostic application in a range of disease models. This chapter summarizes miRNA biogenesis and explores their potential roles in a variety of diseases. miRNAs hold huge potential for diagnostic and prognostic biomarkers and as predictors of drug response.

Exosomal MicroRNA Discovery in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a common condition causing progressive visual impairment, leading to irreversible blindness. Existing diagnostic tools for AMD are limited to clinical signs of drusen deposition in the macula and the visual assessment of the patient. The presence of circulating microRNAs (miRNAs) in the peripheral circulatory system with potential as diagnostic, prognostic, and/or predictive biomarkers has been reported in a number of conditions/diseases. miRNAs are key regulators of several biological processes, and miRNA dysregulation has been linked with numerous diseases, most remarkably cancer. miRNAs have been shown to be involved in AMD pathology, and several miRNA target genes and signalling pathways were associated with AMD pathogenesis. Exosomes are 50-90 nm membrane microvesicles (MVs), released by several cell types. Although exosomal functions are not completely understood, there is much evidence to suggest that exosomes play an essential role in cell-cell communication. They may stimulate target cells by transferring different bioactive molecules such as miRNA. Here we discuss methods to isolate exosome using serum specimens from AMD patients and miRNA profiling for the better understanding of the disease.

Serum MicroRNAs Profiling in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a progressive, degenerative disease of the retina which ultimately results in the irreversible loss of central vision. AMD is one of the foremost causes of blindness in people over the age of 50. Although the precise pathogenesis of AMD has not yet been elucidated, AMD results from a complex interaction between genetic predisposition and environmental provoking factors. These factors might lead to ocular homeostasis dysfunction resulting in inflammation, oxidative stress, and in some cases neovascularization. MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs and are approximately 22 nucleotides long. miRNAs play a central role in several pathophysiological processes such as immune and inflammatory responses, pathological angiogenesis, and the response to oxidative stress, all of which have been suggested to be associated with AMD pathogenesis and progression. Here we discuss methods to isolate miRNAs using serum specimens from AMD patients and miRNA profiling for the better understanding of the pathogenesis and progression of AMD.

Two decades of two-photon lithography: Materials science perspective for additive manufacturing of 2D/3D nano-microstructures.

Two-photon lithography (TPL) is a versatile technology for additive manufacturing of 2D and 3D micro/nanostructures with sub-wavelength resolved features. Recent advancement in laser technology has enabled the application of TPL fabricated structures in several fields such as microelectronics, photonics, optoelectronics, microfluidics, and plasmonic devices. However, the lack of two-photon polymerizable resins (TPPRs) induces bottleneck to the growth of TPL to its true potential, and hence continuous research efforts are focused on developing efficient TPPRs. In this article, we review the recent advancements in PI and TPPR formulation and the impact of process parameters on fabrication of 2D and 3D structures for specific applications. The fundamentals of TPL are described, followed by techniques used for achieving improved resolution and functional micro/nanostructures. Finally, a critical outlook and future prospects of TPPR formulation for specific applications are presented.

Immune System, Inflammation and Autoantigens in Wet Age-Related Macular Degeneration: Pathological Significance and Therapeutic Importance.

Wet age-related macular degeneration (wAMD) is a chronic inflammation-associated neurodegenerative disease affecting the posterior part of the eye in the aging population. Aging results in the reduced functionality of cells and tissues, including the cells of the retina. Initiators of a chronic inflammatory and pathologic state in wAMD may be a result of the accumulation of inevitable metabolic injuries associated with the maintenance of tissue homeostasis from a young age to over 50. Apart from this, risk factors like smoking, genetic predisposition, and failure to repair the injuries that occur, alongside attempts to rescue the hypoxic outer retina may also contribute to the pathogenesis. Aging of the immune system (immunosenescence) and a compromised outer blood retinal barrier (BRB) result in the exposure of the privileged milieu of the retina to the systemic immune system, further increasing the severity of the disease. When immune-privileged sites like the retina are under pathological stress, certain age- and disease-related conditions may necessitate assistance from cells distant from the resident ones to help restore the functionality of the tissue. As a necessary part of tissue repair, inflammation is a major response to disease and recruits immune cells to the site of damage. We suspect that the specific reparative inflammatory responses are controlled by an autoantigen-T cell-mediated mechanism, a process that may be hindered in wAMD.

The use of LC-MS to identify differentially expressed proteins in docetaxel-resistant prostate cancer cell lines.

Docetaxel is a taxane-derived chemotherapy drug that has been approved for treatment of prostate cancer. While docetaxel is frequently used as a treatment for hormone-refractory prostate cancer, a subset of patients either do not respond to this treatment or those that do respond eventually become resistant to the drug over time. Resistance to docetaxel is complex and multi-factoral and further understanding of the cellular biochemistry underlying resistance is vital to improve treatment efficacy. To identify proteins altered in the resistant phenotype, three parental cell lines DU145, 22RV1 and PC-3, as well as their docetaxel resistant sub-lines, were subjected to quantitative label-free LC-MS proteomic profiling. A total of 189 significant (p < 0.05) protein abundance changes were identified in the DU145 resistant sub-lines, 254 in the 22RV1 sub-lines, and 51 and 72 in the 8 and 12 nM resistant PC-3 sub-lines, respectively. From these, 29 proteins demonstrated a significant (p < 0.05) fold change across two or more resistant variants. These included proteins indicative of an epithelial-to-mesenchemyl transition as well as altered heat shock response elements.

MAGE-D4B is a novel marker of poor prognosis and potential therapeutic target involved in breast cancer tumorigenesis.

Melanoma-associated antigen (MAGE) family members are generally described as tumor-specific antigens. An association between MAGE-D4B and breast cancer has yet to be reported and the functional role of the encoded protein has never been established. We performed microarray analysis of 104 invasive breast tumors and matched non-cancerous breast biopsies. qPCR was used for validation in an independent biobank. To investigate the biological relevance of MAGE-D4B in breast tumorigenesis, its phenotypic effects were assessed in vitro. Overall, MAGE-D4B was detected in 43% of tumors while undetected in normal breast tissue. MAGE-D4B was found to correlate with tumor progression and to be an independent prognostic marker for poor outcome in term of relapse-free and overall survival, with potential predictive relevance in relation to response to chemotherapy. RNA interference-mediated knockdown of MAGE-D4B significantly hampered the invasive properties of Hs578T cells by affecting anchorage-independent growth, adhesion, migration and invasion affecting anchorage-independent growth, adhesion, migration and invasion and by modulating expression of invasion-suppressor gene E-cadherin.

Characterisation and manipulation of docetaxel resistant prostate cancer cell lines.

BACKGROUND: There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. RESULTS: The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. CONCLUSION: This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

The Role of Deregulated MicroRNAs in Age-Related Macular Degeneration Pathology.

Purpose: We previously identified three microRNAs (miRNAs) with significantly increased expression in the serum of patients with age-related macular degeneration (AMD) compared with healthy controls. Our objective was to identify potential functional roles of these upregulated miRNAs (miR-19a, miR-126, and miR-410) in AMD, using computational tools for miRNAs prediction and identification, and to demonstrate the miRNAs target genes and signaling pathways. We also aim to demonstrate the pathologic role of isolated sera-derived exosomes from patients with AMD and controls using in vitro models. Methods: miR-19a, miR-126, and miR-410 were investigated using bioinformatic approaches, including DIANA-mirPath and miR TarBase. Data on the resulting target genes and signaling pathways were incorporated with the differentially expressed miRNAs in AMD. Apoptosis markers, human apoptosis miRNAs polymerase chain reaction arrays and angiogenesis/vasculogenesis assays were performed by adding serum-isolated AMD patient or control patient derived exosomes into an in vitro human angiogenesis model and ARPE-19 cell lines. Results: A number of pathways known to be involved in AMD development and progression were predicted, including the vascular endothelial growth factor signaling, apoptosis, and neurodegenerative pathways. The study also provides supporting evidence for the involvement of serum-isolated AMD-derived exosomes in the pathology of AMD, via apoptosis and/or angiogenesis. Conclusions: miR-19a, miR-126, miR-410 and their target genes had a significant correlation with AMD pathogenesis. As such, they could be potential new targets as predictive biomarkers or therapies for patients with AMD. Translational Relevance: The functional analysis and the pathologic role of altered miRNA expression in AMD may be applicable in developing new therapies for AMD through the disruption of individual or multiple pathophysiologic pathways.

Additive-Free All-Carbon Composite: A Two-Photon Material System for Nanopatterning of Fluorescent Sub-Wavelength Structures.

The major bottleneck in fabrication of engineered 3D nanostructures is the choice of materials. Adding functionality to these nanostructures is a daunting task. In order to mitigate these issues, we report a two-photon patternable all carbon material system which can be used to fabricate fluorescent 3D micro/nanostructures using two-photon lithography, with subwavelength resolution. The synthesized material system eliminates the need to use conventional two-photon absorbing materials such as two-photon dyes or two-photon initiators. We have used two different trifunctional acrylate monomers and carbon dots, synthesized hydrothermally from a polyphenolic precursor, to formulate a two-photon processable resin. Upon two-photon excitation, photogenerated electrons in the excited states of the carbon dots facilitate the free radical formation at the surface of the carbon dots. These radicals, upon interaction with vinyl moieties, enable cross-linking of acrylate monomers. Free-radical induced two-photon polymerization of acrylate monomers without any conventional proprietary two-photon absorbing materials was accomplished at an ultrafine subwavelength resolution of 250 nm using 800 nm laser excitation. The effect of critical parameters such as average laser power, carbon dot concentration, and radiation exposure were determined for the fabrication of one-, two-, and three-dimensional functional nanostructures, applicable in a range of domains where fluorescence and toxicity are of the utmost importance. A fabrication speed as high as 100 mm/s was achieved. The ability to fabricate functional 3D micro-/nanostructures is anticipated to instigate a paradigm shift in various areas such as metamaterials, energy storage, drug delivery, and optoelectronics to name a few.

Chitosan-Coated PLGA Nanoparticles Encapsulating Triamcinolone Acetonide as a Potential Candidate for Sustained Ocular Drug Delivery.

The current treatment for the acquired retinal vasculopathies involves lifelong repeated intravitreal injections of either anti-vascular endothelial growth factor (VEGF) therapy or modulation of inflammation with steroids. Consequently, any treatment modification that decreases this treatment burden for patients and doctors alike would be a welcome intervention. To that end, this research aims to develop a topically applied nanoparticulate system encapsulating a corticosteroid for extended drug release. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) supports the controlled release of the encapsulated drug, while surface modification of these NPs with chitosan might prolong the mucoadhesion ability leading to improved bioavailability of the drug. Triamcinolone acetonide (TA)-loaded chitosan-coated PLGA NPs were fabricated using the oil-in-water emulsion technique. The optimized surface-modified NPs obtained using Box-Behnken response surface statistical design were reproducible with a particle diameter of 334 ± 67.95 to 386 ± 15.14 nm and PDI between 0.09 and 0.15. These NPs encapsulated 55-57% of TA and displayed a controlled release of the drug reaching a plateau in 27 h. Fourier-transform infrared spectroscopic (FTIR) analysis demonstrated characteristic peaks for chitosan (C-H, CONH2 and C-O at 2935, 1631 and 1087 cm-1, respectively) in chitosan-coated PLGA NPs. This result data, coupled with positive zeta potential values (ranged between +26 and +33 mV), suggests the successful coating of chitosan onto PLGA NPs. Upon coating of the NPs, the thermal stability of the drug, polymer, surfactant and PLGA NPs have been enhanced. The characteristics of the surface-modified NPs supports their use as potential candidates for topical ocular drug delivery for acquired retinal vasculopathies.

Dexamethasone-Loaded Nanostructured Lipid Carriers for the Treatment of Dry Eye Disease.

Dry eye disease (DED) or keratoconjunctivitis sicca is a chronic multifactorial disorder of the ocular surface caused by tear film dysfunction. Symptoms include dryness, irritation, discomfort and visual disturbance, and standard treatment includes the use of lubricants and topical steroids. Secondary inflammation plays a prominent role in the development and propagation of this debilitating condition. To address this we have investigated the pilot scale development of an innovative drug delivery system using a dexamethasone-encapsulated cholesterol-Labrafac™ lipophile nanostructured lipid carrier (NLC)-based ophthalmic formulation, which could be developed as an eye drop to treat DED and any associated acute exacerbations. After rapid screening of a range of laboratory scale pre-formulations, the chosen formulation was prepared at pilot scale with a particle size of 19.51 ± 0.5 nm, an encapsulation efficiency of 99.6 ± 0.5%, a PDI of 0.08, and an extended stability of 6 months at 4 °C. This potential ophthalmic formulation was observed to have high tolerability and internalization capacity for human corneal epithelial cells, with similar behavior demonstrated on ex vivo porcine cornea studies, suggesting suitable distribution on the ocular surface. Further, ELISA was used to study the impact of the pilot scale formulation on a range of inflammatory biomarkers. The most successful dexamethasone-loaded NLC showed a 5-fold reduction of TNF-α production over dexamethasone solution alone, with comparable results for MMP-9 and IL-6. The ease of formulation, scalability, performance and biomarker assays suggest that this NLC formulation could be a viable option for the topical treatment of DED.

Decreasing Txnip mRNA and protein levels in pancreatic MIN6 cells reduces reactive oxygen species and restores glucose regulated insulin secretion.

BACKGROUND AND AIMS: Recently, thioredoxin-interacting protein (Txnip) expression has been implicated in a number of cellular events associated with diabetes, with increased Txnip levels associated with reduced glucose uptake into peripheral tissues, increased reactive oxygen species (ROS) in endothelial cells, beta cell glucotoxicity and apoptosis. The potential relevance of Txnip with regards to glucose-regulated insulin secretion (GSIS), a fundamentally important characteristic of beta cells and insulin-producing cells being considered as a possible cell therapy for diabetes, has not yet been investigated. METHODS: Here, studying glucose-responsive MIN6 B1(GSIS) and cells which had significantly reduced response to glucose after time in culture i.e. MIN6 B1(Non-GSIS), using ELISAs; qRT-PCR; immunoprecipitation and Western blotting; transient and stable (siRNA/shRNA and cDNA) approaches to achieve Txnip knock-down or over-expression, respectively,we established a direct association between Txnip expression and GSIS. RESULTS: Specifically, increasing Txnip levels correlate with increased intracellular ROS levels and with significant GSIS loss.Conversely, both transient and stable knock-down of Txnip expression was associated with GSIS recovery. CONCLUSION: This, we believe, is another reason in favour of targeting Txnip as a novel approach for diabetes-related therapy.

Identification of Novel Serum MicroRNAs in Age-Related Macular Degeneration.

Purpose: To identify circulating microRNAs (miRNA) associated with age-related macular degeneration (AMD). Thus differentially expressed serum miRNA could be used as AMD biomarkers. Methods: This study involved total RNA isolation from sera from patients with atrophic AMD (n = 10), neovascular AMD (n = 10), and age- and sex-matched controls (n = 10). A total of 377 miRNAs were coanalyzed using array technologies, and differentially regulated miRNAs were determined. Extensive validation studies (n = 90) of serum from AMD patients and controls confirmed initial results. Total RNA isolation was carried out from sera from patients with atrophic AMD (n = 30), neovascular AMD (n = 30), and controls (n = 30). Fourteen miRNAs from the discovery dataset were coanalyzed using quantitative real-time polymerase chain reaction (qRT-PCR) to validate their presence. Results: Unsupervised hierarchical clustering indicated that AMD serum specimens have a different miRNA profile to healthy controls. We successfully identified and validated the differentially regulated miRNAs in serum from AMD patients versus controls. The biomarker potential of three miRNAs (miR-126, miR-19a, and miR-410) was confirmed by qRT-PCR, with significantly increased quantities in serum of AMD patients compared with healthy controls. Conclusions: Increased quantities of miR-126, miR-410, and miR-19a in serum from AMD patients indicate that these miRNAs could potentially serve as diagnostic AMD biomarkers. All three miRNAs significantly correlated with AMD pathogenesis. Translational Relevance: The discovery of new AMD miRNA may act as biomarkers in evaluating AMD diagnosis and prognosis.