Activation of phosphatidylinositol-3 kinase,AMP-activated kinase and Akt substrate-160 kDa by trans-10, cis-12 conjugated linoleic acid mediates skeletal muscle glucose uptake

Conjugated linoleic acid (CLA), a dietary lipid, has been proposed as an antidiabetic agent. However, studies specifically addressing the molecular dynamics of CLA on skeletal muscle glucose transport and differences between the key isomers are limited. We demonstrate that acute exposure of L6 myotubes to cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA isomers mimics insulin action by stimulating glucose uptake and glucose transporter-4 (GLUT4) trafficking. Both c9,t10-CLA and t10,c12-CLA stimulate the phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160), while showing isomer-specific effects on AMP-activated protein kinase (AMPK). CLA isomers showed synergistic effects with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Blocking PI3-kinase and AMPK prevented the stimulatory effects of t10,c12-CLA on AS160 phosphorylation and glucose uptake, indicating that this isomer acts via a PI3-kinase and AMPK-dependent mechanism, whereas the mechanism of c9,t11-CLA remains unclear. Intriguingly, CLA isomers sensitized insulin-Akt-responsive glucose uptake and prevented high insulin-induced Akt desensitisation. Together, these results establish that CLA exhibits isomer-specific effects on GLUT4 trafficking and the increase in glucose uptake induced by CLA treatment of L6 myotubes occurs via pathways that are distinctive from those utilised by insulin.     

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [¹²⁵I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC₅₀ value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.     

Controls of Soil Spatial Variability in a Dry Tropical Forest

We examined the roles of lithology, topography, vegetation and fire in generating local-scale (<1 km²) soil spatial variability in a seasonally dry tropical forest (SDTF) in southern India. For this, we mapped soil (available nutrients, Al, total C, pH, moisture and texture in the top 10cm), rock outcrops, topography, all native woody plants ≥1 cm diameter at breast height (DBH), and spatial variation in fire frequency (times burnt during the 17 years preceding soil sampling) in a permanent 50-ha plot. Unlike classic catenas, lower elevation soils had lesser moisture, plant-available Ca, Cu, Mn, Mg, Zn, B, clay and total C. The distribution of plant-available Ca, Cu, Mn and Mg appeared to largely be determined by the whole-rock chemical composition differences between amphibolites and hornblende-biotite gneisses. Amphibolites were associated with summit positions, while gneisses dominated lower elevations, an observation that concurs with other studies in the region which suggest that hillslope-scale topography has been shaped by differential weathering of lithologies. Neither NO₃⁻-N nor NH₄⁺-N was explained by the basal area of trees belonging to Fabaceae, a family associated with N-fixing species, and no long-term effects of fire on soil parameters were detected. Local-scale lithological variation is an important first-order control over soil variability at the hillslope scale in this SDTF, by both direct influence on nutrient stocks and indirect influence via control of local relief.     

Predominance of Blastocystis sp. Infection among School Children in Peninsular Malaysia

Background

One of the largest cross-sectional study in recent years was carried out to investigate the prevalence of intestinal parasitic infections among urban and rural school children from five states namely Selangor, Perak, Pahang, Kedah and Johor in Peninsula Malaysia. This information would be vital for school authorities to influence strategies for providing better health especially in terms of reducing intestinal parasitism.

Methods and Principal Findings

A total of 3776 stool cups was distributed to 26 schools throughout the country. 1760 (46.61%) responded. The overall prevalence of intestinal parasitic infection in both rural and urban areas was 13.3%, with Blastocystis sp (10.6%) being the most predominant, followed by Trichuris trichiura (3.4%), Ascaris lumbricoides (1.5%) and hook worm infection (0.9%). Only rural school children had helminthic infection. In general Perak had the highest infection (37.2%, total, n = 317), followed by Selangor (10.4%, total, n = 729), Pahang (8.6%, total, n = 221), Kedah (6.2%, total, n = 195) and Johor (3.4%, total, n = 298). School children from rural schools had higher infection (13.7%, total, n = 922) than urban school children (7.2%, total, n = 838). Subtype (ST) 3 (54.3%) is the most predominant ST with persons infected with only ST1 and ST3 showing symptoms. Blastocystis sp infection significantly associated with low household income, low parent’s education and presence of symptoms (p<0.05).

Conclusion

It is critical that we institute deworming and treatment to eradicate the parasite especially in rural school children.     

Blue excitable green emitting Ce3+ doped CaS phosphor for w‐LEDs

CaS:Ce³⁺ is an efficient green‐emitting (535 nm) phosphor, excitable with blue light (450–470 nm) and was synthesized via a solid‐state reaction method by heating under a reducing atmosphere. The luminescent properties, photoluminescent (PL) excitation and emission of the phosphor were analyzed by spectrofluorophotometry. The excitation and emission peaks of the CaS:Ce³⁺ phosphor lay in the visible region, which made them relevant for light‐emitting diode (LED) application for the generation of white light. Judd‐Oflet parameters were calculated and revealed that green light emitted upon blue illumination. The prepared phosphor had strong blue absorption at 470 nm and a broad green emission band range from 490–590 nm with the peak at 537 nm. The characteristics of the CaS:Ce³⁺ phosphor make it suitable for use as a wavelength tunable green emitting phosphor for three band white LEDs pumped by a blue LED (470 nm). The Commission International de l'Eclairage co‐ordinates were calculated by a spectrophotometric method using the spectral energy distribution (0.304, 0.526) and confirm the green emission. The potential application of this phosphor is as a phosphor‐converted white light‐emitting diode. Copyright © 2015 John Wiley & Sons, Ltd.     

Hyaluronic acid modified pH-sensitive liposomes for targeted intracellular delivery of doxorubicin

Context: Surface-modified pH-sensitive liposomal system may be useful for intracellular delivery of chemotherapeutics.

Objective: Achieving site-specific targeting with over-expressed hyaluronic acid (HA) receptors along with using pH sensitive liposome carrier for intracellular drug delivery was the aim of this study.

Materials and methods: Stealth HA-targeted pH-sensitive liposomes (SL-pH-HA) were developed and evaluated to achieve effective intracellular delivery of doxorubicin (DOX) vis–a-vis enhanced antitumor activity.

Results: The in vitro release studies demonstrated that the release of DOX from SL-pH-HA was pH-dependent, i.e. faster at mildly acidic pH ～5, compared to physiological pH ～7.4. SLpH-HA was evaluated for their cytotoxicity potential on CD44 receptor expressing MCF-7 cells. The half maximal inhibitory concentration (IC50) of SL-pH-HA and SL-HA were about 1.9 and 2.5?μM, respectively, after 48?h of incubation. The quantitative uptake study revealed higher localization of targeted liposomes in the receptor positive cells, which was further confirmed by fluorescent microscopy. The antitumor efficacy of the DOX-loaded HA-targeted pH-sensitive liposomes was also verified in a tumor xenograft mouse model.

Discussion: DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction. The major side-effect of conventional DOX formulation, i.e. cardiotoxicity was also estimated by measuring serum enzyme levels of LDH and CPK and found to be minimized with developed formulation. Overall, HA targeted pH-sensitive liposomes were significantly more potent than the non-targeted liposomes in cells expressing high levels of CD44.

Conclusion: Results strongly implies the promise of such liposomal system as an intracellular drug delivery carrier developed for potential anticancer treatment.     

Altered endocrine,cytokine signaling and oxidative stress: A plausible reason for differential changes in testicular cells in diet-induced and genetically-inherited – obesity in adult rats

Obesity is emerging as a potential risk factor for male infertility. It is a multifactorial disorder with primarily genetic and/or environmental factors. Our earlier studies have shown differential effects of genetically inherited-and high fat diet induced-obesity on hormones, fertility and spermatogenesis in adult male rats. In the present study, we assessed the effect of high fat diet induced – and genetically inherited – obesity on the underlying molecular mechanisms affecting spermatogenesis. The expression of hormone receptors, cytokines and markers of oxidative stress as well as cell cycle mediators were affected in both the obese groups, however, the changes were different in the two groups. This could be due to difference in fat distribution between the two types of obese groups. Altered expression of hormone receptors, cytokines, cell cycle mediators and differential effects on oxidative stress could be the plausible reason for differential changes in germ cell population in both the groups.     

Late‐onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1

Late‐onset retinal degeneration (L‐ORD) is an autosomal dominant macular degeneration characterized by the formation of sub‐retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L‐ORD results from mutations in the C1q‐tumor necrosis factor‐5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L‐ORD pathology, we used a human cDNA library yeast two‐hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM‐Ch) from wild‐type (Wt), heterozygous S163R Ctrp5 mutation knock‐in (Ctrp5^S163R/wt), and homozygous knock‐in (Ctrp5^S163R/S163R) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C‐terminal PDZ‐binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R‐CTRP5 protein also binds to HTRA1 but is resistant to HTRA1‐mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM‐Ch of Ctrp5^S163R/S163R and Ctrp5^S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L‐ORD pathology.