Bio-effects and safety of low-intensity, low-frequency ultrasonic exposure

Low-frequency (LF) ultrasound (20-100 kHz) has a diverse set of industrial and medical applications. In fact, high power industrial applications of ultrasound mainly occupy this frequency range. This range is also used for various therapeutic medical applications including sonophoresis (ultrasonic transdermal drug delivery), dentistry, eye surgery, body contouring, the breaking of kidney stones and eliminating blood clots. While emerging LF applications such as ultrasonic drug delivery continue to be developed and undergo translation for human use, significant gaps exist in the coverage of safety standards for this frequency range. Accordingly, the need to understand the biological effects of LF ultrasound is becoming more important. This paper presents a broad overview of bio-effects and safety of LF ultrasound as an aid to minimize and control the risk of these effects. Its particular focus is at low intensities where bio-effects are initially observed. To generate a clear perspective of hazards in LF exposure, the mechanisms of bio-effects and the main differences in action at low and high frequencies are investigated and a survey of harmful effects of LF ultrasound at low intensities is presented. Mechanical and thermal indices are widely used in high frequency diagnostic applications as a means of indicating safety of ultrasonic exposure. The direct application of these indices at low frequencies needs careful investigation. In this work, using numerical simulations based on the mathematical and physical rationale behind the indices at high frequencies, it is observed that while thermal index (TI) can be used directly in the LF range, mechanical index (MI) seems to become less reliable at lower frequencies. Accordingly, an improved formulation for the MI is proposed for frequencies below 500 kHz.     

Digestion of restriction enzyme for the detection of single-base mismatch in DNA

DNA hybridization and enzymatic digestion for the detection of mutation was investigated on the gold nanoparticles-calf thymus DNA (AuNPs-ctDNA) modified glassy carbon electrode (GCE). The thiol modified probe oligonucleotides (SH-ssDNA) were assembled on the surface of AuNPs-ctDNA modified GCE. The electrochemical response of the electrode was measured by differential pulse voltammetry and cyclic voltammetry. Methylene blue (MB) was used as the electroactive indicator. AuNPs were then dispersed effectively on the GCE surface in the presence of ct-DNA. When hybridization occurred, a decrease in the signal of MB current was observed. The modified electrode was used for the detection of mutations during the enzymatic digestion reaction in DNA. During this reaction, an increase in the signal of MB current was observed. So, the modified SH-ssDNA had a higher electrochemical response on the AuNPs-ctDNA/GCE because of the strong affinity of MB for guanine residues in it. The electrochemical detection of restriction enzyme digestion can provide a simple and practical method for observing single-base mismatches that can help in distinguishing mismatch sequences of DNA from the complementary ones.     

In Vitro Insulin Release from Thermosensitive Chitosan Hydrogel

Recently, great attention has been paid to in situ gel-forming chitosan/glycerol-phosphate (chitosan/Gp) solution due to their good biodegradability and thermosensitivity. This in situ gel-forming system is injectable fluid that can be introduced into the body in a minimally invasive manner prior to solidifying within the desired tissue. At the present study, insulin release from chitosan/Gp solution has been investigated. Insulin in different concentrations was loaded in two formulations of chitosan/Gp solution and in vitro drug release was studied over a period of 3 weeks. Results indicated that the release of insulin from chitosan/Gp gel decreases by increasing in Gp salt and initial insulin concentration. Stability of released insulin was investigated by 8-anilino-1-naphthalenesulfonate probe. Results proved that insulin have been released in its native form. Because of simple preparation and administration, prolonged release of insulin and stability of released insulin, this in situ gel-forming system could be used as a controlled release delivery system for insulin.KEY WORDS: biodegradable, chitosan, controlled release, in situ forming, insulin     

Exosomes as Biomarker Enriched Microvesicles: Characterization of Exosomal Proteins Derived from a Panel of Prostate Cell Lines with Distinct AR Phenotypes

Prostate cancer is the leading type of cancer diagnosed in men. In 2010, ∼217,730 new cases of prostate cancer were reported in the United States. Prompt diagnosis of the disease can substantially improve its clinical outcome. Improving capability for early detection, as well as developing new therapeutic targets in advanced disease are research priorities that will ultimately lead to better patient survival. Eukaryotic cells secrete proteins via distinct regulated mechanisms which are either ER/Golgi dependent or microvesicle mediated. The release of microvesicles has been shown to provide a novel mechanism for intercellular communication. Exosomes are nanometer sized cup-shaped membrane vesicles which are secreted from normal and cancerous cells. They are present in various biological fluids and are rich in characteristic proteins. Exosomes may thus have potential both in facilitating early diagnosis via less invasive procedures or be candidates for novel therapeutic approaches for castration resistance prostate cancer. Because exosomes have been shown previously to have a role in cell-cell communication in the local tumor microenvironment, conferring activation of numerous survival mechanisms, we characterized constitutive lipids, cholesterol and proteins from exosomes derived from six prostate cell lines and tracked their uptake in both cancerous and benign prostate cell lines respectively. Our comprehensive proteomic and lipidomic analysis of prostate derived exosomes could provide insight for future work on both biomarker and therapeutic targets for the treatment of prostate cancer.Prostate cancer (PCa)¹ is the leading type of cancer diagnosed in men. The American Cancer Society reported 217,730 new cases of PCa in the United States last year. Death from PCa follows its incidence profile closely as the third leading cause of cancer-related death in men (1). In the early stages, the disease is locally confined to the prostate and is hormone or androgen-dependent. It can be managed at this stage by surgical intervention or radiation treatment. However, over time (varying from months to years), many prostate cancers metastasize and, even with aggressive hormone deprivation therapy, progress to castration resistant prostate cancer (CRPC), which ultimately results in death. During early metastasis, a response to androgen deprivation therapy (ADT) is usually observed. Nonetheless, despite the reduction in androgen levels after ADT, androgen receptor (AR) remains active and contributes to CRPC progression (2–4).The routine screening test for PCa diagnosis in North America includes measurement of prostate specific antigen (PSA) in the blood, digital rectal examination and a prostate biopsy (5). PSA screening for PCa detection is controversial because certain activities can induce the production of PSA, unrelated to the presence of cancer (6). Consequently prostate biopsy, albeit an invasive procedure, remains the only definitive diagnostic test for PCa. There is an urgent current need, therefore, for the discovery of relevant biomarkers to replace the existing diagnostic tests for better and earlier detection of PCa (7).One possible source of biomarkers which could be used as part of a diagnostic test are exosomes. All cells produce and release exosomes, which are often found in different body fluids such as plasma (8), serum (9, 10) malignant ascites (11, 12) urine (13), amniotic fluid (14), bronchoalveolar lavage fluid (15, 16), and breast milk (17, 18). Recent studies suggest however that cancer cells produce exosomes, which may be differentiated from those derived from normal cells primarily based upon their cargo. Exosomes are cup-shaped (19) encapsulated by a bi-layer lipid membrane (20) with a membrane-bound compartment varying between 30–100 nm in size (19). As mentioned above, they are secreted from both normal cells and tumor cells (21) and although the underlying mechanism of exosome function is not fully understood it is known that exosomes are formed in the endosomal compartment of cells and are secreted upon fusion of multivesicular bodies (MVB) with the plasma membrane (21). The schematic cartoon in Fig. 1 depicts early endosome (EE) formation as a result of the invagination of specific regions of the plasma membrane. In addition, endocytotic cargo transported out of the cell is sorted from EE into intraluminal vesicles (ILV). Mechanisms involved in protein sorting into ILVs are still under investigation however there is evidence supporting the involvement of ubiquitin and endosomal sorting complex required for transport (ESCRT machinery) in this process. Finally, fusion of late endosome or MVB with plasma membrane releases ILVs into the extracellular matrix or the tissue microenvironment. Accumulating evidence suggests that induction of intracellular calcium (22–25), overexpression of Rab11 or citron kinase (26) as well as a reduction in membrane cholesterol, or inhibition of cholesterol biosynthesis (27), could stimulate the release of exosomes into the microenvironment.Open in a separate windowFig. 1.Mechanism involved in exosome formation and trafficking in the microenvironment.As shown in Fig. 1, once released, exosomes will interact with recipient target cells via different mechanisms such as fusion with the plasma membrane or adhesion to corresponding receptors on the plasma membrane (25).Although, the mechanisms underlying exosome formation and secretion is still under investigation, it is well-known that factors such as cell type, cell cycle, and stage of cancer, could affect the amount and composition of exosomes formed and secreted from various cells (19). It has been shown that exosomes are secreted in a multitude of cell types and though it is postulated that they are involved in membrane trafficking as communication vesicles, their relevance in cancer initiation and specifically prostate tumor growth and progression has yet to be determined (28–30). Studies on tumor-related microvesicles suggest that exosomes play a significant role in cell communication thus potentially influencing cancer progression via different mechanisms (31). Exosomes contain and protect the integrity of various proteins and an array of lipids, mRNA and miRNA which would otherwise be hydrolytically or enzymatically broken down if they existed as free soluble molecules in the extracellular microenvironment. The presence of differential exosomal protein markers involved in cancer progression combined with the presence of exosomes in accessible biological fluids highlights a potential role of exosomes as clinical biomarkers for PCa at diagnosis and progression (32, 33). Therefore isolation, purification and characterization of exosomes derived from different body fluids is an essential first step in identifying novel biomarkers from this source.In addition, exosomes may also present novel therapeutic strategies. If in fact implicated in cancer progression, exosomes present a new target set for development of novel therapeutics. Hence, a better understanding of the mechanisms involved in formation and secretion of exosomes for therapeutic targeting as well as investigating the relevance of the presence of different proteins in these membrane vesicles is required.Therefore the main purpose of the present study was to observe the release of exosomes by prostate cells, and determine characteristic differences between exosomes released by parent cells of different characteristic AR phenotypes. In order to answer this question, in addition to one nonmalignant cell line, we used five different PCa cell lines which contain/lack AR and were representative of different stages of PCa.We then confirmed the transfer of exosomes to target cells in culture using confocal microscopy of fluorescence labeled exosomes. We subsequently performed a comprehensive proteomic analysis of all six different prostate cell lines using mass spectrometry to understand differences between the protein profiles released via exosome externalization in different prostate cell lines. The final part of this study was to investigate the difference in broad classes of lipids and cholesterol as constituents of different prostate cell lines and their exosomes.Taken together the comprehensive characterization of exosomes derived from prostate cell lines which have distinct AR ±ve phenotypes, provides a basis for evaluating transfer of identified composite exosome proteins between different PCa cells as part of a recognized cell communication phenomenon. In addition this study forms a platform for future clinical validation research using exosomes as biomarkers for PCa diagnosis as well as potential therapeutic targets which could be important in the treatment of CRPC.     

Study of DNA-deltamethrin binding by voltammetry, competitive fluorescence, thermal denaturation, circular dichroism, and atomic force microscopy techniques

The interaction of deltamethrin (DM), a synthetic insecticide, with calf thymus DNA was studied. The cyclic voltammetric (CV) results revealed that DM has two irreversible cathodic peaks. The first peak (a) was devoted to reduction of -CN by 4 electrons and the second peak (b) was devoted to reduction of the -C = C- moiety by two electrons. By using non-linear regression analysis of CV data of peak (a), the binding constant, binding site size, and diffusion coefficient for free DM (D(f)) and DNA-DM (D(b)) were calculated as: 2.6 × 10(4), 1.6, 3.2 × 10(-4)Cm(2) S(-1), and 8.5 × 10(-6)Cm(2) S(-1), respectively. The thermal denaturation, competitive fluorescence, and AFM results revealed that the mode of interaction may be non-intercalative. Also the circular dichroism spectra showed that the conformation of CT DNA was converted from right-handed B-DNA to A-DNA due to the destacking of the adjacent guanine bases in pH 7.3 solution.     

Cation-π interaction of alkali metal ions with C24 fullerene: a DFT study

Using first principle calculations, we investigated cation-π interactions between alkali cations (Li(+), Na(+), and K(+)) and pristine C(24) or doped fullerenes of BC(23), and NC(23). The most suitable adsorption site is found to be atop the center of a six-membered ring of the exterior surface of C(24) molecule. Interaction energies of these cations decreased in the order: Li(+)?>?Na(+)?>?K(+), with values of -31.82, -22.36, and -15.68 kcal mol(-1), respectively. It was shown that the interaction energies are increased and decreased by impurity doping of B and N atoms in adjacent wall of adsorption site, depending on electron donating or receptivity of the doping atoms.     

Cardiac glycogen accumulation after dexamethasone is regulated by AMPK

Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (Dex)-treated animals, glycogen accumulation was enhanced. We examined the influence of 5'-AMP-activated protein kinase (AMPK) on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. After Dex, cardiac tissue had a limited contribution toward the development of whole body insulin resistance. Measurement of glucose transporter 4 (GLUT4) at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated with increased GLUT4 mRNA. Both total and phosphorylated AMPK increased after Dex. Immunoprecipitation of Akt substrate of 160 kDa (AS160) followed by Western blot analysis demonstrated no change in Akt phosphorylation at Ser(473) and Thr(308) in Dex-treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr(172), which correlated well with AS160 phosphorylation. In Dex-treated hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas glycogen synthase kinase-3-beta phosphorylation was augmented. Our data suggest that AMPK-mediated glucose entry combined with the activation of glycogen synthase and a reduction in glucose oxidation (Qi et al., Diabetes 53: 1790-1797, 2004) act together to promote glycogen storage. Should these effects persist chronically in the heart, they may explain the increased morbidity and mortality observed with long-term excesses in endogenous or exogenous glucocorticoids.     

Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.