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A complete surgical excision with negative tumor margins is the single most important factor in the prediction of long-term survival for most cancer patients with solid tumors. We hypothesized that image-guided surgery using nanoparticle-enhanced photoacoustic and fluorescence imaging could significantly reduce the rate of local recurrence.A murine model of invasive mammary carcinoma was utilized. Three experimental groups were included: (1) control; (2) tumor-bearing mice injected with non-targeted nanoprobe; and (3) tumor-bearing mice injected with targeted nanoprobe. The surgeon removed the primary tumor following the guidance of photoacoustic imaging (PAI), then inspected the surgical wound and removed the suspicious tissue using intraoperative near-infrared (NIR) fluorescence imaging. The mice were followed with bioluminescence imaging weekly to quantify local recurrence.Nanoprobe-enhanced photoacoustic contrast enabled PAI to map the volumetric tumor margins up to a depth of 31 mm. The targeted nanoparticles provided significantly greater enhancement than non-targeted nanoparticles. Seven mice in the group injected with the targeted nanoprobes underwent additional resections based upon NIR fluorescence imaging. Pathological analysis confirmed residual cancer cells in the re-resected specimens in 5/7 mice. Image-guided resection resulted in a significant reduction in local recurrence; 8.7 and 33.3 % of the mice in the targeted and control groups suffered recurrence, respectively.These results suggest that photoacoustic and NIR intraoperative imaging can effectively assist a surgeon to locate primary tumors and to identify residual disease in real-time. This technology has promise to overcome current clinical challenges that result in the need for second surgical procedures.

In this report, we present a breast imaging technique combining high-resolution near-infrared (NIR) light induced photoacoustic tomography (PAT) with NIR dye-labeled amino-terminal fragments of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (NIR830-ATF-IONP) for breast cancer imaging using an orthotopic mouse mammary tumor model. We show that accumulation of the targeted nanoparticles in the tumor led to photoacoustic contrast enhancement due to the high absorption of iron oxide nanoparticles (IONP). NIR fluorescence images were used to validate specific delivery of NIR830-ATF-IONP to mouse mammary tumors. We found that systemic delivery of the targeted IONP produced 4- and 10-fold enhancement in photoacoustic signals in the tumor, compared to the tumor of the mice that received non-targeted IONP or control mice. The use of targeted nanoparticles allowed imaging of tumors located as deep as 3.1 cm beneath the normal tissues. Our study indicates the potential of the combination of photoacoustic tomography and receptor-targeted NIR830-ATF-IONP as a clinical tool that can provide improved specificity and sensitivity for breast cancer detection.

Clinical applications of the indocyanine green (ICG) dye, the only near infrared (NIR) imaging dye approved by the Food and Drug Administration (FDA) in the USA, are limited due to rapid protein binding, fast clearance, and instability in physiologically relevant conditions. Encapsulating ICG in silica particles can enhance its photostability, minimize photobleaching, increase the signal-to-noise (S/N) ratio and enable in vivo studies. Furthermore, a combined magnetic resonance (MR) and NIR imaging particulate can integrate the advantage of high-resolution 3D anatomical imaging with high-sensitivity deep-tissue in-vivo fluorescent imaging. In this report, a novel synthesis technique that can achieve these goals is presented. A reverse-microemulsion-based synthesis protocol is employed to produce 25 nm ICG-doped silica nanoparticles (NPs). The encapsulation of ICG is achieved by manipulating coulombic attractions with bivalent ions and aminated silanes and carrying out silica synthesis in salt-catalyzed, mildly basic pH conditions using dioctyl sulfosuccinate (AOT)/heptane/water microemulsion system. Furthermore, paramagnetic properties are imparted by chelating paramagnetic Gd to the ICG-doped silica NPs. Aqueous ICG-dye-doped silica NPs show increased photostability (over a week) and minimal photobleaching as compared to the dye alone. The MR and optical imaging capabilities of these particles are demonstrated through phantom, in vitro and in vivo experiments. The described particles have the potential to act as theranostic agents by combining photodynamic therapy through the absorption of NIR irradiated light.

Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a β-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, that form protein conjugates in the archaeon Haloferax volcanii. The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the e-amino group of lysines from a number of protein targets and Lys 58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage. Ubiquitination is an important protein modification process in eukaryotes, but its role in prokaryotes is less certain. Now structural homologues of ubiquitin (small archaeal modifier proteins or SAMPs) are shown to form a large number of protein conjugates in the archaeon Haloferax volcanii. Although Archaea encode proteasomes highly related to those of eukaryotes, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation, complicating our understanding of the origins of ubiquitination. Two small archaeal modifier proteins, SAMP1 and SAMP2, structurally similar to ubiquitin, are now reported to form protein conjugates in the archaeon Haloferax volcanii.

Candida albicans is the most commonly isolated fungus associated with biofilms, which are extracellular ma- trix (ECM)-encased, drug-resistant microbial communities formed on indwelling medical devices. Protein profiles of fun- gal biofilms have not been investigated in detail, although such profiles are believed to play critical roles in fungal biofilm formation. In this study, we used two-dimensional difference-in-gel electrophoresis (DIGE)-based proteomics to identify differentially expressed proteins in C. albicans biofilms grown to early and mature phases, compared to planktonic cells. The resulting proteomic data set was subjected to pathway mapping to reveal phase-specific pathways that were differen- tially expressed in biofilm cell walls and extracellular matrix (ECM). Our analyses showed 107 proteins to be differen- tially expressed in ECM, while 44 were differentially expressed in cell walls during biofilm formation, compared to planktonic controls. Furthermore, 95% (102/107) and 68% (30/44) of these differentially expressed proteins were upregu- lated in ECM and cell walls of biofilms, respectively. These proteins were mapped to cellular pathways, which revealed that these differentially expressed proteins were associated with several metabolic pathways, in a phase-dependent man- ner. For example, among ECM-associated proteins, proteins within 18 pathways were differentially expressed, with two pathways (glutamate and nitrogen metabolism) unique to early phase, and four pathways (purine, Gly/Ser/Thr, and inosi- tol metabolism, and carbon fixation) unique to mature phase biofilms. Such differences were also observed in cell wall- associated proteins, where proteins associated with 14 specific pathways were differentially regulated. We also found gly- colytic enzymes including the key enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were overexpressed in biofilms at both early and mature phases, compared to planktonic controls. Iodoacetate-mediated inhibition of this enzyme completely abrogated the ability of C. albicans to form biofilms, indicating the role of glycolysis/gluconeogenesis path- ways in biofilm formation. Taken together, we demonstrate that ECM and cell walls of C. albicans biofilms express in- creased levels of specific proteins within pathways in a phase-dependent manner, suggesting that these pathways, espe- cially glycolysis/gluconeogenesis, play critical roles in fungal biofilm formation and maintenance.

Device-related infections in most nosocomial diseases can be traced to the formation of biofilms (microbial communities encased within a polysaccharide-rich extracellular matrix) by pathogens on surfaces of these devices. Candida species are the most common causative agents of these infections, and biofilms formed by these fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents. This enhanced resistance contributes to the persistence of this fungus despite antifungal therapy. Recent studies showed that Candida biofilms exhibit antifungal resistance against most antifungal agents with the exception of echinocandins and lipid formulations of AMB. This review discusses methods used to evaluate biofilm resistance and provide information on susceptibility pattern of candidal biofilm as well as studies investigating the mechanisms underlying biofilm resistance.

Candida albicans biofilms form on indwelling medical devices (e.g., denture acrylic or intravenous catheters) and are associated with both oral and invasive candidiasis. Here, we determined whether surface modifications of polyetherurethane (Elasthane 80A [E80A]), polycarbonateurethane, and poly(ethyleneterephthalate) (PET) can influence fungal biofilm formation. Polyurethanes were modified by adding 6% polyethylene oxide (6PEO), 6% fluorocarbon, or silicone, while the PET surface was modified to generate hydrophilic, hydrophobic, cationic, or anionic surfaces. Formation of biofilm was quantified by determining metabolic activity and total biomass (dry weight), while its architecture was analyzed by confocal scanning laser microscopy (CSLM). The metabolic activity of biofilm formed by C. albicans on 6PEO-E80A was significantly reduced (by 78%) compared to that of biofilm formed on the nonmodified E80A (optical densities of 0.054 ± 0.020 and 0.24 ± 0.10, respectively; P = 0.037). The total biomass of Candida biofilm formed on 6PEO-E80A was 74% lower than that on the nonmodified E80A surface (0.46 ± 0.15 versus 1.76 ± 0.32 mg, respectively; P = 0.003). Fungal cells were easily detached from the 6PEO-E80A surface, and we were unable to detect C. albicans biofilm on this surface by CSLM. All other surface modifications allowed formation of C. albicans biofilm, with some differences in thearchitecture. Correlation between contact angle and biofilm formation was observed for polyetherurethane substrates ( r = 0.88) but not for PET biomaterials ( r = −0.40). This study illustrates that surface modification is a viable approach for identifying surfaces that have antibiofilm characteristics. Investigations into the clinical utility of the identified surfaces are warranted.

Biofilms are colonies of microbial cells encased in a self-produced organic polymeric matrix and represent a common mode of microbial growth. Microbes growing as biofilm are highly resistant to commonly used antimicrobial drugs. Recently, microbial biofilms have gained prominence because of the increase in infections related to indwelling medical devices (IMD). Candida albicans, the pathogenic fungus which is a major cause of morbidity and mortality in blood stream infections, is the most common fungal pathogen isolated from patients with IMD-associated infections. Biofilm formation by Candida species is believed to contribute to invasiveness of these fungal species. We discuss experimental methods used to study fungal biofilms as well as the biology of biofilm formation by clinically relevant Candida species. Recent advances that are discussed in this review include the role of specific, differentially expressed genes and proteins, quorum sensing molecule in C. albicans biofilms, and the correlation between biofilm formation and fungal pathogenesis.

Adriamycin (ADR) is a powerful and widely used antitumor drug, but its dose dependent cardiotoxicity limits its application. This side effect is believed to be caused by the adriamycin semiquinone free radical (ASFR). The primary focus of this work is to test effects of sodium tanshinone IIA sulfonate (STS) on ASFR and adriamycin–induced lipid peroxidation. It was found that ADR, whether in the system of heart homogenate, heart mitochondria or heart submitochondria, with NADH as the substrate or in xanthine/xanthine oxidase under anaerobic conditions, all produced ASFR rapidly. STS was shown to effectively scavenge ASFR in all these systems and postpone the appearance of ASFR. The delayed time was proportional to the amount of STS. Under aerobic conditions, ASFR could be oxidized to generate oxygen free radicals. STS could not scavenge these oxygen free radicals, but it could effectively scavenge lipid free radicals generated from membrane lipid peroxidation of heart mitochondria. STS could significantly reduce mitochondrial swelling and lipid peroxidation induced by ADR. Animal experiments show that treatment of STS could inhibit endogenous lipid peroxidation caused by ADR. Here, a protective mechanism of STS is suggested that STS can rapidly and univalently oxidize ASFR, causing the cycle of adriamycin between its quinone form and semiquinone form and inhibiting the accumulation of ASFR. Under aerobic condition, STS can protect heart mitochondria by scavenging lipid free radicals generated from adriamycin-induced mitochondrial lipid peroxidation. This investigation shows that STS may be a physiological drug to antagonize the cardiotoxicity of ADR.

In this paper, we demonstrate that the use of nanoparticle-enhanced photoacoustic and fluorescence imaging can effectively assist a surgeon to locate primary tumors and identify residual disease, thus resulting in the reduction of tumor recurrence.

A modified method based on EPR spin trapping and triacetylglycerol extraction was used for tissue nitric oxide (NO) detection at room temperature. NO signal intensity was stable for about 1.5 h and the detection limit of this method was less than 200 pmol g−1 tissue. Using this method, we report evidence that NO production in vivo can be inhibited by adriamycin in mice livers.

Pyrochlore-type Bi2Sn2O7(BSO) nanoparticles have been prepared by a hydrothermal method assisted with a cationic surfactant cetyltrimethylammonium bromide (CTAB). These BSO products were characterized by powder X-ray diffraction (XRD), infrared spectroscopy (IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET), and UV-visible diffuse reflectance spectroscopy (DRS). The results indicated that CTAB alters the surface parameters and the morphology and enhances the photoinduced charge separation rate of BSO. The photocatalytic degradation test using rhodamine B as a model pollutant showed that the photocatalytic activity of the BSO assisted with CTAB was two times that of the reference BSO. Close investigation revealed that the size, the band gap, the structure, and the existence of impurity level played an important role in the photocatalytic activities.

Praseodymium doped Bi2Sn2O7(BSO), as a visible-light responsive photocatalyst, was prepared by a hydrothermal method with different dopant contents. The as-prepared photocatalysts were investigated by X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscope (TEM), N2adsorption-desorption isotherm, X-ray photoelectron spectroscopy analysis (XPS), and UV-Vis diffuse reflectance spectroscopy (DRS). The photocatalytic activity of prepared catalysts was evaluated by the degradation of Rhodamine Bextra (RhB) and 2,4-dichlorophenol (2,4-DCP) in aqueous solution under visible light irradiation. It was found that Pr doping inhibited the growth of crystalline size and the as-prepared materials were small in size (10–20 nm). In our experiments, Pr-doped BSO samples exhibited enhanced visible-light photocatalytic activity compared to the undoped BSO, and the optimal dopant amount of Pr was 1.0 mol% for the best photocatalytic activity. On the basis of the calculated PL spectra, the mechanism of enhanced photocatalytic activity has been discussed.

Breast cancer represents a major ongoing public health problem as the most common non-cutaneous malignancy among U.S. women. While significant progress has been made in improving loco-regional treatments for breast cancer, relatively little progress has been made in diagnosing and treating patients with metastatic breast cancer. At present there are limited curative options for patients with breast cancer metastatic beyond regional nodes. Emerging nanotechnologies promise new approaches to early detection and treatment of metastatic breast cancer. Fulfilling the promise of nanotechnologies for patients with metastatic breast cancer will require delivery of nanomaterials to sites of metastatic disease. Future translational approaches will rely on an ever increasing understanding of the biology of breast cancer subtypes and their metastases. These important concepts will be highlighted and elucidated in this manuscript.

We report a microelectromechanical systems based intraoperative PAT technique, and demonstrate its ability for accurately mapping tumors in three-dimension and inspecting the completeness of tumor resection during surgery in an animal model.

Proteasomes are composed of 20S core particles (CPs) ofα- andβ-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeonHaloferax volcaniias a model. Indicative of phosphorylation, phosphatase-sensitive isoforms ofα1andα2were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped includingα1Thr147,α2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped toα1, thus, revealing a new type of proteasomal modification. Probing the biological role ofα1and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation forα1variants including Thr147Ala, Thr158Ala and Ser58Ala. AnH. volcaniiRio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer toα1. Theα1variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N α acetylation of the initiator methionine of the α1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of α1 in H. volcanii cells. To further examine this, α1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP “gate” formed by the α1 N terminus were examined for their N α acetylation. Both the “gate” deletion and Q2A substitution completely altered the N α -acetylation pattern of α1, with the deletion rendering α1 unavailable for N α acetylation and the Q2A modification apparently enhancing cleavage of α1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two α1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although α1 proteins with Q2D, Q2P, and Q2T substitutions were N α acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of α1 as rings in N α -acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these α1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N α acetylation of α1 is important in CP assembly and activity, high levels of α1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP “gate” impairs the ability of cells to overcome salt stress.

Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core α1 ( psmA ), β ( psmB ), and α2 ( psmC ) subunits as well as genes ( panA and panB ) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either α1 or α2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either β alone or α1 and α2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either α1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of l -canavanine. Abolishment of α1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.

Despite the quinone-dependent copper amine oxidases being described as having the ability to metabolize unbranched primary amines to the corresponding aldehydes, we previously showed that the secondary amines 3-pyrrolines are metabolized as mechanism-based inactivators of bovine plasma amine oxidase (BPAO), and that the 3-(3-nitro-4-methoxyphenyl)-substituted analog was a particularly potent and efficient inactivator. We now show that additional 3-aryl-3-pyrrolines containing highly electron-withdrawing aryl groups (pyridyl, quinolyl, isoquinolyl, and pentafluorophenyl) are some of the most potent inactivators of BPAO reported to date. We also provide mass spectroscopic confirmation of the proposed mechanism of inhibition involving pyrrolylation of the active-site cofactor, through identification by MALDI-TOF and LC–ESI-MS/MS of the (3-arylpyrrol-1-yl)resorcinol derivatives of the cofactor-containing thermolytic peptides.

Insight into the world of proteolysis has expanded considerably over the past decade. Energy-dependent proteases, such as the proteasome, are no longer viewed as nonspecific degradative enzymes associated solely with protein catabolism but are intimately involved in controlling biological processes that span life to death. The proteasome maintains this exquisite control by catalyzing the precisely timed and rapid turnover of key regulatory proteins. Proteasomes also interplay with chaperones to ensure protein quality and to readjust the composition of the proteome following stress. Archaea encode proteasomes that are highly related to those of eukaryotes in basic structure and function. Investigations of archaeal proteasomes coupled with those of eukaryotes has greatly facilitated our understanding of the molecular mechanisms that govern regulated protein degradation by this elaborate nanocompartmentalized machine.

In this paper, an electron transfer reaction mediated by sodium tanshinone IIA sulfonate (STS) was studied in rat heart mitochondria. It was found that STS could stimulate mitochondrial NADH oxidation dose-dependently and partly restore NADH oxidation in the presence of respiratory inhibitor (rotenone or antimycin A or KCN). It was likely that STS could accept electrons from complex I similar to ferricyanide and could be converted to its semiquinone form that could then reduce oxygen molecule. The data also showed that cytochrome c (Cyt c) could be reduced by STS in the presence of KCN, or STS could transfer the electron to oxygen directly. Free radicals were involved in the process. The results suggest that STS may protect ischemia-reperfusion injury through an electron transfer reaction in mitochondria against forming reactive oxygen radicals.

Protective effects of sodium tanshinone IIA sulphonate against adriamycin-induced lipid peroxidation were investigated. Data showed that treatment with sodium tanshinone IIA sulphonate could prevent mice from decrease in body weight caused by adriamycin. It was found that myocardial lipid peroxidation in sodium tanshinone IIA sulphonate-treated mice was lower compared with that in adriamycin-treated ones. The activities of some endogenous antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase and catalase, were higher in the sodium tanshinone IIA sulphonate group than that in the adriamycin group. In vitro experiments showed that sodium tanshinone IIA sulphonate could inhibit adriamycin-induced mitochondrial lipid peroxidation and swelling. Sodium tanshinone IIA sulphonate could scavenge adriamycin semiquinone free radical in heart homogenate dose-dependently. Thus, protective effects of sodium tanshinone IIA sulphonate may not only be related to its antioxidant activity but also to its regulation of antioxidant enzyme activities in the heart.pc 1999 Academic Press@p$hr

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