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Tajemství vládnutí spočívá totiž v tom, jak spojit víru ve vlastní neomylnost se schopností učit se z minulých chyb.

George Orwell

Tisková zpráva
docx / www / www

mendel
mendel

2014

Zprávy z laboratoře / Laboratory information

liposon liposon liposon

Picture of the week

Pravidelný seminář (29 T) Strasferic baloon sonda for analysis DNA demage. Harware and electronic situaction in 35 week

NEW PROJECTS in the LAB

Pravidelný seminář (29 T) Nové dva evropské projekty vstupují do naší laboratoře. ID 120 Green Learning Network http://www.greenlearningnetwork.eu/ a projekt ID 134 Young Innovation brokers for Visegrad countries http://visegradfund.org/. Oba projekty jsou primárně zaměřeny na networking mezi evropskými státy.

SEMINAR

Pravidelný seminář (29 T)

RESEARCH IN LAB

An Assay for Isolation of Zinc(II)-Containing Bacterial Proteins Using Microfluidic Coupled with Paramagnetic Particles
Pravidelný seminář (40 T)
Staphylococcus aureus has become a looming threat due to its increasingly multiple drug resistance to many antibiotics. In 1947, four years after mass production of penicillin began S. aureus penicillin resistant strains were identified. In 1959, methicillin, a new antibiotic, was found to be effective against penicillin-resistant staphylococci. The bacteria eventually became resistant to methicillin which consequently led to the discovery of the first methicillin resistant strain of S. aureus (MRSA) in 1961. Currently, approximately 65% of staphylococcal strains have been identified with MRSA phenotype (resistance to beta-lactam antibiotics and the cephalosporins). Emergence and the increase number of antibiotic resistance bacteria have resulted in exploring alternatives to antibiotics such as heavy metals complexes, heavy metal nanomaterials and nanoparticles. Heavy metals, namely zinc based complexes seem to be promising antibacterial agents; however, bacteria pathogens are able to develop or acquire resistance to heavy metals. In 1991, loss of methicillin resistance due to elimination of resistance to heavy metals and tetracycline was reported. Studies found that exposure to nutraceutics, namely zinc derivatives, echinacea and garlic products exhibited resistance to antibiotics in S. aureus.

Již před 10 lety se Česká republika spolu s dalšími devíti evropskými zeměmi staly členy Evropské unie. Ačkoliv si Česká republika oficiálně připomene svůj vstup do Evropské unie 1. května, Úřad vlády ČR ve spolupráci se Zastoupením Evropské komise v ČR a dalšími partnery přichystaly řadu akcí, které budou probíhat po celé jaro. Europe Day is observed annually on May 05, 2014. There are two separate designations of Europe Day: The Council of Europe's day was established in 1949, while the European Union's (EU) day celebrates the day the EU's predecessor was proposed in 1950. For the EU, the day is also known as Schuman Day, comme morating the historical declaration by French foreign minister Robert Schuman.

Journal of Metallomics and Nanotechnologies

První číslo vychází v průběhu května 2014 Časopis Journal of Metallomics and Nanotechnologies vychází pouze elektronicky, čtvrtletně. Jeho obsahové zaměření je v oblastí nano-biochemie, nanotechonologie, biomedicína a nanomedicína. Časopis vychází bez regionálních mutací v českém, slovenském a anglickém jazyce. Vydavatel: Laboratoř metalomiky a nanotechnologií Mendelova univerzita v Brně, Zemědělská 1, 613 00, Brno, Česká republika http://web2.mendelu.cz/af_239_nanotech/J_Met_Nano/index.html

Zprávy z výzkumu/ Scientific Reports




X-rays From A Young Supernova Remnant Located in the constellation of Perseus, this star is in the early stages of its life and is still forming into a fully-grown star. In this view from Hubble?s Advanced Camera for Surveys(ACS) it appears to have a murky chimney of material emanating outwards and downwards, framed by bright bursts of gas flowing from the star itself. This fledgling star is actually surrounded by a bright disk of material swirling around it as it forms ? a disc that we see edge-on from our perspective. However, this small bright speck is dwarfed by its cosmic neighbor towards the bottom of the frame, a clump of bright, wispy gas swirling around as it appears to spew dark material out into space. The bright cloud is a reflection nebula known as [B77] 63, a cloud of interstellar gas that is reflecting light from the stars embedded within it. There are actually a number of bright stars within [B77] 63, most notably the emission-line star LkHA 326, and it nearby neighbor LZK 18. These stars are lighting up the surrounding gas and sculpting it into the wispy shape seen in this image. However, the most dramatic part of the image seems to be a dark stream of smoke piling outwards from [B77] 63 and its stars ? a dark nebula called Dobashi 4173. Dark nebulae are incredibly dense clouds of pitch-dark material that obscure the patches of sky behind them, seemingly creating great rips and eerily empty chunks of sky. The stars speckled on top of this extreme blackness actually lie between us and Dobashi 4173. European Space Agency Credit: ESA/NASA

W3 Star-forming Region NASA has successfully tested the most complex rocket engine parts ever designed by the agency and printed with additive manufacturing, or 3-D printing, on a test stand at NASA's Marshall Space Flight Center in Huntsville, Alabama. NASA has successfully tested the most complex rocket engine parts ever designed by the agency and printed with additive manufacturing, or 3-D printing, on a test stand at NASA's Marshall Space Flight Center in Huntsville, Alabama. NASA engineers pushed the limits of technology by designing a rocket engine injector --a highly complex part that sends propellant into the engine -- with design features that took advantage of 3-D printing. To make the parts, the design was entered into the 3-D printer's computer. The printer then built each part by layering metal powder and fusing it together with a laser, a process known as selective laser melting. The additive manufacturing process allowed rocket designers to create an injector with 40 individual spray elements, all printed as a single component rather than manufactured individually. The part was similar in size to injectors that power small rocket engines and similar in design to injectors for large engines, such as the RS-25 engine that will power NASA's Space Launch System (SLS) rocket, the heavy-lift, exploration class rocket under development to take humans beyond Earth orbit and to Mars. Youtube Override: 3-D Printed Rocket Injector Roars to Life: The most complex 3-D printed rocket injector ever built by NASA roars to life on the test stand at NASA’s Marshall Space Flight Center in Huntsville, Alabama. "We wanted to go a step beyond just testing an injector and demonstrate how 3-D printing could revolutionize rocket designs for increased system performance," said Chris Singer, director of Marshall's Engineering Directorate. "The parts performed exceptionally well during the tests."Using traditional manufacturing methods, 163 individual parts would be made and then assembled. But with 3-D printing technology, only two parts were required, saving time and money and allowing engineers to build parts that enhance rocket engine performance and are less prone to failure. "One of our goals is to collaborate with a variety of companies and establish standards for this new manufacturing process," explained Marshall propulsion engineer Jason Turpin. "We are working with industry to learn how to take advantage of additive manufacturing in every stage of space hardware construction from design to operations in space. We are applying everything we learn about making rocket engine components to the Space Launch System and other space hardware." Additive manufacturing not only helped engineers build and test a rocket injector with a unique design, but it also enabled them to test faster and smarter. Using Marshall's in-house capability to design and produce small 3-D printed parts quickly, the propulsion and materials laboratories can work together to apply quick modifications to the test stand or the rocket component."Having an in-house additive manufacturing capability allows us to look at test data, modify parts or the test stand based on the data, implement changes quickly and get back to testing," said Nicholas Case, a propulsion engineer leading the testing. "This speeds up the whole design, development and testing process and allows us to try innovative designs with less risk and cost to projects."Marshall engineers have tested increasingly complex injectors, rocket nozzles and other components with the goal of reducing the manufacturing complexity and the time and cost of building and assembling future engines. Additive manufacturing is a key technology for enhancing rocket designs and enabling missions into deep space.
NASA Probes Studying Earth’s Radiation Belts to Celebrate Two Year Anniversary
W3 Star-forming Region NASA's twin Van Allen Probes will celebrate on Saturday two years of studying the sun’s influence on our planet and near-Earth space. The probes, shortly after launch in August 2012, discovered a third radiation belt around Earth when only two had previously been detected. The radiation belts are layers of energetic charged particles held in place by the magnetic field surrounding our planet. The new third belt occurred only occasionally but persisted for as long as a month. This revealed to scientists the dynamic and variable nature of the radiation belts and provided new insight into how they respond to solar activity. "The primary science objective of the Van Allen Probes is to provide understanding of how particles in the radiation belts form and change in response to energy input from the sun," said Mona Kessel, the mission’s program scientist at NASA Headquarters in Washington. "The discoveries and understanding gained have far exceeded expectations." The probes, each weighing just less than 1,500 pounds, were specifically designed to withstand and study the harsh radiation belt region around Earth. The belts are critical regions that have a connection to Earth’s atmosphere and space-based technologies. The belts are affected by solar storms and space weather events and as a result, can swell dramatically. When this occurs, they can pose dangers to communications and GPS satellites, as well as humans in low-Earth orbit. Formerly known as the Radiation Belt Storm Probes, the mission was renamed Van Allen Probes in November 2012 in honor of Dr. James Van Allen, who discovered the two radiation belts in 1958. The twin spacecraft have also revealed how particles in the heart of the belts can be accelerated to nearly the speed of light; proven that electrons in the belts are undergoing acceleration from very low frequency plasma waves; and shown persistent stripe-like structures are a common feature of the inner belt, and are caused by Earth’s rotation, a mechanism previously thought to be incapable of such an effect.
PEG — A versatile conjugating ligand for drugs and drug delivery systems
W3 Star-forming Region Polyethylene glycol (PEG) conjugation is a rapidly evolving strategy to solve hurdles in therapeutic delivery and is being used as an add-on tool to the traditional drug delivery methods. Chemically, PEGylation is a term used to denote modification of therapeutic molecules by conjugation with PEG. Efforts are constantly being made to develop novel strategies for conjugation of PEG with these molecules in order to increase its current applications. These strategies are specific to the therapeutic system used and also depend on the availability of activated PEGylating agents. Therefore, a prior knowledge is essential in selecting appropriate method for PEGylation. Once achieved, a successful PEGylation can amend the pharmacokinetic and pharmacodynamic outcomes of therapeutics. Specifically, the primary interest is in their ability to decrease uptake by reticuloendothelial system, prolong blood residence, decrease degradation by metabolic enzymes and reduce protein immunogenicity. The extensive research in this field has resulted into many clinical studies. The knowledge of outcome of these studies gave a good feedback and lessons which helped researchers to redesign PEG conjugates with improved features which can increase the chance of hitting the market. In light of this, the current paper highlights the approaches, novel strategies and the utilization of modern concept for PEG conjugation with respect to various bioactive components of clinical relevance. Moreover, this review also discusses potential clinical outcomes of the PEG conjugation, regulatory approved PEGylated product, clinical trials for newer formulations, and also provides future prospects of this technology.
Targeting strategies for delivery of anti-HIV drugs
W3 Star-forming Region Human Immunodeficiency Virus (HIV) infection remains a significant cause of mortality globally. Though antiretroviral therapy has significantly reduced AIDS-related morbidity and mortality, there are several drawbacks in the current therapy, including toxicity, drug–drug interactions, development of drug resistance, necessity for long-term drug therapy, poor bio-availability and lack of access to tissues and reservoirs. To circumvent these problems, recent anti-HIV therapeutic research has focused on improving drug delivery systems through drug delivery targeted specifically to host cells infected with HIV or could potentially get infected with HIV. In this regard, several surface molecules of both viral and host cell origin have been described in recent years, that would enable targeted drug delivery in HIV infection. In the present review, we provide a comprehensive overview of the need for novel drug delivery systems, and the successes and challenges in the identification of novel viral and host-cell molecules for the targeted drug delivery of anti-HIV drugs. Such targeted anti-retroviral drug delivery approaches could pave the way for effective treatment and eradication of HIV from the body.
Translocation of cell-penetrating peptides across the plasma membrane is controlled by cholesterol and microenvironment created by membranous proteins
W3 Star-forming Region Despite the extensive research in the field of CPPs' cell entry the exact mechanisms underlying their cellular uptake and the role of involved cell surface molecules in the internalization process have remained controversial. The present study focused on the interactions between CPPs and plasma membrane compounds using giant plasma membrane vesicles (GPMVs) . GPMVs have shown to be a suitable model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis. Our results show that higher cholesterol content and tighter packing of membrane predominantly reduce the accumulation of transportan, TP10 and model amphipathic peptide (MAP) in vesicles, indicating that the internalization of CPPs takes place preferentially via the more dynamic membrane regions. The partial digestion of membrane proteins from GPMVs' surface, on the other hand, drastically reduced the accumulation of nona-arginine and Tat peptide into vesicles, suggesting that proteins play a crucial role in the uptake of arginine-rich CPPs.
Suppression in mice of immunosurveillance against PEGylated liposomes by encapsulated doxorubicin
W3 Star-forming Region PEGylated liposomes (PEG-lip) can escape from recognition by immune system and show a longer half-life in the blood than non-PEGylated liposomes. In this study, we investigated the influence of injected PEG-lip encapsulating doxorubicin (PEG-lip-DOX) on the biodistribution of subsequently injected PEG-lip in mice. PEG-lip-DOX, free doxorubicin or empty PEG-lip were initially injected into BALB/c mice via a tail vein, and 3 days later [3H]-labeled PEG-lip ([3H] PEG-lip) were injected into these same mice. At 24 h after the injection, the distribution of [3H] PEG-lip in the liver and spleen was significantly reduced in the PEG-lip-DOX group compared with that in the free doxorubicin or PEG-lip group. Consequently, the plasma concentration of [3H] PEG-lip was significantly elevated by the pretreatment with PEG-lip-DOX. Altered pharmacokinetics was observed at least until 72 h after the injection of [3H] PEG-lip. The influence of the injected PEG-lip-DOX on the pharmacokinetics of the subsequently injected [3H] PEG-lip was clearly observed from 1 to 14 days, and slightly observed on days 21 and 28, after the injection of the PEG-lip-DOX. Flow cytometric analysis showed that the number of liver Kupffer cells was significantly reduced after the treatment with PEG-lip-DOX. On the other hand, a similar alteration in the distribution of the subsequently injected [3H] PEG-lip was observed in immunodeficient mice such as BALB/c nu/nu and severe combined immunodeficiency (SCID) mice. These findings suggest that immune cells including liver Kupffer cells responsible for recognizing PEG-lip were selectively damaged by the encapsulated doxorubicin in PEG-lip injected initially, which damage led to prolongation of the half-life of subsequently injected [3H] PEG-lip in the blood.
Efficient in vitro gene delivery by hybrid biopolymer/virus nanobiovectors
W3 Star-forming Region Recombinant retroviruses provide highly efficient gene delivery and the potential for sustained gene expression, but suffer from significant disadvantages including low titer, expensive production, poor stability and limited flexibility for modification of tropism. In contrast, polymer-based vectors are more robust and allow cell- and tissue-specific deliveries via conjugation of ligands, but are comparatively inefficient. The design of hybrid gene delivery agents comprising both virally derived and synthetic materials (nanobiovectors) represents a promising approach to development of safe and efficient gene therapy vectors. Non-infectious murine leukemia virus-like particles (M-VLPs) were electrostatically complexed with chitosan (?) to replace the function of the viral envelope protein. At optimal fabrication conditions and compositions, ranging from 6 to 9 µg chitosan/109 M-VLPs at 10 × 109 M-VLPs/ml to 40 µg chitosan/109 M-VLPs at 2.5 × 109 M-VLPs/ml, ?/M-VLPs were ~ 300–350 nm in diameter and exhibited efficient transfection similar to amphotropic MLV vectors. In addition, these nanobiovectors were non-cytotoxic and provided sustained transgene expression for at least three weeks in vitro. This combination of biocompatible synthetic agents with inactive viral particles to form a highly efficient hybrid vector is a significant extension in the development of novel gene delivery platforms.
Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles
W3 Star-forming Region There is no effective clinical therapy yet for triple-negative breast cancer (TNBC) without particular human epidermal growth factor receptor-2, estrogen and progesterone receptor expression. In this study, we report a molecularly targeted and synthetic lethality-based siRNA therapy for TNBC treatment, using cationic lipid assisted poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles as the siRNA carrier. It is demonstrated that only in c-Myc overexpressed TNBC cells, while not in normal mammary epithelial cells, delivery of siRNA targeting cyclin-dependent kinase 1 (CDK1) with the nanoparticle carrier (NPsiCDK1) induces cell viability decreasing and cell apoptosis through RNAi-mediated CDK1 expression inhibition, indicating the synthetic lethality between c-Myc with CDK1 in TNBC cells. Moreover, systemic delivery of NPsiCDK1 is able to suppress tumor growth in mice bearing SUM149 and BT549 xenograft and cause no systemic toxicity or activate the innate immune response, suggesting the therapeutic promise with such nanoparticles carrying siCDK1 for c-Myc overexpressed triple negative breast cancer.
Peptide-functionalized nanoparticles for selective targeting of pancreatic tumor
W3 Star-forming Region Chemotherapy for pancreatic cancer is hampered by the tumor's physio-pathological complexity. Here we show a targeted nanomedicine using a new ligand, the CKAAKN peptide, which had been identified by phage display, as an efficient homing device within the pancreatic pathological microenvironment. Taking advantage of the squalenoylation platform, the CKAAKN peptide was conjugated to squalene (SQCKAAKN) and then co-nanoprecipitated with the squalenoyl prodrug of gemcitabine (SQdFdC) giving near monodisperse nanoparticles (NPs) for safe intravenous injection. By interacting with a novel target pathway, the Wnt-2, the CKAAKN functionalization enabled nanoparticles: (i) to specifically interact with both tumor cells and angiogenic vessels and (ii) to simultaneously promote pericyte coverage, thus leading to the normalization of the vasculature likely improving the tumor accessibility for therapy. All together, this approach represents a unique targeted nanoparticle design with remarkable selectivity towards pancreatic cancer and multiple mechanisms of action.
Altered immune proteome of Staphylococcus aureus under iron-restricted growth conditions
W3 Star-forming Region Staphylococcus aureus is one of the major causative agents of severe infections, and is responsible for a high burden of morbidity and mortality. Strains of increased virulence have emerged (e.g. USA300) that can infect healthy individuals in the community and are difficult to treat. To add to the knowledge about the pathophysiology of S. aureus, the adaption to iron restriction, an important in vivo stressor, was studied and the corresponding immune response of the human host characterized. Using a combination of 1D and 2D immune proteomics, the human antibody response to the exoproteomes of S. aureus USA300?spa grown under iron restriction or with excess iron was compared. Human antibody binding to the altered exoproteome under iron restriction showed a 2.7- to 6.2-fold increase in overall signal intensity, and new antibody specificities appeared. Quantification of the secreted bacterial proteins by gel-free proteomics showed the expected strong increase in level of proteins involved in iron acquisition during iron-restricted growth compared to iron access. This was accompanied by decreased levels of superantigens and hemolysins. The latter was corroborated by functional peripheral blood mononuclear cell proliferation assays. The present data provide a comprehensive view of S. aureus exoproteome adaptation to iron restriction. Adults have high concentrations of serum antibodies specific for some of the newly induced proteins. We conclude that iron restriction is a common feature of the microenvironment, where S. aureus interacts with the immune system of its human host. The present study reports a comparative proteome cataloging of a bovine mastitis and a human-associated Staphylococcus epidermidis strain with a specific focus on surfome (cell-wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC-MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house-keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy-metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein- and DNA-mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO2 conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404
Mycobacterial and HIV Infections Up-Regulated Human Zinc Finger Protein 134
W3 Star-forming Region This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1-infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano-ultra performance LC-MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1-infected group as compared with the control group. Of 14 up- and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine-rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers

W3 Star-forming Region Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the presence of competitive microbiota makes the isolation of Y. enterocolitica and Y. pseudotuberculosis from naturally contaminated foods difficult. We attempted to evaluate the performance of a modified Cefsulodin-Irgasan-Novobiocin (CIN) agar in the differentiation of Y. enterocolitica from non-Yersinia species, particularly the natural intestinal microbiota. The modified CIN enabled the growth of Y. enterocolitica colonies with the same efficiency as CIN and Luria-Bertani agar. The detection limits of the modified CIN for Y. enterocolitica in culture medium (10 cfu/ml) and in artificially contaminated pork (104 cfu/ml) were also comparable to those of CIN. However, the modified CIN provided a better discrimination of Yersinia colonies from other bacteria exhibiting Yersinia-like colonies on CIN (H2S- producing Citrobacter freundii, C. braakii, Enterobacter cloacae, Aeromonas hydrophila, Providencia rettgeri, and Morganella morganii). The modified CIN exhibited a higher recovery rate of Y. enterocolitica from artificially prepared bacterial cultures and naturally contaminated samples compared with CIN. Our results thus demonstrated that the use of modified CIN may be a valuable means to increase the recovery rate of food borne Yersinia from natural samples, which are usually contaminated by multiple types of bacteria
Circulating miR-19a and miR-205 in Serum May Predict the Sensitivity of Luminal A Subtype of Breast Cancer Patients to Neoadjuvant Chemotherapy with Epirubicin Plus Paclitaxele
W3 Star-forming Region The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.

W3 Star-forming Region Altered expression and function of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been associated with several diseases such as endothelial dysfunction, atherosclerosis and obesity. In these pathologies, oxLDL/LOX-1 activates signaling pathways that promote cell proliferation, cell motility and angiogenesis. Recent studies have indicated that olr1 mRNA is over-expressed in stage III and IV of human prostatic adenocarcinomas. However, the function of LOX-1 in prostate cancer angiogenesis remains to be determined. Our aim was to analyze the contribution of oxLDL and LOX-1 to tumor angiogenesis using C4-2 prostate cancer cells. We analyzed the expression of pro-angiogenic molecules and angiogenesis on prostate cancer tumor xenografts, using prostate cancer cell models with overexpression or knockdown of LOX-1 receptor. Our results demonstrate that the activation of LOX-1 using oxLDL increases cell proliferation, and the expression of the pro-angiogenic molecules VEGF, MMP-2, and MMP-9 in a dose-dependent manner. Noticeably, these effects were prevented in the C4-2 prostate cancer model when LOX-1 expression was knocked down. The angiogenic effect of LOX-1 activated with oxLDL was further demonstrated using the aortic ring assay and the xenograft model of tumor growth on chorioallantoic membrane of chicken embryos. Consequently, we propose that LOX-1 activation by oxLDL is an important event that enhances tumor angiogenesis in human prostate cancer cells.

W3 Star-forming Region Sulfurimonas denitrificans was originally isolated from coastal marine sediments. It can grow with thiosulfate and nitrate or sulfide and oxygen. Recently sequencing of its genome revealed that it encodes periplasmic and cytoplasmic [NiFe]-hydrogenases but the role of hydrogen for its metabolism has remained unknown. We show the first experimental evidence that S. denitrificans can indeed express a functional hydrogen uptake active hydrogenase and can grow on hydrogen. In fact, under the provided conditions it grew faster and denser on hydrogen than on thiosulfate alone and even grew with hydrogen in the absence of reduced sulfur compounds. In our experiments, at the time points tested, the hydrogen uptake activity appeared to be related to the periplasmic hydrogenase and not to the cytoplasmic hydrogenase. Our data suggest that under the provided conditions S. denitrificans can grow more efficiently with hydrogen than with thiosulfate.

W3 Star-forming Region Influenza A viruses (IAVs) are a major cause of infectious respiratory human diseases and their transmission is dependent upon the environment. However, the role of environmental factors on IAV survival outside the host still raises many questions. In this study, we used lentiviral pseudotypes to study the influence of the hemagglutinin protein in IAV survival. High-titered and cleaved influenza-based lentiviral pseudoparticles, through the use of a combination of two proteases (HAT and TMPRSS2) were produced. Pseudoparticles bearing hemagglutinin proteins derived from different H1N1, H3N2 and H5N1 IAV strains were subjected to various environmental parameters over time and tested for viability through single-cycle infectivity assays. We showed that pseudotypes with different HAs have different persistence profiles in water as previously shown with IAVs. Our results also showed that pseudotypes derived from H1N1 pandemic virus survived longer than those derived from seasonal H1N1 virus from 1999, at high temperature and salinity, as previously shown with their viral counterparts. Similarly, increasing temperature and salinity had a negative effect on the survival of the H3N2 and H5N1 pseudotypes. These results showed that pseudotypes with the same lentiviral core, but which differ in their surface glycoproteins, survived differently outside the host, suggesting a role for the HA in virus stability.

W3 Star-forming Region Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been presented as a superior method for the detection of microorganisms in body fluid samples (e.g., blood, saliva, pus, etc.) However, the performance of MALDI-TOF MS in routine identification of caries-related Lactobacillus isolates from saliva of adult patients with caries has not been determined. In the present study, we introduced a new MALDI-TOF MS system for identification of lactobacilli. Saliva samples were collected from 120 subjects with caries. B acteria were isolated and cultured, and each isolate was identified by both 16S rRNA sequencing and MALDI-TOF MS. The identification results obtained by MALDI-TOF MS were concordant at the genus level with those of conventional 16S rRNA-based sequencing for 88.6% of lactobacilli (62/70) and 95.5% of non-lactobacilli (21/22). Up to 96 results could be obtained in parallel on a single MALDI target, suggesting that this is a reliable high-throughput approach for routine identification of lactobacilli. However, additional reference strains are necessary to increase the sensitivity and specificity of species-level identification.

W3 Star-forming Region Zinc plays a critical role in many biological processes. However, it is toxic at high concentrations and its homeostasis is strictly regulated by metal-responsive transcription factor 1 (MTF-1) together with many other proteins to protect cells against metal toxicity and oxidative stresses. In this paper, we used high-resolution two-dimensional gel electrophoresis (2DE) to profile global changes of the whole soluble proteome in human lung adenocarcinoma (A549) cells in response to exogenous zinc treatment for 24 h. Eighteen differentially expressed proteins were identified by MALDI TOF/TOF and MASCOT search. In addition, we used Western blotting and RT-PCR to examine the time-dependent changes in expression of proteins regulated by MTF-1 in response to Zn treatment, including the metal binding protein MT-1, the zinc efflux protein ZnT-1, and the zinc influx regulator ZIP-1. The results indicated that variations in their mRNA and protein levels were consistent with their functions in maintaining the homeostasis of zinc. However, the accumulation of ZIP-1 transcripts was down-regulated while the protein level was up-regulated during the same time period. This may be due to the complex regulatory mechanism of ZIP-1, which is involved in multiple signaling pathways. Maximal changes in protein abundance were observed at 10 h following Zn treatment, but only slight changes in protein or mRNA levels were observed at 24 h, which was the time-point frequently used for 2DE analyses. Therefore, further study of the time-dependent Zn-response of A549 cells would help to understand the dynamic nature of the cellular response to Zn stress. Our findings provide the basis for further study into zinc-regulated cellular signaling pathways.

W3 Star-forming Region The amino acid substitution of aspartic acid to glycine in hemagglutinin (HA) in position 222 (HA-D222G) as well as HA-222D/G polymorphism of pandemic (H1N1) 2009 influenza viruses (A(H1N1)pdm09) were frequently reported in severe influenza in humans and mice. Their impact on viral pathogenicity and the course of influenza has been discussed controversially and the underlying mechanism remained unclarified. In the present study, BALB/c mice, infected with the once mouse lung- and cell-passaged A(H1N1)pdm09 isolate A/Jena/5258/09 (mpJena/5258), developed severe pneumonia. From day 2 to 3 or 4 post infection (p.i.) symptoms (body weight loss and clinical score) continuously worsened. After a short disease stagnation or even recovery phase in most mice, severity of disease further increased on days 6 and 7 p.i. Thereafter, surviving mice recovered. A 45 times higher virus titer maximum in the lung than in the trachea on day 2 p.i. and significantly higher tracheal virus titers compared to lung on day 6 p.i. indicated changes in the organ tropism during infection. Sequence analysis revealed an HA-222D/G polymorphism. HA-D222 and HA-G222 variants co-circulated in lung and trachea. Whereas, HA-D222 variant predominated in the lung, HA-G222 became the major variant in the trachea after day 4 p.i. This was accompanied by lower neutralizing antibody titers and broader receptor recognition including terminal sialic acid ?-2,3-linked galactose, which is abundant on mouse trachea epithelial cells. Plaque-purified HA-G222-mpJena/5258 virus induced severe influenza with maximum symptom on day 6 p.i. These results demonstrated for the first time that HA-222D/G quasispecies of A(H1N1)pdm09 caused severe biphasic influenza because of fast viral intra-host evolution, which enabled partial antibody escape and minor changes in receptor binding.

W3 Star-forming Region Avian influenza viruses are circulating continuously in ducks, inducing a mostly asymptomatic infection, while chickens are accidental hosts highly susceptible to respiratory disease. This discrepancy might be due to a different host response to the virus between these two bird species and in particular to a different susceptibility to reinfection. In an attempt to address this question, we analyzed, in ducks and in chickens, the viral load in infected tissues and the humoral immune response after experimental primary and secondary challenge infections with either homologous or heterologous low pathogenicity avian influenza viruses (LPAIV). Following homologous reinfection, ducks were only partially protected against viral shedding in the lower intestine in conjunction with a moderate antibody response, whereas chickens were totally protected against viral shedding in the upper respiratory airways and developed a stronger antibody response. On the contrary, heterologous reinfection was not followed by a reduced viral excretion in the upper airways of chickens, while ducks were still partially protected from intestinal excretion of the virus, with no correlation to the antibody response. Our comparative study provides a comprehensive demonstration of the variation of viral tropism and control of the host humoral response to LPAIV between two different bird species with different degrees of susceptibility to avian influenza.

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