A comparison of gold nanoparticle surface co-functionalization approaches using Polyethylene Glycol (PEG) and the effect on stability, non-specific protein adsorption and internalization

To create clinically useful gold nanoparticle (AuNP) based cancer therapeutics it is necessary to co-functionalize the AuNP surface with a range of moieties; e.g. Polyethylene Glycol (PEG), peptides and drugs. AuNPs can be functionalized by creating either a mixed monolayer by attaching all the moieties directly to the surface using thiol chemistry, or by binding groups to the surface by means of a bifunctional polyethylene glycol (PEG) linker. The linker methodology has the potential to enhance bioavailability and the amount of functional agent that can be attached. While there is a large body of published work using both surface arrangements independently, the impact of attachment methodology on stability, non-specific protein adsorption and cellular uptake is not well understood, with no published studies directly comparing the two most frequently employed approaches. This paper compares the two methodologies by synthesizing and characterizing PEG and Receptor Mediated Endocytosis (RME) peptide co-functionalized AuNPs prepared using both the mixed monolayer and linker approaches. Successful attachment of both PEG and RME peptide using the two methods was confirmed using Dynamic Light Scattering, Fourier Transform Infrared Spectroscopy and gel electrophoresis. It was observed that while the 'as synthesized' citrate capped AuNPs agglomerated under physiological salt conditions, all the mixed monolayer and PEG linker capped samples remained stable at 1M NaCl, and were stable in PBS over extended periods. While it was noted that both functionalization methods inhibited non-specific protein attachment, the mixed monolayer samples did show some changes in gel electrophoresis migration profile after incubation with fetal calf serum. PEG renders the AuNP stable in-vivo however, studies with MDA-MB-231 and MCF 10A cell lines indicated that functionalization with PEG, blocks cellular uptake. It was observed that co-functionalization with RME peptide using both the mixed monolayer and PEG linker methods greatly enhanced cellular internalization compared to PEG capped AuNPs.

Anti-tumoral effect of the non-nucleoside DNMT inhibitor RG108 in human prostate cancer cells

Background: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. Methods: The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-β2, by quantitative methylation-specific PCR and RT-PCR, respectively. Results: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-β2 promoter hypermethylation levels, although mRNA re-expression was only attained GSTP1 and APC. Conclusions: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.

Non-cell autonomous neuroprotective effect of carbon monoxide : microglia-to-neuron communication

RESUMO: A isquémia cerebral é uma das doenças mais predominantes a nivel mundial, sendo uma das principais causas de mortalidade e invalidez. Parte da propagação de dano no cérebro é causado por inflamação descontrolada, causada principalmente por disfunção da microglia. Desta forma, existe a necessidade de tentar desenvolver estratégias para melhor compreender e modular as acções destas células. O monóxido de carbono (CO), é uma molécula endógena com provas dadas como anti-neuroinflamatório em vários modelos. Assim, o principal objectivo do trabalho foi o estudo do CO como um modulador da acção da microglia, com principal foco dado à comunicação entre estas células e neurónios, tentando entender se existe um efeito neuroprotector por inibição da inflamação. Um protocolo de meio condicionado foi estabelecido usando as linhas celulares BV2 e SH-SY5Y, de microglia e neurónio. A molécula CORM-A1, que liberta expontaniamente CO, foi usada como método de entrega da molécula às celulas. Demonstrámos que o pre-tratamento de células BV2 com CORM-A1 gera neuroprotecção já que reduz a morte celular de neurónios SH-SY5Y quando são incubados com meio condicionado de microglia activada em conjunto com o pró-oxidante t-BHP (tert-butil hidroperóxido). Assim, considerámos que o CO promove neuroprotecção ao inibir as acções inflamatórias da microglia. O papel anti-inflamatório da molécula CORM-A1 foi confirmado quando se verificou que pré-tratamento desta molécula em microglia BV2 limita a secreção de TNF-α mas estimula a secreção de IL-10. Por último, a CORM-A1 induziu a expressão do receptor da microglia CD200R1, molécula que participa na comunicação neurónio-microglia e fundamental para a modulação das acções inflamatórias destas últimas. Em suma, o nosso trabalho reforçou as propriedades anti-neuroinflamatórias do CO e uma capacidade de modular viabilidade neuronal através do seu efeito a nível de comunicação célula-célula. ---------------------------- ABSTRACT: Brain ischemia is a widespread disease worldwide, being one of the main causes of mortality and permanent disability. A portion of the damage that ensues following the ischemic event is caused by unrestrained inflammation, which is mainly orchestrated by exacerbated microglial activity. Hence, developing strategies for modulating microglial inflammation is a major concern nowadays. The endogenous molecule carbon monoxide (CO) has been shown to possess anti-neuroinflammatory properties using in vitro and in vivo approaches. Thus, our objective was to study CO as modulator of microglial activity, in particular in what concerns their communication with neurons, by promoting neuronal viability and limiting inflammatory output of activated microglia. A conditioned media strategy was established with BV2 microglia and SH-SY5Y neurons as cell models. CO-releasing molecule A1 (CORM-A1), a compound that releases CO spontaneously, was used as method of CO delivery to cells. We found that CORM-A1 pre-treatment in BV2 cells yields neuroprotective results, as it limits cell death when SH-SY5Y neurons are challenged with conditioned media from LPS-activated microglia and the pro-oxidant t-BHP (tert-butyl-hydroperoxide). Thus, we assumed carbon monoxide promotes neuroprotection via inhibition of microglial inflammation, displaying a non-cell autonomous role. CORM-A1 pre-treatment limited inflammation by inhibiting BV2 secretion of TNF-α and stimulating IL-10 production. These results reinforce that CO’s anti-inflammatory role confers neuroprotection, as the alterations in these cytokines occur concurrently with the increase in SH-SY5Y viability. Finally, we showed for the first time that carbon monoxide promotes the expression of CD200R1, a microglial receptor involved in neuron-glia communication and modulation of microglia inflammation. Further studies are necessary to clarify this role. Altogether, other than just highlighting CO as an anti-inflammatory and neuroprotective molecule, this work set the foundation for disclosing its involvement in cell-to-cell communication.

Orientation of Non-Native 2-Monosubstituted and 2,3-Disubstituted 1,4-Naphthoquinones in the A1 binding site of PSI and the effect on the rate of electron transfer : an electron paramagnetic resonance spectroscopy study

The proce-ss ofoxygenic photosynthesis is vital to life on Earth. the central event in photosynthesis is light induced electron transfer that converts light into energy for growth. Ofparticular significance is the membrane bound multisubunit protein known as Photosystem I (PSI). PSI is a reaction centre that is responsible for the transfer of electrons across the membrane to reduce NADP+ to NADPH. The recent publication ofa high resolution X-ray structure of PSI has shown new information about the structure, in particular the electron transfer cofactors, which allows us to study it in more detail. In PSI, the secondary acceptor is crucial for forward electron transfer. In this thesis, the effect of removing the native acceptor phylloquinone and replacing it with a series of structurally related quinones was investigated via transient electron paramagnetic resonance (EPR) experiments. The orientation of non native quinones in the binding site and their ability to function in the electron transfer process was determined. It was found that PSI will readily accept alkyl naphthoquinones and anthraquinone. Q band EPR experiments revealed that the non-native quinones are incorporated into the binding site with the same orientation of the headgroup as in the native system. X band EPR spectra and deuteration experiments indicate that monosubstituted naphthoquinones are bound to the Al site with their side group in the position occupied by the methyl group in native PSI (meta to the hydrogen bonded carbonyl oxygen). X band EPR experiments show that 2, 3- disubstituted methyl naphthoquinones are also incorporated into the Al site in the same orientation as phylloquinone, even with the presence of a halogen- or sulfur-containing side chain in the position normally occupied by the phytyl tail ofphylloquinone. The exception to this is 2-bromo-3-methyl --.- _. -. - -- - - 4 _._ _ _ - _ _ naphthoquinone which has a poorly resolved spectrum, making determination of the orientation difficuh. All of the non-native quinones studied act as efficient electron acceptors. However, forward electron transfer past the quinone could only be demonstrated for anthraquinone, which has a more negative midpoint potential than phylloquinone. In the case of anthraquinone, an increased rate of forward electron transfer compared to native PSI was found. From these results we can conclude that the rate ofelectron transfer from Al to Fx in native PSI lies in the normal region ofthe Marcus Curve.

Effect of pH on non-photochemical quenching in spinach photsystem 2 particles as measured by picosecond fluorescence decay kinetics

Single photon timing was used to study picosecond chlorophyll a fluorescence decay kinetics of pH induced non-photochemical quenching in spinach photosystem 2 particles. The characteristics of this quenching are a decrease in chlorophyll a fluorescence yield as well as a decrease in photochemistry at low pH. Picosecond kinetics of room temperature fluorescence temporally resolve the individual components of the steady state fluorescence yield into components that are related to primary energy conversion processes in photosystem 2. Four components were resolved for dark adapted (Fo), light saturated (Fm), and chemically reduced (Nadithionite) photosystem 2 reaction centres. The fastest and slowest components, indicative of energy transfer to and energy capture by the photosystem 2 reaction centre and uncoupled ("dead") chlorophyll, respectively, were not affected by changing pH from 6.5 to 4.0. The two intermediate components, indicative of electron transfer processes within the reaction centre of photosystem 2, were affected by the pH change. Results indicate that the decrease in the steady state fluorescence yield at low pH was primarily due to the decrease in lifetime and amplitude of the slower of the intermediate components. These results imply that the decrease in steady state fluorescence yield at low pH is not due to changes in energy transfer to and energy capture by the photosystem 2 reaction centre, but is related to changes in charge stabilization and charge recombination in the photosystem 2 reaction centre.

A systematic analysis of the detrimental effect of hormonotherapy on skeletal and non-skeletal morbidities in male with prostate cancer

Plusieurs patients atteints d’un cancer de la prostate (CaP) se verront prescrire l'hormonothérapie (HT) en raison du stade avancé ou en cas d’une récidive de la maladie, pour freiner la progression de la maladie et améliorer leur survie. Cependant, l’HT présente des effets pouvant nuire significativement à la santé du patient.

Chicken Caecal Microbiome Modifications Induced by Campylobacter jejuni Colonization and by a Non-Antibiotic Feed Additive

Campylobacter jejuni is an important zoonotic foodborne pathogen causing acute gastroenteritis in humans. Chickens are often colonized at very high numbers by C. jejuni, up to 109 CFU per gram of caecal content, with no detrimental effects on their health. Farm control strategies are being developed to lower the C. jejuni contamination of chicken food products in an effort to reduce human campylobacteriosis incidence. It is believed that intestinal microbiome composition may affect gut colonization by such undesirable bacteria but, although the chicken microbiome is being increasingly characterized, information is lacking on the factors affecting its modulation, especially by foodborne pathogens. This study monitored the effects of C. jejuni chicken caecal colonization on the chicken microbiome in healthy chickens. It also evaluated the capacity of a feed additive to affect caecal bacterial populations and to lower C. jejuni colonization. From day-0, chickens received or not a microencapsulated feed additive and were inoculated or not with C. jejuni at 14 days of age. Fresh caecal content was harvested at 35 days of age. The caecal microbiome was characterized by real time quantitative PCR and Ion Torrent sequencing. We observed that the feed additive lowered C. jejuni caecal count by 0.7 log (p<0.05). Alpha-diversity of the caecal microbiome was not affected by C. jejuni colonization or by the feed additive. C. jejuni colonization modified the caecal beta-diversity while the feed additive did not. We observed that C. jejuni colonization was associated with an increase of Bifidobacterium and affected Clostridia and Mollicutes relative abundances. The feed additive was associated with a lower Streptococcus relative abundance. The caecal microbiome remained relatively unchanged despite high C. jejuni colonization. The feed additive was efficient in lowering C. jejuni colonization while not disturbing the caecal microbiome.

The effect of the year of wheat variety release on productivity and stability of performance on two organic and two non-organic farms

Nineteen wheat cultivars, released from 1934 to 2000, were grown at two organic and two non-organic sites in each of 3 years. Assessments included grain yield, grain protein concentration, protein yield, disease incidence and green leaf area. The superiority of each cultivar (the sum of the squares of the differences between its mean in each environment and the mean of the best cultivar there, divided by twice the number of environments; CS) was calculated for yield, grain protein concentration and protein yield, and ranked in each environment. The yield and grain protein concentration CS were more closely correlated with cultivar release date at the non-organic sites than at organic sites. This difference may be attributed to higher yield levels with larger differences among cultivars at the non-organic sites, rather than to improved stability (i.e. similar ranks) across sites. The significant difference in the correlation of protein yield CS and cultivar age between organic and non-organic sites would support evidence that the ability to take up mineral nitrogen (N) compared to soil N has been a component of the selection conditions of more modern cultivars (released after 1989). This is supported by assessment of green leaf area (GLA), where more modern cultivars in the non-organic systems had greater late-season GLA, a trend that was not identified in organic conditions. This effect could explain the poor correlation between age and protein yield CS in organic compared to non-organic conditions where modern cultivars are selected to benefit from later nitrogen (N) availability which includes the spring nitrogen applications tailored to coincide with peak crop demand. Under organic management, N release is largely based on the breakdown of fertility-building crops incorporated (ploughed-in) in the previous autumn. The release of nutrients from these residues is dependent on the soil conditions, which includes temperature and microbial populations, in addition to the potential leaching effect of high winter rainfall in the UK. In organic cereal crops, early resource capture is a major advantage for maximizing the utilization of nutrients from residue breakdown. It is concluded that selection of cultivars under conditions of high agrochemical inputs selects for cultivars that yield well under maximal conditions in terms of nutrient availability and pest, disease and weed control. The selection conditions for breeding have a tendency to select cultivars which perform relatively better in non-organic compared to organic systems.

Hyper-parallel tempering Monte Carlo simulations of Ar adsorption in new models of microporous non-graphitizing activated carbon: effect of microporosity

The adsorption of gases on microporous carbons is still poorly understood, partly because the structure of these carbons is not well known. Here, a model of microporous carbons based on fullerene- like fragments is used as the basis for a theoretical study of Ar adsorption on carbon. First, a simulation box was constructed, containing a plausible arrangement of carbon fragments. Next, using a new Monte Carlo simulation algorithm, two types of carbon fragments were gradually placed into the initial structure to increase its microporosity. Thirty six different microporous carbon structures were generated in this way. Using the method proposed recently by Bhattacharya and Gubbins ( BG), the micropore size distributions of the obtained carbon models and the average micropore diameters were calculated. For ten chosen structures, Ar adsorption isotherms ( 87 K) were simulated via the hyper- parallel tempering Monte Carlo simulation method. The isotherms obtained in this way were described by widely applied methods of microporous carbon characterisation, i. e. Nguyen and Do, Horvath - Kawazoe, high- resolution alpha(a)s plots, adsorption potential distributions and the Dubinin - Astakhov ( DA) equation. From simulated isotherms described by the DA equation, the average micropore diameters were calculated using empirical relationships proposed by different authors and they were compared with those from the BG method.

The effect of non-uniform radiative damping on the zonal-mean dynamics of the extratropical middle atmosphere

The effect of spatial and temporal variations in the radiative damping rate on the response to an imposed forcing or diabatic heating is examined in a zonal-mean model of the middle atmosphere. Attention is restricted to the extratropics, where a linear approach is viable. It is found that regions with weak radiative damping rates are more sensitive in terms of temperature to the remote influence of the diabatic circulation. The delay in the response in such regions can mean that ‘downward’ control is not achieved on seasonal time-scales. A seasonal variation in the radiative damping rate modulates the evolution of the response and leaves a transient-like signature in the annual mean temperature field. Several idealized examples are considered, motivated by topical questions. It is found that wave drag outside the polar vortex can significantly affect the temperatures in its interior, so that high-latitude, high-altitude gravity-wave drag is not the only mechanism for warming the southern hemisphere polar vortex. Diabatic mass transport through the 100 hPa surface is found to lag the seasonal evolution of the wave drag that drives the transport, and thus cannot be considered to be in the downward control regime. On the other hand, the seasonal variation of the radiative damping rate is found to make only a weak contribution to the annual mean temperature increase that has been observed above the ozone hole. Copyright © 2002 Royal Meteorological Society.

Observation impact in data assimilation: the effect of non-Gaussian observation error.

Data assimilation methods which avoid the assumption of Gaussian error statistics are being developed for geoscience applications. We investigate how the relaxation of the Gaussian assumption affects the impact observations have within the assimilation process. The effect of non-Gaussian observation error (described by the likelihood) is compared to previously published work studying the effect of a non-Gaussian prior. The observation impact is measured in three ways: the sensitivity of the analysis to the observations, the mutual information, and the relative entropy. These three measures have all been studied in the case of Gaussian data assimilation and, in this case, have a known analytical form. It is shown that the analysis sensitivity can also be derived analytically when at least one of the prior or likelihood is Gaussian. This derivation shows an interesting asymmetry in the relationship between analysis sensitivity and analysis error covariance when the two different sources of non-Gaussian structure are considered (likelihood vs. prior). This is illustrated for a simple scalar case and used to infer the effect of the non-Gaussian structure on mutual information and relative entropy, which are more natural choices of metric in non-Gaussian data assimilation. It is concluded that approximating non-Gaussian error distributions as Gaussian can give significantly erroneous estimates of observation impact. The degree of the error depends not only on the nature of the non-Gaussian structure, but also on the metric used to measure the observation impact and the source of the non-Gaussian structure.