A facile system to evaluate in vitro drug release from dissolving microneedle arrays

The use of biological tissues in the in vitro assessments of dissolving (?) microneedle (MN) array mechanical strength and subsequent drug release profiles presents some fundamental difficulties, in part due to inherent variability of the biological tissues employed. As a result, these biological materials are not appropriate for routine used in industrial formulation development or quality control (QC) tests. In the present work a facile system using Parafilm M�� (PF) to test drug permeation performance using dissolving MN arrays is proposed. Dissolving MN arrays containing 196 needles (600 ��m needle height) were inserted into a single layer of PF and a hermetic ���pouch��� was created including the array inside. The resulting system was placed in a dissolution bath and the release of model molecules was evaluated. Different MN formulations were tested using this novel setup, releasing between 40 and 180 ��g of their cargos after 6 hours. The proposed system is a more realistic approach for MN testing than the typical performance test described in the literature for conventional transdermal patches. Additionally, the use of PF membrane was tested either in the hermetic ���pouch��� and using Franz Cell methodology yielding comparable release curves. Microscopy was used in order to ascertain the insertion of the different MN arrays in the PF layer. The proposed system appears to be a good alternative to the use of Franz cells in order to compare different MN formulations. Given the increasing industrial interest in MN technology, the proposed system has potential as a standardised drug/active agent release test for quality control purposes.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells

<p>NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.</p>

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

<p>Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.</p>

Sustained release of the candidate antiretroviral peptides T-1249 and JNJ54310516-AFP from a rod insert vaginal ring

<p>Administration of biomacromolecular drugs in effective quantities from conventional vaginal rings is hampered by poor drug permeability in the polymers from which rings are commonly constructed. Here, we report the formulation development and testing of rod insert rings for sustained release of the candidate antiretroviral peptides T-1249 and JNJ54310516-AFP (JNJ peptide), both of which have potential as HIV microbicides. Rod inserts were prepared comprising antiviral peptides T-1249 or JNJ peptide in combination with a hydrophilic excipient (sodium chloride, sodium glutamate, lactose or zinc acetate) dispersed at different loadings within a medical grade silicone elastomer. The inserts were tested for weight change and swelling when immersed in simulated vaginal fluid (SVF). Dye migration into the inserts was also assessed visually over 28 days. In vitro release of T-1249 and JNJ peptide from rings containing various insert types was tested. Weight change and degree of swelling of rods immersed in SVF was dependent on the type and concentration of excipient present. The rods displayed the following rank order in terms of weight change: sodium glutamate &gt; zinc acetate���������sodium chloride &gt; lactose. The weight change and degree of swelling of the inserts did not correlate with the level of dye uptake observed. In vitro release of T-1249 was improved through addition of lactose, sodium chloride and sodium glutamate, while release of JNJ peptide was improved through addition of sodium chloride or sodium glutamate. Sustained release of hydrophobic peptides can be achieved using a rod insert ring design formulated to include a hydrophilic excipient. Release rates were dependent upon the type of excipient used. The degree of release improvement with different inserts partially reflects their ability to imbibe surrounding fluid and swell in aqueous environments.</p>

Molecular mechanisms mediating the beneficial metabolic effects of [Arg4]tigerinin-1R in mice with diet-induced obesity and insulin resistance

<p>The frog skin host-defense peptide tigerinin-1R stimulates insulin release in vitro and improves glucose tolerance and insulin sensitivity in animal models of type 2 diabetes. This study extends these observation by investigating the molecular mechanisms of action underlying the beneficial metabolic effects of the analogue [Arg4]tigerinin-1R in mice with diet induced obesity, glucose intolerance and insulin resistance. The study also investigates the electrophysiological effects of the peptide on KATP and L-type Ca2+ channels in BRINBD11 clonal �� cells. Non-fasting plasma glucose and glucagon concentrations were significantly (P&lt;0.05) decreased and plasma insulin increased by twice daily treatment with [Arg4]tigerinin-1R (75 nmol.kg-1 body weight) for 28 days. Oral and intraperitoneal glucose tolerance were significantly (P &lt; 0.05) improved accompanied by enhanced secretion and action of insulin. The peptide blocked KATP channels and, consistent with this, improved beta cell responses of isolated islets to a range of secretagogues. Peptide administration resulted in up-regulation of key functional genes in islets involved insulin secretion (Abcc8, Kcnj11, Cacna1c and Slc2a2) and in skeletal muscle involved with insulin action (Insr, Irs1, Pdk1, Pik3ca, and Slc2a4). These observations encourage further development of tigerinin-1R analogues for the treatment of patients with type 2 diabetes.</p>

Accidental release of hazardous gases: modelling and assessing risk

The renewed concern in assessing risks and consequences from technological hazards in industrial and urban areas continues emphasizing the development of local-scale consequence analysis (CA) modelling tools able to predict shortterm pollution episodes and exposure effects on humans and the environment in case of accident with hazardous gases (hazmat). In this context, the main objective of this thesis is the development and validation of the EFfects of Released Hazardous gAses (EFRHA) model. This modelling tool is designed to simulate the outflow and atmospheric dispersion of heavy and passive hazmat gases in complex and build-up areas, and to estimate the exposure consequences of short-term pollution episodes in accordance to regulatory/safety threshold limits. Five main modules comprising up-to-date methods constitute the model: meteorological, terrain, source term, dispersion, and effects modules. Different initial physical states accident scenarios can be examined. Considered the main core of the developed tool, the dispersion module comprises a shallow layer modelling approach capable to account the main influence of obstacles during the hazmat gas dispersion phenomena. Model validation includes qualitative and quantitative analyses of main outputs by the comparison of modelled results against measurements and/or modelled databases. The preliminary analysis of meteorological and source term modules against modelled outputs from extensively validated models shows the consistent description of ambient conditions and the variation of the hazmat gas release. Dispersion is compared against measurements observations in obstructed and unobstructed areas for different release and dispersion scenarios. From the performance validation exercise, acceptable agreement was obtained, showing the reasonable numerical representation of measured features. In general, quality metrics are within or close to the acceptance limits recommended for ���non-CFD models���, demonstrating its capability to reasonably predict hazmat gases accidental release and atmospheric dispersion in industrial and urban areas. EFRHA model was also applied to a particular case study, the Estarreja Chemical Complex (ECC), for a set of accidental release scenarios within a CA scope. The results show the magnitude of potential effects on the surrounding populated area and influence of the type of accident and the environment on the main outputs. Overall the present thesis shows that EFRHA model can be used as a straightforward tool to support CA studies in the scope of training and planning, but also, to support decision and emergency response in case of hazmat gases accidental release in industrial and built-up areas.

Micro/nanoreservoirs for controlled release of active species in smart functional coatings

This work reports one possible way to develop new functional coatings used to increase the life time of metallic structures. The functionalities selected and attributed to model coatings in the frame of this work were corrosion protection, self-sensing and prevention of fouling (antifouling). The way used to confer those functionalities to coatings was based on the encapsulation of active compounds (corrosion inhibitors, pH indicators and biocides) in micro and nanocontainers followed by their incorporation into the coating matrices. To confer active corrosion protection, one corrosion inhibitor (2-mercaptobenzothiazole, MBT) was encapsulated in two different containers, firstly in silica nanocapsules (SiNC) and in polyurea microcapsules (PU-MC). The incorporation of both containers in different models coatings shows a significant improvement in the corrosion protection of aluminum alloy 2024 (AA2024). Following the same approach, SiNC and PU-MC were also used for the encapsulation of phenolphthalein (one well known pH indicator) to introduce sensing properties in polymeric coatings. SiNC and PU-MC containing phenolphthalein acted as corrosion sensor, showing a pink coloration due to the beginning of cathodic reaction, resulting in a pH increase identified by those capsules. Their sensing performance was proved in suspension and when integrated in coatings for aluminium alloy 2024 and magnesium alloy AZ31. In a similar way, the biocide activity (antifouling) was assigned to two polymeric matrices using SiNC for encapsulation of one biocide (Dichloro-2-octyl-2H-isothiazol-3-one, DCOIT) and also SiNC-MBT was tested as biocide. The antifouling activity of those two encapsulated compounds was assessed through inhibition and consequent decrease in the bioluminescence of modified E. coli. That effect was verified in suspension and when incorporated in coatings for AISI 1008 carbon steel. The developed micro and nanocontainers presented the desired performance, allowing the introduction of new functionalities to model coatings, showing potential to be used as functional additives in the next generation of multifunctional coatings.

In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure���activity study

Hymenochirin-1b (Hym-1B; IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is a cationic, ��-helical amphibian host-defense peptide with antimicrobial, anticancer, and immunomodulatory properties. This study investigates the abilities of the peptide and nine analogues containing substitutions of Pro5, Glu6, and Asp9 by either l-lysine or d-lysine to stimulate insulin release in vitro using BRIN-BD11 clonal �� cells or isolated mouse islets and in vivo using mice fed a high-fat diet to produce obesity and insulin resistance. Hym-1B produced a significant and concentration-dependent increase in the rate of insulin release from BRIN-BD11 cells without cytotoxicity at concentrations up to 1 ��M with a threshold concentration of 1 nM. The threshold concentrations for the analogues were: [P5K], [E6K], [D9K], [P5K, E6K] and [E6K, D9k] 0.003 nM, [E6K, D9K] and [D9k] 0.01 nM, [P5K, D9K] 0.1 nM and [E6k] 0.3 nM. All peptides displayed cytotoxicity at concentrations ���1 ��M except the [P5K] and [D9k] analogues which were non-toxic at 3 ��M. The potency and maximum rate of insulin release from mouse islets produced by the [P5K] peptide were significantly greater than produced by Hym-1B. Neither Hym-1B nor the [P5K] analogue at 1 ��M concentration had an effect on membrane depolarization or intracellular Ca2+. The [P5K] analogue (1 ��M) produced a significant increase in cAMP concentration in BRIN-BD11 cells and stimulated GLP-1 secretion from GLUTag cells. Down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of [P5K]hym-1B. Intraperitoneal administration of the [P5K] and [D9k] analogues (75 nmol/kg body weight) to high-fat-fed mice with insulin resistance significantly enhanced glucose tolerance with a concomitant increase in insulin secretion. We conclude that [P5K]hym-1B and [D9k]hym-1B show potential for development into anti-diabetic agents.

Estudo da interac����o de complexos de van��dio com a ca2+-atpase de ret��culo sarcoplasm��tico: an��lise estrutural e funcional

A bomba de c��lcio de ret��culo sarcoplasm��tico �� uma das prote��nas mais extensivamente estudadas, capaz de interagir com v��rias esp��cies e compostos de van��dio. Combinando-se estudos de fluoresc��ncia com ensaios cin��ticos de transporte e liga����o de 45Ca �� ATPase, avaliou-se o efeito de tr��s complexos de van��dio na fun����o bioenerg��tica e estrutural de Ca2+-ATPase. Demonstrou-se que concentra����es pr��ximas dos valores de IC50 (para a hidr��lise de ATP) de BMOV-V(IV) e de PDC-V(V) n��o inibem significativamente a acumula����o de 45Ca por sarcoves��culas. Por outro lado, o complexo PDC-V(V) mostrou ser capaz de estimular a liga����o de 45Ca ao ret��culo sarcoplasm��tico, sugerindo que este complexos pode interagir com o dom��nio de liga����o de c��lcio �� bomba. V��rios estudos de fluoresc��ncia mostraram que BMOV-V(IV) e PDC-V(V) poder��o ser capazes de induzir a conforma����o E2 (tal como solu����es de ���decavanadato��� e de ���monovanadato���) e E1 de Ca2+-ATPase (tal como solu����es de ���metavanadato���), respectivamente. Apesar de o composto HAIDA-V(IV) previlegiar a conforma����o E1 (devido �� sua elevada afinidade para i��es Ca2+), inibiu significativamente o transporte e a liga����o de 45Ca, o que sugere que possa interagir com os locais de uni��o de c��lcio. Os resultados obtidos s��o consistentes com a forma����o de um aducto entre o composto de van��dio e a prote��na, o que sugere um efeito na homeostasia intracelular de c��lcio, nos sistemas de contrac����o muscular e, inclusive, nas vias de ac����o de insulina. Cada um dos tr��s complexos promoveu respostas distintas na Ca2+-ATPase, sugerindo uma evidencia de actividades biol��gicas diversas em fun����o da esp��cie qu��mica de V e do ambiente de coordena����o.

TAG & RELEASE Volume 19, No. 1 March 2014

Tag & Release is the newsletter for the South Carolina Governor's Cup Billfishing Series, an official program of the South Carolina Department of Natural Resources in cooperation with the South Carolina Department of Parks, Recreation and Tourism and the Harry R.E. Hampton Memorial Wildlife Fund.

TAG & RELEASE Volume 19, No. 2 May 2014

Tag & Release is the newsletter for the South Carolina Governor's Cup Billfishing Series, an official program of the South Carolina Department of Natural Resources in cooperation with the South Carolina Department of Parks, Recreation and Tourism and the Harry R.E. Hampton Memorial Wildlife Fund.

TAG & RELEASE Volume 20, No. 1 March 2015

Tag & Release is the newsletter for the South Carolina Governor's Cup Billfishing Series, an official program of the South Carolina Department of Natural Resources in cooperation with the South Carolina Department of Parks, Recreation and Tourism and the Harry R.E. Hampton Memorial Wildlife Fund.

TAG & RELEASE Volume 19, No. 4 Nov. 2014

Tag & Release is the newsletter for the South Carolina Governor's Cup Billfishing Series, an official program of the South Carolina Department of Natural Resources in cooperation with the South Carolina Department of Parks, Recreation and Tourism and the Harry R.E. Hampton Memorial Wildlife Fund.

Release of Bet v 1 from Birch Pollen 1 from 5 European 2 Countries. Results from the HIALINE Study

Exposure to allergens is pivotal in determining sensitization and allergic symptoms in individuals. Pollen grain counts in ambient air have traditionally been assessed to estimate airborne allergen exposure. However, the exact allergen content of ambient air is unknown. We therefore monitored atmospheric concentrations of birch pollen grain and the matched major birch pollen allergen Bet v 1 simultaneously across Europe within the EU-funded project HIALINE (Health Impacts of Airborne Allergen Information Network). Pollen count was assessed with Hirst type pollen traps at 10 l/min at sites in France, United Kingdom, Germany, Italy and Finland. Allergen concentrations in ambient air were sampled at 800l/min with a Chemvol high-volume cascade impactor equipped with stages PM>10��m, 10 ��m>PM>2.5��m, and in Germany also 2.5 ��m>PM>0.12��m. The major birch pollen allergen Bet v 1 was determined with an allergen specific ELISA. Bet v 1 isoform patterns were analyzed by 2D-SDS-PAGE blots and mass spectrometric identification. Basophil activation was tested in an Fc��R1-humanized rat basophil cell line passively sensitized with serum of a birch pollen lmptomatic patient. Compared to 10 previous years, 2009 was a representative birch pollen season for all stations. About 90% of the allergen was found in the PM>10��m fraction at all stations. Bet v 1 isoforms pattern did not varied substantially neither during ripening of pollen nor between different geographical locations. The average European allergen release from birch pollen was 3.2 pg Bet v 1/pollen and did not vary much between the European countries. However, in all countries a >10-fold difference in daily allergen release per pollen was measured which could be explained by long range transport of pollen with a deviating allergen release. Basophil activation by ambient air extracts correlated better with airborne allergen than with pollen concentration. Although Bet v 1 is a mixture of different isoforms, its fingerprint is constant across Europe. Bet v 1 was also exclusively linked to pollen. Pollen from different days varied >10-fold in allergen release. Thus exposure to allergen is inaccurately monitored by only monitoring birch pollen grains. Indeed, a humanized basophil activation test correlated much better with allergen concentrations in ambient air than with pollen count. Monitoring the allergens themselves together with pollen in ambient air might be an improvement in allergen exposure assessment.

Pollen Season and Climate: Is the Timing of Birch Pollen Release in the UK Approaching its Limit?

In light of heightened interest in the response of pollen phenology to temperature, we investigated recent changes to the onset of Betula (birch) pollen seasons in central and southern England, including a test of predicted advancement of the Betula pollen season for London. We calculated onset of birch pollen seasons using daily airborne pollen data obtained at London, Plymouth and Worcester, determined trends in the start of the pollen season and compared timing of the birch pollen season with observed temperature patterns for the period 1995���2010. We found no overall change in the onset of birch pollen in the study period although there was evidence that the response to temperature was nonlinear and that a lower asymptotic start of the pollen season may exist. The start of the birch pollen season was strongly correlated with March mean temperature. These results reinforce previous findings showing that the timing of the birch pollen season in the UK is particularly sensitive to spring temperatures. The climate relationship shown here persists over both longer decadal-scale trends and shorter, seasonal trends as well as during periods of ���sign-switching��� when cooler spring temperatures result in later start dates. These attributes, combined with the wide geographical coverage of airborne pollen monitoring sites, some with records extending back several decades, provide a powerful tool for the detection of climate change impacts, although local site factors and the requirement for winter chilling may be confounding factors.