Free Patches Research Articles

Innovative transdermal doxorubicin patches prepared using greenly synthesized iron oxide nanoparticles for breast cancer treatment

ABSTRACT An innovative strategy was formulated to overcome the challenges in delivering doxorubicin (DOX) for breast cancer treatment. Transdermal patches were prepared using carbopol 971 (CP) and polyvinyl alcohol (PVA) loaded with starch-coated iron oxide nanoparticles conjugated to DOX (DOX@St-IONPs). The patches’ physicochemical properties, in vitro drug diffusion, alongside the physical properties of DOX@St-IONPs, and their impact on breast cancer cell lines’ viability were examined. The M1 patch, with a 1:1 CP:PVA ratio and DOX@St-IONPs, showed unique properties, indicating effective delivery. It maintained consistent thickness, weight, and 97% DOX loading. Demonstrated flexibility and acceptable surface pH. M1 displayed fivefold increased drug penetration compared to free DOX patches (flux of 14.85 µg/cm2/h and permeability of 0.95 cm/h), with higher cytotoxicity against triple-negative breast cancer. These results propose promising non-invasive DOX delivery through the developed patches.

Investigation of factors that influence the temperature sensitivity of the TRPM2 cation channel.

TRPM2 is a cation channel gated by Ca2+, ADPR and PiP2. The channel plays important role in the central and peripheral heat detection. To understand the physiological relevance of the channel in the regulation of body temperature we conducted electrophysiological studies in cell free patches at various temperatures and agonist concentrations. In the temperature range of 15-40°C we found that heating alone cannot open the channel, gating is strictly agonist dependent. Rising temperature increases channel conductance, open probability and apparent agonist binding affinities. We identified a strongly temperature dependent positive feedback on channel activity caused by the Ca2+ influx through the channel's pore. This temperature dependent Ca2+ influx generates a supraphysiological intracellular [Ca2+] nanodomain that tunes channel activity. These findings explain the physiological relevance of the channel in the regulation of body temperature. Based on our measured data we constructed a mechanistic gating model that allows the calculation of agonist concentrations in the channel's direct environment from measured open probabilities at given temperatures. This model allows us to further investigate the TRPM2 channel's temperature dependent function in different cellular environments and study how the resting membrane potential or the intracellular Ca2+ buffering properties modulate the channel's temperature threshold in different cell types.

Ground-nesting bees prefer bare ground areas on calcareous grasslands

Although most of the wild bee species are ground-nesting, little is known about their nesting requirements and the conservation measures to promote ground-nesting bees. Calcareous grasslands are one of the most species-rich habitats in Central Europe and therefore essential for protecting bee diversity. The management practices of calcareous grasslands are so far often focused on plant populations, but in order to support declining insect populations, additional measures have to be considered for nature conservation. As wild bees depend on the availability of nesting sites, we studied the effects of locally increased bare ground availability on ground-nesting bees on four large and four small calcareous grasslands in Central Germany. Vegetation cover of 24 experimental plots (1 m²) was removed. The number of bee nests as well as the presence of bees on these plots (here: nesting activity) were compared to 24 control plots during six sampling runs. Results showed that the number of bee nests on experimental plots was fourteen times higher compared to control plots, positively related to the surrounding flower cover and higher on steeper slopes, independent of grassland area. Moreover, the observed nesting activity on experimental plots was 2.5 times higher compared to the control and positively related to the currently prevailing soil surface temperature. Bee abundance quantified during transect walks increased on grasslands with a generally higher bare ground availability and a higher flower cover. In conclusion, our study emphasizes the need to consider the availability of nesting resources to promote the vast majority of wild bees, which are ground-nesting. They benefited from bare ground availability as well as adjacent floral resources, and experimental removal of the vegetation cover appeared to be a major, so far underestimated conservation practice. • Ground-nesting bees prefer artificially created vegetation free patches on calcareous grasslands. • The abundance of (ground-nesting) bees is enhanced by a higher bare ground availability. • Nearby floral resources and steeper slopes increase the nest number of ground-nesting bees. • Higher soil temperatures increased the nesting activity of ground nesting bees. • Habitat size has no influence on bee abundance, the proportion of ground-nesting bees or colonization.

Quitline Promotion to Medicaid Members Who Smoke: Effects of COVID-19–Specific Messaging and a Free Patch Offer

People who smoke are at increased risk of serious COVID-19-related disease but have had reduced access to cessation treatment during the pandemic. This study tested 2 approaches to promoting quitline services to Medicaid members who smoke at high rates: using COVID-19-specific messaging and offering free nicotine patches. The hypotheses were that both would increase enrollment. A California Medicaid mailing from October 2020 to January 2021 (N=7,489,093) included 4 versions of a flyer following a 2×2 design comparing generic with COVID-19-specific messaging and a no-patch with free-patch offer. The main outcome measure was quitline enrollments. Quit outcomes (attempted quitting, quit ≥7 days, quit ≥30 days) were assessed at 2 months. A subsequent free-patch offer was sent to all members (N=7,577,198) from April 2021 to June 2021. Data were collected in 2020-2021 and analyzed in 2022. The first mailing generated 1,753 enrollments. Response rates were 0.023% and 0.024% for generic and COVID-19-specific messaging, respectively (p=0.538), and 0.006% and 0.041% for no-patch and free-patch offers, respectively, the latter being 6.7 times more effective than the former (p<0.0001). Quit outcomes were comparable across conditions. The subsequent free-patch offer generated 3,546 enrollments at $40.28 per enrollee. In a Medicaid mailing during COVID-19, offering free patches generated more than 6 times as many quitline enrollments as offering generic help. COVID-19-specific messaging was no more effective than generic messaging. Offering free patches was highly cost-effective. Medicaid programs partnering with quitlines should consider using similar strategies, especially during a pandemic when regular health care is disrupted.

Mesh Free Radial Point Interpolation Based Displacement Recovery Techniques for Elastic Finite Element Analysis

The study develops the displacement error recovery method in a mesh free environment for the finite element solution employing the radial point interpolation (RPI) technique. The RPI technique uses the radial basis functions (RBF), along with polynomials basis functions to interpolate the displacement fields in a node patch and recovers the error in displacement field. The global and local errors are quantified in both energy and L2 norms from the post-processed displacement field. The RPI technique considers multi-quadrics/gaussian/thin plate splint RBF in combination with linear basis function for displacement error recovery analysis. The elastic plate examples are analyzed to demonstrate the error convergence and effectivity of the RPI displacement recovery procedures employing mesh free and mesh dependent patches. The performance of a RPI-based error estimators is also compared with the mesh dependent least square based error estimator. The triangular and quadrilateral elements are used for the discretization of plates domains. It is verified that RBF with their shape parameters, choice of elements, and errors norms influence considerably on the RPI-based displacement error recovery of finite element solution. The numerical results show that the mesh free RPI-based displacement recovery technique is more effective and achieve target accuracy in adaptive analysis with the smaller number of elements as compared to mesh dependent RPI and mesh dependent least square. It is also concluded that proposed mesh free recovery technique may prove to be most suitable for error recovery and adaptive analysis of problems dealing with large domain changes and domain discontinuities.

Free, easy and effective: how young adults used 8 weeks of mailed nicotine patches and to what effect

AbstractIntroductionResearch shows the mass distribution of free nicotine replacement therapy (NRT) is a high-impact, population-level strategy for smoking cessation; but underrepresentation of younger, and/or lighter, smokers challenges generalisability of findings to young adult smokers.AimsThis naturalistic study examined how and with what effect young adult smokers used free nicotine patches provided through a mass mailout programme.MethodsIn total, 5,025 eligible 18–29 year-old smokers who accessed an online ordering platform received self-help materials and an 8-week course of patches matched to their consumption level (&lt;10 cigarettes per day (cpd); ≥10 cpd). No other behavioural support occurred. Whether participants used patches correctly and achieved 30-day continuous abstinence at 6-month follow-up were assessed.ResultsAmong 694 participants with complete data: 89% used some patches; 8% used the patches correctly for 8 weeks; 31.0% (95% confidence interval (CI) = 27.6, 34.7) achieved abstinence. Adjusted logistic regression analysis showed the highest odds of abstinence was associated with the correct use of patches (odds ratio = 2.8, 95% CI = 1.5, 5.1).ConclusionsMass distribution of free patches may be an effective public health measure for supporting younger, lighter smokers to attempt cessation, reduce consumption, or achieve abstinence. Emphasising why and how to use NRT for the entire treatment course may enhance outcomes.

Conserving bees in destroyed landscapes: The potentials of reclaimed sand mines

Sand mines represent anthropogenically impacted habitats found worldwide, which bear potential for bee conservation. Although floral resources can be limited at these habitats, vegetation free patches of open sandy soils and embankments may offer good nesting possibilities for sand restricted and other bees. We compared bee communities as found in three reclaimed sand mines and at adjacent roadside meadows in Maryland, USA, over two years. Both sand mines and roadsides hosted diverse bee communities with 111 and 88 bee species, respectively. Bee abundances as well as richness and Shannon diversity of bee species were higher in sand mines than at roadsides and negatively correlated with the percentage of vegetational ground cover. Species composition also differed significantly between habitats. Sand mines hosted a higher proportion of ground nesters, more uncommon and more ‘sand loving’ bees similar to natural sandy areas of Maryland. Despite the destruction of the original pre-mining habitat, sand mines thus appear to represent a unique habitat for wild bees, particularly when natural vegetation and open sand spots are encouraged. Considering habitat loss, the lack of natural disturbance regimes, and ongoing declines of wild bees, sand mines could add promising opportunities for bee conservation which has hitherto mainly focused on agricultural and urban habitats.

Effects of Offering Nicotine Patches, Incentives, or Both on Quitline Demand

Previous studies found that offering free nicotine patches significantly increases calls to quitlines, although most used pre-post designs and did not directly compare the effects of patches and other incentives. The current study with California Medicaid members used a 2 × 2 design to directly assess the effects of offering free patches and incentives on calls to a quitline. The hypotheses were that offering either would make members more likely to call, and that offering both would increase demand even further. Flyers were inserted into a mailing sent to 4,268,696 Medicaid households, with one of four offers: (1) free counseling; (2) counseling plus patches; (3) counseling plus a $20 gift card; and (4) counseling plus patches and gift card. Ninety percent received the first offer and 10% received one of the other three offers, in equal proportions. The mailers shipped late 2013 to early 2014. Data were collected 2013-2015 and analyzed 2018. Response rates were 0.029% for counseling, 0.115% for counseling plus patches, 0.122% for counseling plus gift card, and 0.200% for counseling, patches, and gift card. Both patches and gift cards had statistically significant effects (both p<0.001). Promotional costs were 59%-75% lower with an incentive. Non-whites responded more strongly than whites to a gift card offer. Offering either free patches or a $20 gift card quadrupled the likelihood of Medicaid smokers calling a quitline; offering both had a nearly additive effect. Incentive offers dramatically increased the cost-effectiveness of promotions. Piggybacking on existing Medicaid communications to promote cessation proved very successful. This article is part of a supplement entitled Advancing Smoking Cessation in California's Medicaid Population, which is sponsored by the California Department of Public Health.

PREPARATION AND CHARACTERIZATION OF POLY (VINYL ALCOHOL)–POLY (VINYL PYRROLIDONE) MUCOADHESIVE BUCCAL PATCHES FOR DELIVERY OF LIDOCAINE HCL

Objective: The objectives of this study were to prepare and characterize a buccal mucoadhesive patch using poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP) as a mucoadhesive matrix, Eudragit S100 as a backing layer, and lidocaine HCl as a model drug.Methods: Lidocaine HCl buccal patches were prepared using double casting technique. Molecular interactions in the polymer matrices were studied using attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and X-ray diffractometry. Mechanical and mucoadhesive properties were measured using texture analyzer. In vitro permeation of lidocaine HCl from the patch was conducted using Franz diffusion cell.Results: Both of the free and lidocaine HCl patches were smooth and transparent, with good flexibility and strength. ATR-FTIR, DSC and X-ray diffractometry studies confirmed the interaction of PVA and PVP. Mechanical properties of matrices containing 60% PVP were significantly lower than those containing 20% PVP (*P&lt;0.05). Mucoadhesive properties had a tendency to decrease with the concentration of PVP in the patch. The patch containing 60% PVP had significantly lower muco-adhesiveness than those containing 20% PVP (*P&lt;0.05). In vitro permeation revealed that the pattern of lidocaine HCl permeation started with an initial fast permeation, followed by a slower permeation rate. The initial permeation fluxes follow the zero-order model of which rate was not affected by the PVP concentrations in the PVA/PVP matrix.Conclusion: Mucoadhesive buccal patches fabricated with PVA/PVP were successfully prepared. Incorporation of PVP in PVA/PVP matrix affected the strength of polymeric matrix and mucoadhesive property of patches.

An Ideal Software Release Policy for an Improved Software Reliability Growth Model Incorporating Imperfect Debugging with Fault Removal Efficiency and Change Point

This paper presents a general software reliability growth model (SRGM) based on non-homogeneous Poisson process (NHPP) and optimal software release policy with cost and reliability criteria. The main motive of this study is to develop a software release time decision model considering maintenance cost and warranty cost under fuzzy environment. In previous studies, maintenance cost has been defined either in terms of warranty cost or fault debugging cost. In reality, maintenance cost includes the cost of free patches, updates, technical support and future enhancement. Also, it is possible that maintenance process causes the removal of software faults in the operational phase including the faults which occur outside the warranty period or warranty definition. In other words, warranty action may be included the maintenance action, but not the converse. Considering this fact, maintenance cost and warranty cost are defined separately in the proposed study. Initially, an SRGM has been proposed with the revised concept of imperfect debugging phenomenon considering fault removal efficiency (FRE). Furthermore, the effect of changes in various environmental factors on models parameters has been taken into account. Numerical examples based on real software failure data sets have been given to analyze the performance of the proposed models.

Modelling and Simulation of Impedance-Based Damage Monitoring of Structures

Electromechanical impedance (EMI) based monitoring techniques are successfully in use in current engineering structures. With the help of piezoelectric sensors, the EMI technique is used for monitoring the health of such structures. Generally, potential damage to the host structure is detected by examining the EMI signature and identifying changes in that unique signature. Since this technique has the potential to offer greater safety and reliability while lowering maintenance costs, it is becoming increasingly popular. This paper investigates the use of finite element method (FEM) to simulate the electro-mechanical impedance technique. A numerical analysis of simple models, such as free piezoelectric patches of various shapes and thicknesses is used to comprehend the fundamentals of this technique. Then, studies on different parts of the structure are conducted to find the effect on the output of system when both damage and loading co-exist, and investigate the effect of temperature for structural health monitoring based on EMI. The simulation results are then compared to experimental data and a very good agreement is observed.

Structural and electronic properties of Pt induced nanowires on Ge(110)

The structural and electronic properties of Pt induced nanowires on Ge(110) surfaces have been studied by scanning tunneling microscopy and low energy electron microscopy. The deposition of a sub-monolayer amount of Pt and subsequent annealing at 1100 (±30) K results into nanowires which are aligned along the densely packed [1–10] direction of the Ge(110) surface. With increasing Pt coverage the nanowires form densely packed arrays with separations of 1.1±0.1nm, 2.0±0.1nm and 3.4±0.1nm. Ge pentagons reside in the troughs for nanowire separations of 3.4nm, however for smaller nanowire separations no pentagons are found. Spatially resolved scanning tunneling spectroscopy measurements reveal a filled electronic state at −0.35eV. This electronic state is present in the troughs as well as on the nanowires. The −0.35eV state has the strongest intensity on the pentagons. For Pt depositions exceeding two monolayers, pentagon free nanowire patches are found, that coexist with Pt/Ge clusters. Upon annealing at 1040K these Pt/Ge clusters become liquid-like, indicating that we are dealing with eutectic Pt0.22Ge0.78 clusters. Low energy electron microscopy videos reveal the formation and spinodal decomposition of these eutectic Pt/Ge clusters.

Understanding clonal plant competition for space over time: a fine‐scale spatial approach based on experimental communities

AbstractAimsCompetitive interactions are a determinant driver of plant community structure in temperate grasslands. In such dense vegetation cover, competition mostly occurs over free space, which conditions niche availability. Clonal growth determines how plants exploit horizontal space, by (1) exploring and colonizing free patches (guerilla form) or (2) resisting competitors through space consolidation (phalanx form), with possible intermediate strategies. Few studies have explored the dynamics of co‐existing plants belonging to different clonal strategies. Models predict that guerilla forms may be advantageous during the early stages of succession, whereas phalanx forms are expected to be better competitors over time. We investigated whether these predictions are true under experimental conditions and explored possible mechanisms that promote clonal plant co‐existence.LocationExperimental garden of the University of Rennes 1, France.MethodsWe set up a large‐scale mesocosm, in which we manipulated different mixtures of three clonal growth forms (guerilla, intermediate and phalanx) over 5 yr (2009–2014), which impeded all types of reproduction other than clonal spread of the initial planted individuals. We analysed the spatio‐temporal dynamics of the communities using linear mixed models to compare the change in relative cover and spatial association (differentiating between associations with similar and different species) for each clonal form.ResultsGuerilla forms dominated the early community, but decreased in dominance at later stages, at which point intermediate forms benefitted. Furthermore, guerilla forms were more likely to co‐occur with other guerrillas, with this pattern remaining consistent over time. In contrast, and contrary to our expectations, phalanx forms had the lowest cover throughout the experiment, and exhibited clear segregation from intermediate or other phalanx species. Intermediate growth forms between guerilla and phalanx were the most variable over time, displaying both consolidative and explorative patterns depending on the clonal forms of the other species which they coexisted with, suggesting plastic variation in their traits.ConclusionsOur results highlight the key role of clonal forms in driving competitive interactions and, hence, determining the spatio‐temporal dynamics of grassland communities.

Coevolution of bed surface patchiness and channel morphology: 2. Numerical experiments

In gravel bed rivers, bed topography and the bed surface grain size distribution evolve simultaneously, but it is not clear how feedbacks between topography and grain sorting affect channel morphology. In this, the second of a pair of papers examining interactions between bed topography and bed surface sorting in gravel bed rivers, we use a two‐dimensional morphodynamic model to perform numerical experiments designed to explore the coevolution of both free and forced bars and bed surface patches. Model runs were carried out on a computational grid simulating a 200 m long, 2.75 m wide, straight, rectangular channel, with an initially flat bed at a slope of 0.0137. Over five numerical experiments, we varied (a) whether an obstruction was present, (b) whether the sediment was a gravel mixture or a single size, and (c) whether the bed surface grain size feeds back on the hydraulic roughness field. Experiments with channel obstructions developed a train of alternate bars that became stationary and were connected to the obstruction. Freely migrating alternate bars formed in the experiments without channel obstructions. Simulations incorporating roughness feedbacks between the bed surface and flow field produced flatter, broader, and longer bars than simulations using constant roughness or uniform sediment. Our findings suggest that patches are not simply a by‐product of bed topography, but they interact with the evolving bed and influence morphologic evolution.

Comparing noisy patches for image denoising: a double noise similarity model.

This paper presents a concept of noise similarity (NS), which can be used to refine the comparison of noisy patch and enhance the denoising power of the nonlocal means (NLM) filter. The fact behind this concept is that the similarity of noisy patch should depend on not only the underlying signal (noise free patches), but also the noise. Based on the concept of noise similarity, we derived a double NS (DNS) model, which converts the denoising problem into the problem of reducing two kinds of noise: one is the superimposed additive noise; the other is the deviation error, defined as another kind of noise denoting the difference between similar pixels on their true intensities. The former corresponds to noise suppression, while the latter corresponds to the restoration of image details. To evaluate the effectiveness of the DNS model, we proposed an iterative version of the NLM filter, where the two noise similarities can work collaboratively in the framework of maximum a posterior. Finally, the experimental results demonstrate that the proposed approach can provide competitive performance when compared with other state-of-the-art NLM filters.

THE EFFECT OF PLASTICIZER CONCENTRATION ON POLYMERIC TRANSDERMAL PATCH

An effort was made to formulate drug free transdermal patches by using different polymers like ethyl cellulose, poly vinyl pyrrolidone and eudragit. Four groups of twelve formulations of drug free transdermal patches were prepared by solvent evaporation technique in which each group have different plasticizer concentration and evaluated for flatness, tensile strength, folding endurance, moisture content, Water vapor transmission rate (WVTR), percent elongation. The tensile strength and folding endurance of the patches prepared with 20% Di-n-butylphthalate as plasticizer was high compared to patches prepared by 10% and 15 % Di-n-butylphthalate. The result of 20% plasticizer indicated that the patches would not break and would maintain their integrity with general skin folding when used. All the formulations show 100 % flatness. The WVTR was not significantly affected by varying the concentration of plasticizer (Di-n-butylphthalate). At concentration of 25 % of plasticizer the tensile strength and percent elongation not shows significant result due to soft and sticky formulation. On the basis of above observations we can easily concluded that the Di-n-butylphthalate at concentration 20% of polymers used as plasticizer for further developmental studies. Key words: Di-n-butylphthalate, plasticizer, ethyl cellulose, tensile strength, transdermal patch.

Sodium Alginate Microneedle Arrays Mediate the Transdermal Delivery of Bovine Serum Albumin

BackgroundThe “poke and release” strategy for the delivery of macromolecules using polymeric microneedle (MN) is of great importance because it eliminates microneedle reuse, the risks of biohazardous sharps and cross contamination, and it requires no special disposal mechanism. The main objective of this study was the determination of the stability and delivery of bovine serum albumin (BSA) that was transported across human skin via sodium alginate (SA) microneedle arrays (MNs) and SA needle free patches using two different analytical methods.Methodology and FindingsThe capability of two analytical methods, the bicinchoninic acid (BCA) assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), to precisely detect and quantify BSA within different types of polymeric MNs was assessed. The ex vivo protein release of BSA across dermatomed human abdominal skin from 10 w/w SA MNs was compared to that from needle-free patches using Franz diffusion cells. The developed applicator was mechanically characterized using a Texture Analyzer. The patch mold and its components were fabricated using a rapid prototyping machine.Conclusions/SignificanceThe BCA method was able to precisely detect BSA that had been loaded into SA MNs. However, the use of SDS-PAGE as the analytical method resulted in significantly different amounts of BSA recovered from differently conditioned polymeric MNs. The permeation of BSA across dermatomed human abdominal skin by SA MNs, which were composed of 100 pyramidal needles, increased by approximately 15.4 fold compared to the permeation obtained with SA needle-free patches. The ease of use of the applicator during the release studies was also demonstrated, as was its mechanical characterization.

A New Weight for Nonlocal Means Denoising Using Method Noise

Method noise, i.e., the difference between the noisy image and its denoised version, often contains residual image information due to imperfect denoising methods. In this letter, a novel weight is derived for nonlocal means (NLM) denoising, which tries to exploit the nonlocal similarities of residual image in method noise, and estimates directly the similarities between noise free patches instead of the commonly used similarity measure based on noisy observations or their pre-denoised versions. The denoising scheme is implemented in two stages: the first stage is the original NLM, and the second stage is the NLM with the new weight that incorporates the role of both the pre-denoised result and the method noise produced in the previous stage. Experimental results demonstrate the robustness of the proposed weight and its potential with respect to classical NLM methods and other state-of-the-art methods.

Abstract 9723: Calcium Loading Induces a PKC Isoform Switch That Activates Constitutive Acetylcholine Regulated Potassium Current in Atrial Fibrillation-Related Remodeling

Background: AF causes atrial tachycardia remodeling (ATR), with enhanced constitutive acetylcholine regulated K + current (I KAChc ) contributing to ATR-induced APD shortening and AF promotion. The present study examined the mechanisms underlying I KAChc activation; based on preliminary evidence for an essential role of phosphorylation, we evaluated the participation of protein kinase C (PKC). Methods: Cultured dog atrial cardiomyocytes in vitro tachypaced (TP) at 3 Hz for 24 hours were compared to parallel 1 Hz paced cells and cells from ATR dogs (paced in vivo at 400 bpm x 1 week). I KAChc single channel activity was assessed in cell attached and cell free (I-O) patches. Protein expression was assessed by Western blot. Results: In vitro TP for 24 hours activated I KAChc (Fig A), mimicking effects of in vivo ATR. The classical PKC isoform PKCα inhibited, whereas the novel isoform PKCε enhanced, I KAChc when applied to the intracellular side of I-O patches. TP and ATR downregulated PKCα (by 33, 37% respectively) and caused membrane translocation of PKCε, switching PKC-predominance to the stimulatory novel isoform. TP increased [Ca 2+ ] i at 2 hours by 30% (Fig B), with subsequent return to baseline at 24 hrs. Ca 2+ activates calpain, which breaks down proteins. Decreasing calpain activity during TP with PD150606 (20 µM) prevented TP enhancement of I KAChc (Fig C). Decreasing [Ca 2+ ] i during TP with BAPTA-AM (1 µM), a cell permeable Ca 2+ chelator, also prevented TP-enhancement of I KAChc (Fig D). Both PD150606 and BAPTA-AM prevented downregulation of PKCα protein expression by TP. Exposure of TP cells to a PKCε inhibitor (Fig E) suppressed TP-induced I KAChc activation, whereas a PKCα inhibitor (Fig F) enhanced I KAChc activity in 1 Hz cells. Conclusions: Rate-related [Ca 2+ ] i loading due to ATR causes a PKC isoform switch, with downregulation of inhibitory PKCα and membrane translocation (activation) of stimulatory PKCε, which enhances I KAChc causing APD shortening and AF promotion.

Amelogenin and enamelysin localization in human dental germs

Odontogenesis is extensively studied in animal models but less understood in human. In early amelogenesis, amelogenin constitutes 90% of enamel organic matrix, which is degraded by enamelysin and replaced by hydroxyapatite crystals. Here, amelogenin and enamelysin distribution changes during amelogenesis were shown by co-localization experiments by confocal microscopy. Early bell stage showed more amelogenin labeling than enamelysin, as free immune-reactive granular patches towards basal membrane between ameloblast and odontoblast. Increased amelogenin expression and secretion towards extracellular matrix formation region was found. Enamelysin distribution was perinuclear in early bell stage. During late bell stage, a decreasing amelogenin labeling in contrast with enamelysin increasing along the cells was found, suggesting specific temporal amelogenin degradation. Enamelysin was located initially around nuclei and later was found in all the ameloblast and stellate reticulum cytoplasm. Amelogenin was observed inside ameloblast, stellate reticulum, and intermediate stratum cells in the enamel as well as in the newly formed dentin extracellular matrix. In contrast, in dentin more amelogenin than enamelysin was found located close to the periphery.