Long non-coding RNA exploration for mesenchymal stem cell characterisation.

BACKGROUND: The development of RNA sequencing (RNAseq) and the corresponding emergence of public datasets have created new avenues of transcriptional marker search. The long non-coding RNAs (lncRNAs) constitute an emerging class of transcripts with a potential for high tissue specificity and function. Therefore, we tested the biomarker potential of lncRNAs on Mesenchymal Stem Cells (MSCs), a complex type of adult multipotent stem cells of diverse tissue origins, that is frequently used in clinics but which is lacking extensive characterization. RESULTS: We developed a dedicated bioinformatics pipeline for the purpose of building a cell-specific catalogue of unannotated lncRNAs. The pipeline performs ab initio transcript identification, pseudoalignment and uses new methodologies such as a specific k-mer approach for naive quantification of expression in numerous RNAseq data. We next applied it on MSCs, and our pipeline was able to highlight novel lncRNAs with high cell specificity. Furthermore, with original and efficient approaches for functional prediction, we demonstrated that each candidate represents one specific state of MSCs biology. CONCLUSIONS: We showed that our approach can be employed to harness lncRNAs as cell markers. More specifically, our results suggest different candidates as potential actors in MSCs biology and propose promising directions for future experimental investigations.

Peptide Technologies at Energypolis.

The new Energypolis campus brings together the skills of EPFL Valais-Wallis, HES-SO Valais-Wallis, and the Ark Foundation's services. Together these partners respond to today's major concerns in the domains of energy, health, and the environment cutting-edge technology. The spirit of this new campus is to foster innovation in these disciplines and emulate the creation of start-up companies. The HES-SO hosts the School of Engineering (HEI) at this campus, which includes the following degree programmes: Life Technologies, Systems Engineering and Energy and Environmental Engineering, as well as their corresponding applied research institutes. Peptide technologies belong to the many activities that are carrying out in the Institute of Life Technologies. The present review summarizes the peptide technologies that are currently under development, that is, the regioselective labeling of therapeutic antibodies for cancer imaging, the development of peptide antivirals and antimicrobials for the treatment of infectious diseases, targeting of drugs conjugated to peptidic scaffolds as well as engineering of biomaterials.

Phenolic compounds of grape stems and their capacity to precipitate proteins from model wine.

Reintegration of grape stem, a by-product from wine production, into the food chain is of high interest from an economic and environmental perspective. Therefore, an investigation of stems was undertaken and is described here. It is known that quality of stems is of high variability. In this study the stems from four grapevine varieties (Syrah, Cabernet Sauvignon, Merlot and Chasselas) cultivated in Switzerland were treated in following ways: drying, cutting and separation into fractions based on particle size. All fractions were then characterised for their phenolic compounds content. It was found that Chasselas fractions contained most phenolic compounds. The addition of grape stems of the four different varieties allowed reduction of the protein content of a model wine. The extent of protein precipitation was highly correlated with the amount of phenolic compounds in stems added. Among the examined varieties, Chasselas brought most promising results, with the high reduction of the protein at low level of stem addition.

High mobility group box 1 (HMGB1): dual functions in the cochlear auditory neurons in response to stress?

High mobility group box 1 (HMGB1) is a DNA-binding protein that facilitates gene transcription and may act extracellularly as a late mediator of inflammation. The roles of HMGB1 in the pathogenesis of the spiral ganglion neurons (SGNs) of the cochlea are currently unknown. In the present study, we tested the hypothesis that early phenotypical changes in the SGNs of the amikacin-poisoned rat cochlea are mediated by HMGB1. Our results showed that a marked downregulation of HMGB1 had occurred by completion of amikacin treatment, coinciding with acute damage at the dendrite extremities of the SGNs. A few days later, during the recovery of the SGN dendrites, the protein was re-expressed and transiently accumulated within the nuclei of the SGNs. The phosphorylated form of the transcription factor c-Jun (p-c-Jun) was concomitantly detected in the nuclei of the SGNs where it often co-localized with HMGB1, while the anti-apoptotic protein BCL2 was over-expressed in the cytoplasm. In animals co-treated with amikacin and the histone deacetylase inhibitor trichostatin A, both HMGB1 and p-c-Jun were exclusively found within the cytoplasm. The initial disappearance of HMGB1 from the affected SGNs may be due to its release into the external medium, where it may have a cytokine-like function. Once re-expressed and translocated into the nucleus, HMGB1 may facilitate the transcriptional activity of p-c-Jun, which in turn may promote repair mechanisms. Our study therefore suggests that HMGB1 can positively influence the survival of SGNs following ototoxic exposure via both its extracellular and intranuclear functions.

Involvement of angiopoietin-like 4 in matrix remodeling during chondrogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are considered for cartilage engineering given their ability to differentiate into chondrocytes. Chondrogenic differentiation of MSCs is currently triggered by micromass culture in the presence of a member of the TGF-ß superfamily. However, the main constituents of the cartilaginous matrix, aggrecan and type II collagen, are degraded at the end of the differentiation process through induction of matrix metallopeptidase (MMP)13. We hypothesized that MSCs undergoing chondrogenic differentiation produce an intermediate cytokine that triggers this matrix remodeling. Analysis of transcriptomic data identified angiopoietin-like 4 (ANGPTL4) as one of the most strongly up-regulated gene encoding a secreted factor during TGF-ß-induced chondrogenesis. To gain insight into the role of ANGPTL4 during chondrogenesis, we used recombinant ANGPTL4 as well as a RNA interference approach. Addition of exogenous ANGPTL4 during the course of TGF-ß-induced differentiation reduced the mRNA levels of aggrecan and type II collagen, although it increased those of MMP1 and MMP13. Accordingly, deposition of aggrecan and total collagens was diminished, whereas release of MMP1 and MMP13 was increased. Conversely, transfection of MSCs with an siRNA targeting ANGPTL4 prior to induction of chondrogenesis increased expression of type II collagen and aggrecan, whereas it repressed that of MMP1, MMP3, and MMP13. A neutralizing antibody against integrin αVß5, a known receptor for ANGPTL4, mimicked some of the effects observed after siRNA-mediated ANGPTL4 silencing. Our data provide evidence that ANGPTL4 promotes cartilage matrix remodeling by inhibiting expression of its two key components and by up-regulating the level of certain MMPs.

Chemical Modification of Polyhydroxyalkanoates (PHAs) for the Preparation of Hybrid Biomaterials.

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by bacteria as intracellular granules under metabolic stress conditions. Many carbon sources such as alkanes, alkenes, alcohols, sugars, fatty acids can be used as feedstock and thus a wide variety of polyesters and monomer units can be potentially synthetized. The work presented here describes the process to chemically modify such biopolymers in order to render them readily available for the preparation of bio-molecular conjugates as promising new classes of biocompatible biomaterials. Such hybrid biomaterials belong to the rapidly growing class of biocompatible polymers, which are of great interest for medical and therapeutic applications. In this work, the biosynthesis of a new PHA homopolymer and the chemical modification, an epoxidation reaction, are described.

The nNOS-p38MAPK pathway is mediated by NOS1AP during neuronal death.

Neuronal nitric oxide synthase (nNOS) and p38MAPK are strongly implicated in excitotoxicity, a mechanism common to many neurodegenerative conditions, but the intermediary mechanism is unclear. NOS1AP is encoded by a gene recently associated with sudden cardiac death, diabetes-associated complications, and schizophrenia (Arking et al., 2006; Becker et al., 2008; Brzustowicz, 2008; Lehtinen et al., 2008). Here we find it interacts with p38MAPK-activating kinase MKK3. Excitotoxic stimulus induces recruitment of NOS1AP to nNOS in rat cortical neuron culture. Excitotoxic activation of p38MAPK and subsequent neuronal death are reduced by competing with the nNOS:NOS1AP interaction and by knockdown with NOS1AP-targeting siRNAs. We designed a cell-permeable peptide that competes for the unique PDZ domain of nNOS that interacts with NOS1AP. This peptide inhibits NMDA-induced recruitment of NOS1AP to nNOS and in vivo in rat, doubles surviving tissue in a severe model of neonatal hypoxia-ischemia, a major cause of neonatal death and pediatric disability. The highly unusual sequence specificity of the nNOS:NOS1AP interaction and involvement in excitotoxic signaling may provide future opportunities for generation of neuroprotectants with high specificity.

Supporting cells regulate the remodelling of aminoglycoside-injured organ of Corti, through the release of high mobility group box 1.

To examine whether an inflammatory process occurs in the amikacin-poisoned cochlea, we investigated the presence of the cytokines tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-10. No TNF-α, IL-1ß or IL-10 was detected in the cochlear perilymph after the loss of most auditory hair cells, indicating the absence of severe inflammation. In contrast, we observed a significant and temporary increase in the level of extracellular high mobility group box 1 (HMGB1), a late mediator of inflammation that also functions as a signal of tissue damage. This increase coincided with epithelial remodelling of the injured organ of Corti, and occurred concomitantly with robust and transient cytoplasmic expression of acetylated HMGB1 within the non-sensory supporting cells, Deiters cells. Here, HMGB1 was found to be enclosed within vesicles, a number of which carried the secretory vesicle-associated membrane-bound protein Rab 27A. In addition, transient upregulation of receptor for advanced glycation end-products (RAGE), an HMGB1 membrane receptor, was found in most epithelial cells of the scarring organ of Corti when extracellular levels of HMGB1 were at their highest. Altogether, these results strongly suggest that, in stressful conditions, Deiters cells liberate HMGB1 to regulate the epithelial reorganization of the injured organ of Corti through engagement of RAGE in neighbouring epithelial cells.

Cartilage regeneration in zebrafish depends on Nrg1/ErbB signaling pathway.

Objective: Cartilage, as the majority of adult mammalian tissues, has limited regeneration capacity. Cartilage degradation consecutive to joint injury or aging then leads to irreversible joint damage and diseases. In contrast, several vertebrate species such as the zebrafish have the remarkable capacity to spontaneously regenerate skeletal structures after severe injuries. The objective of our study was to test the regenerative capacity of Meckel's cartilage (MC) upon mechanical injury in zebrafish and to identify the mechanisms underlying this process. Methods and Results: Cartilage regenerative capacity in zebrafish larvae was investigated after mechanical injuries of the lower jaw MC in TgBAC(col2a1a:mCherry), to visualize the loss and recovery of cartilage. Confocal analysis revealed the formation of new chondrocytes and complete regeneration of MC at 14 days post-injury (dpi) via chondrocyte cell cycle re-entry and proliferation of pre-existing MC chondrocytes near the wound. Through expression analyses, we showed an increase of nrg1 expression in the regenerating lower jaw, which also expresses Nrg1 receptors, ErbB3 and ErbB2. Pharmacological inhibition of the ErbB pathway and specific knockdown of Nrg1 affected MC regeneration indicating the pivotal role of this pathway for cartilage regeneration. Finally, addition of exogenous NRG1 in an in vitro model of osteoarthritic (OA)-like chondrocytes induced by IL1ß suggests that Nrg1/ErbB pathway is functional in mammalian chondrocytes and alleviates the increased expression of catabolic markers characteristic of OA-like chondrocytes. Conclusion: Our results show that the Nrg1/ErbB pathway is required for spontaneous cartilage regeneration in zebrafish and is of interest to design new therapeutic approaches to promote cartilage regeneration in mammals.

Multi-modal ECG Holter system for sleep-disordered breathing screening: a validation study.

BACKGROUND: The high prevalence of sleep disordered breathing (SDB) among heart diseases patients becomes increasingly recognized. A reliable exploring tool of SDB well adapted to cardiologists practice would be very useful for the management of these patients. METHODS: We assessed a novel multi-modal electrocardiogram (ECG) Holter which incorporated both thoracic impedance and pulse oximetry signals. We compared in a home setting, a standard condition for Holter recordings, results from the novel device to a classical ambulatory polygraph in subjects with suspected SDB. The analysis of cardiac arrhythmias in relationship with SDB is also presented. A total of 118 patients clinically suspected of having SDB were evaluated (mean age 57 ± 14 years, mean body mass index [BMI] 32 ± 6 kg/m(2)). The new device allows calculating a new index called thoracic impedance (TI) disturbance index (TIDI+) evaluated from TI and SpO(2) signals recorded from a Holter monitor. RESULTS: In the population under study, 93% had more than 70% of usable TI signal and 95% had more than 90% for SpO(2) during sleep time recording. Screening performance results based on automatic analysis is accurate: TIDI + demonstrates a high level of sensitivity (96.8%), specificity (72.3%) as well as positive (82.4%) and negative (94.4%) predictive value for the detection of SDB. Moreover, detection of SDB periods permits us to observe a possible respiratory association of several nocturnal arrhythmias. CONCLUSIONS: The multi-modal Holter should be considered as a valuable evaluating tool for SDB screening and as a case selection technique for facilitating access to a full polysomnography for severe cases. Moreover, it offers a unique opportunity to study arrhythmia consequences with both respiratory and hypoxia disturbances.

Novel mathematical processing method of nocturnal oximetry for screening patients with suspected sleep apnoea syndrome.

AIM: Polysomnography (PSG) is the current standard for diagnosing sleep apnea syndrome (SAS). A reliable test would be useful to reduce the considerable resources required for PSG. METHODS: We developed a new mathematical analysis, which quantifies amplitude variations of pulse oximetry (SpO(2)) and heart rate (HR) throughout the night, allowing measurement of the total time in which ΔSpO(2) >4% and presented as a new oximetric index ventilatory hypoxemic index (VHI). VHI was compared prospectively with standard PSG parameters apnea-hypopnea index (AHI) and oxygen desaturation index (ODI) in 106 patients (aged, 57 ± 14 years; BMI, 29 ± 5 kg/m(2)) suspected of having SAS. The criterion for diagnosis of SAS was AHI >15/h of sleep during PSG. The relationship between ΔSpO(2) and ΔΗR was also investigated. RESULTS: We observed a significant correlation between the AHI and VHI (R = 0.87, p < 0.0001). Using VHI >15 as the criterion for SpO(2), oximetry had a sensitivity of 81%, specificity of 98%, positive predictive value (PPV) of 98%, and negative predictive value (NPV) of 84% as a screening test for SAS. We also observed a clear linear relationship between ΔSpO(2) and ΔΗR, and concomitant use of VHI and the ΔSpO(2)/ΔHR slope improved the NPV to 90%. CONCLUSIONS: The present findings from wavelet-aggregate processing of oximeter data and the relationship between ΔSpO(2) and ΔΗR show promise as a useful summary prediction of screening SAS.

NRG1/ErbB signalling controls the dialogue between macrophages and neural crest-derived cells during zebrafish fin regeneration.

Fish species, such as zebrafish (Danio rerio), can regenerate their appendages after amputation through the formation of a heterogeneous cellular structure named blastema. Here, by combining live imaging of triple transgenic zebrafish embryos and single-cell RNA sequencing we established a detailed cell atlas of the regenerating caudal fin in zebrafish larvae. We confirmed the presence of macrophage subsets that govern zebrafish fin regeneration, and identified a foxd3-positive cell population within the regenerating fin. Genetic depletion of these foxd3-positive neural crest-derived cells (NCdC) showed that they are involved in blastema formation and caudal fin regeneration. Finally, chemical inhibition and transcriptomic analysis demonstrated that these foxd3-positive cells regulate macrophage recruitment and polarization through the NRG1/ErbB pathway. Here, we show the diversity of the cells required for blastema formation, identify a discrete foxd3-positive NCdC population, and reveal the critical function of the NRG1/ErbB pathway in controlling the dialogue between macrophages and NCdC.

Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

Whole embryo culture, transcriptomics and RNA interference identify TBX1 and FGF11 as novel regulators of limb development in the mouse.

Identifying genes involved in vertebrate developmental processes and characterizing this involvement are daunting tasks, especially in the mouse where viviparity complicates investigations. Attempting to devise a streamlined approach for this type of study we focused on limb development. We cultured E10.5 and E12.5 embryos and performed transcriptional profiling to track molecular changes in the forelimb bud over a 6-hour time-window. The expression of certain genes was found to diverge rapidly from its normal path, possibly reflecting the activation of a stress-induced response. Others, however, maintained for up to 3 hours dynamic expression profiles similar to those seen in utero. Some of these resilient genes were known regulators of limb development. The implication of the others in this process was either unsuspected or unsubstantiated. The localized knockdown of two such genes, Fgf11 and Tbx1, hampered forelimb bud development, providing evidence of their implication. These results show that combining embryo culture, transcriptome analysis and RNA interference could speed up the identification of genes involved in a variety of developmental processes, and the validation of their implication.

Multipotent mesenchymal stromal cells and rheumatoid arthritis: risk or benefit?

Multipotent mesenchymal stromal cells (MSCs) have raised interest mainly because of cartilage/bone differentiation potential which is now partly eclipsed by their capacity to counteract inflammation and suppress host immune responses as well as to prevent fibrosis. MSCs have been identified within joint tissues including synovium, cartilage, subchondral bone, periosteum or adipose tissue. They are characterized by their phenotype and their ability to differentiate into three lineages, chondrocytes, osteoblasts and adipocytes. MSCs have also paracrine effects through the secretion of a number of cytokines and growth factors. This may explain the trophic effects that may be of therapeutic value for rheumatic diseases including OA and RA. On the other hand, MSCs have been associated with tumour growth. MSCs migrate to the tumour stroma, express chemokines involved in the attraction of carcinoma cells in metastasis. Indeed, the aim of this review is not only to focus on new potential therapeutic applications in osteo-articular diseases, but also to assess the potential risk of MSC-based cell therapy.

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Secreted α-Klotho maintains cartilage tissue homeostasis by repressing NOS2 and ZIP8-MMP13 catabolic axis.

Progressive loss of tissue homeostasis is a hallmark of numerous age-related pathologies, including osteoarthritis (OA). Accumulation of senescent chondrocytes in joints contributes to the age-dependent cartilage loss of functions through the production of hypertrophy-associated catabolic matrix-remodeling enzymes and pro-inflammatory cytokines. Here, we evaluated the effects of the secreted variant of the anti-aging hormone α-Klotho on cartilage homeostasis during both cartilage formation and OA development. First, we found that α-Klotho expression was detected during mouse limb development, and transiently expressed during in vitro chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Genome-wide gene array analysis of chondrocytes from OA patients revealed that incubation with recombinant secreted α-Klotho repressed expression of the NOS2 and ZIP8/MMP13 catabolic remodeling axis. Accordingly, α-Klotho expression was reduced in chronically IL1ß-treated chondrocytes and in cartilage of an OA mouse model. Finally, in vivo intra-articular secreted α-Kotho gene transfer delays cartilage degradation in the OA mouse model. Altogether, our results reveal a new tissue homeostatic function for this anti-aging hormone in protecting against OA onset and progression.

A reliable and validated LC-MS/MS method for the simultaneous quantification of 4 cannabinoids in 40 consumer products.

In the past 50 years, Cannabis sativa (C. sativa) has gone from a substance essentially prohibited worldwide to one that is gaining acceptance both culturally and legally in many countries for medicinal and recreational use. As additional jurisdictions legalize Cannabis products and the variety and complexity of these products surpass the classical dried plant material, appropriate methods for measuring the biologically active constituents is paramount to ensure safety and regulatory compliance. While there are numerous active compounds in C. sativa the primary cannabinoids of regulatory and safety concern are (-)-Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and their respective acidic forms THCA-A and CBDA. Using the US Food and Drug Administration (FDA) bioanalytical method validation guidelines we developed a sensitive, selective, and accurate method for the simultaneous analysis CBD, CBDA, THC, and THCA-A in oils and THC & CBD in more complex matrices. This HPLC-MS/MS method was simple and reliable using standard sample dilution and homogenization, an isocratic chromatographic separation, and a triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) for analytes was 0.195 ng/mL over a 0.195-50.0 ng/mL range of quantification with a coefficient of correlation of >0.99. Average intra-day and inter-day accuracies were 94.2-112.7% and 97.2-110.9%, respectively. This method was used to quantify CBD, CBDA, THC, and THCA-A in 40 commercial hemp products representing a variety of matrices including oils, plant materials, and creams/cosmetics. All products tested met the federal regulatory restrictions on THC content in Canada (<10 µg/g) except two, with concentrations of 337 and 10.01 µg/g. With respect to CBD, the majority of analyzed products contained low CBD levels and a CBD: CBDA ratio of <1.0. In contrast, one product contained 8,410 µg/g CBD and a CBD: CBDA ratio of >1,000 (an oil-based product). Overall, the method proved amenable to the analysis of various commercial products including oils, creams, and plant material and may be diagnostically indicative of adulteration with non-hemp C. sativa, specialized hemp cultivars, or unique manufacturing methods.

Comparative pharmacokinetics and safety assessment of transdermal berberine and dihydroberberine.

The natural alkaloid berberine has been ascribed numerous health benefits including lipid and cholesterol reduction and improved insulin sensitivity in diabetics. However, oral (PO) administration of berberine is hindered by poor bioavailability and increasing dose often elicits gastro-intestinal side effects. To overcome the caveats associated with oral berberine, we developed transdermal (TD) formulations of berberine (BBR) and the berberine precursor dihydroberberine (DHB). These formulations were compared to oral BBR using pharmacokinetics, metabolism, and general safety studies in vivo. To complete this work, a sensitive quantitative LC-MS/MS method was developed and validated according the FDA guidelines for bioanalytical methods to simultaneously measure berberine, simvastatin, and simvastatin hydroxy acid with relative quantification used for the berberine metabolite demethylene berberine glucuronide (DBG). Acute pharmacokinetics in Sprague-Dawley rats demonstrated a statistically relevant ranking for berberine bioavailability based upon AUC0-8 as DHB TD > BBR TD >> BBR PO with similar ranking for the metabolite DBG, indicating that transdermal administration achieves BBR levels well above oral administration. Similarly, chronic administration (14 days) resulted in significantly higher levels of circulating BBR and DBG in DHB TD treated animals. Chronically treated rats were given a single dose of simvastatin with no observed change in the drugs bioavailability compared with control, suggesting the increased presence of BBR had no effect on simvastatin metabolism. This observation was further supported by consistent CYP3A4 expression across all treatment groups. Moreover, no changes in kidney and liver biomarkers, including alanine aminotransferase and alkaline phosphatase, were observed between treatment formats, and confirming previous reports that BBR has no effect on HMG-CoA expression. This study supports the safe use of transdermal compositions that improve on the poor bioavailability of oral berberine and have the potential to be more efficacious in the treatment of dyslipidemia or hypercholesterolemia.

Differential regulation of RANTES and IL-8 expression in lung adenocarcinoma cells.

In lung adenocarcinoma, expression of Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) is a predictor of survival while that of interleukin (IL)-8 is associated with a poor prognosis. In several models, tumorigenesis is abolished by RANTES, while it is facilitated by IL-8. We studied the regulation of RANTES and IL-8 expression in A549 lung adenocarcinoma cells. The effects of tumor necrosis factor (TNF)-alpha and regulators of protein kinases C (PKC)alpha/beta were tested because these have been shown to modulate cancer development and progression. TNF-alpha stimulated expression of both chemokines, while the PKCalpha/beta activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced only expression of IL-8 and inhibited TNF-alpha-induced RANTES expression. The PKCalpha/beta inhibitor Gö 6976 increased TNF-alpha-induced RANTES production and prevented its down-regulation by TPA. In contrast, it decreased TNF-alpha or TPA-induced IL-8 release. The differential regulation of RANTES and IL-8 expression was further analyzed. Site-directed mutagenesis indicated that regulation of RANTES promoter activity required two nuclear factor (NF)-kappaB response elements but not its activator protein (AP)-1 binding sites. An AP-1 and a NF-kappaB recognition sites were necessary for full induction of IL-8 promoter activity by TNF-alpha and TPA. Moreover, electrophoretic mobility shift assays demonstrated that NF-kappaB response elements from the RANTES promoter were of lower affinity than that from the IL-8 promoter. Immunoblotting experiments showed that TPA was more potent than TNF-alpha to induce in a PKCalpha/beta dependent manner the p44/p42 mitogen-activated protein kinases (MAPK) signaling cascade which controls AP-1 activity. Conversely, TPA inhibited TNF-alpha-induced NF-kappaB signaling and was a weak activator of this pathway. Thus, TPA did not sufficiently activate NF-kappaB to increase transcription through the low affinity NF-kappaB binding sites on RANTES promoter and its inhibitory effect on TNF-alpha-induced NF-kappaB signaling resulted in a reduced transcription rate. On IL-8 promoter, increased transcription through the high affinity NF-kappaB binding site occurred even with poorly activated NF-kappaB and the functional AP-1 response element compensated any loss of transcription rate. These data provide a mechanistic insight into the differential regulation of IL-8 and RANTES expression by PKCalpha/beta in lung adenocarcinoma cells.