Background
==========

The immunomodulatory potential of probiotic bacteria is of increasing interest for prophylactic and therapeutic options in various complex disorders, ranging from diarrhea to allergy. However, clinical and intervention studies with probiotics are not universally effective. For instance, the well-known probiotic *Lactobacillus rhamnosus* GG (LGG) shows promising results in the prevention of atopic disease \[[@B1]-[@B3]\], prevention and relief of diarrhea \[[@B4]\] and prevention of respiratory tract infections in children \[[@B5]\], while the results in Crohn's disease are less promising \[[@B6]-[@B8]\]. Clearly, the mode and timing of delivery and severity of the conditions are important factors in determining probiotic efficacy. To select the optimal strain for each application, detailed molecular knowledge would be of great help. Many immunomodulatory molecules of bacteria are microbe-associated molecular patterns or MAMPs that can interact with pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). PRRs expressed on host cells such as intestinal epithelial cells (IECs) and dendritic cells (DCs) in the gastro-intestinal tract appear to be especially crucial for probiotic-host interactions. The interaction between a MAMP and a PRR on host cells results in the induction of signalling cascades that mount a molecular response. This response can include the production of pro- or anti-inflammatory cytokines and chemokines, antimicrobial molecules and cytoprotective factors \[[@B9]\].

Many MAMPs are surface located molecules, such as lipopolysaccharides interacting with TLR4/MD2 and flagellin interacting with TLR5. Lipoteichoic acid (LTA) is a widely studied MAMP of the low G+C subdivision of the Gram-positive bacteria such as the *Firmicutes*. LTA is an amphiphilic molecule of which the hydrophilic region is generally composed of a 1,3-phosphodiester-linked polymer of glycerol-phosphate or ribitol-phosphate variously substituted with D-alanine or sugars. The hydrophobic region is a glycolipid, often Glc(β1-6)Glc(β1-3)diacylglycerol \[[@B10]\]. LTA of several pathogens such as *Staphylococcus aureus*, *Streptococcus pyogenes* and *Streptococcus pneumoniae* has been shown to trigger the production of pro-inflammatory cytokines, nitric oxide (NO) and other pro-inflammatory mediators in various cell types \[[@B10],[@B11]\]. Similar to LPS, LTA appears to be an important endotoxin capable of causing sepsis. However, whereas LPS is active in the nanomolar range, biological activities of LTA are mostly observed in the micromolar range \[[@B11]\]. Moreover, the activity of LTA is often debated due to problems with contaminants such as lipoproteins and loss of bio-active residues during isolation and purification \[[@B12]\]. Some studies have indicated that TLR2 is an important PRR for pathogenic LTA of *Streptococcus pneumoniae*\[[@B13]\] and *Staphylococcus aureus*\[[@B14]\], but generally without detailed mechanistic insights.

Recently, LTA molecules of probiotic lactobacilli have gained interest as important immune modulators based on studies with LTA mutants showing enhanced efficacy in experimental colitis models \[[@B15]\]. We observed previously that LGG wild-type can aggravate colitic symptoms in a DSS-induced colitis model in mice, suggesting that pro-inflammatory molecules of LGG itself can trigger an enhanced inflammatory response in certain conditions such as in severely progressed colitis \[[@B16]\]. In contrast to LGG wild-type, adding the *dltD* mutant of LGG seemed to improve colitic symptoms compared to the PBS control. This *dltD* mutant was previously shown to lack D-Ala on its LTA, but also expresses a modified glycolipid anchor and a shorter polyglycerol-P chain \[[@B17]\]. Similarly, a *dlt* mutant with modified D-alanylation of LTA was shown to have a higher anti-inflammatory potential in *L. plantarum* NCIMB8826 \[[@B18]\], while LTA removal in *L. acidophilus* NCFM conferred improved anti-inflammatory properties in several mouse models for IBD \[[@B19]\]. However, only very limited data exist on the structure-activity relationship of LTA of probiotic lactobacilli. In this study, we aimed to provide molecular insights in the biological activity of LTA of the widely used and model probiotic strain LGG, by investigating its potential pro-inflammatory activity and its interaction with PRRs in cell models.

Results
=======

Isolation and chemical characterization of native and modified LTA of LGG
-------------------------------------------------------------------------

Highly purified LTA of LGG was isolated as described in Methods. In order to verify its structure, purified LTA was analysed with ^1^H-NMR. Hereto, 5 mg of purified LTA was dissolved in D~2~O. The obtained spectra were compared to ^1^H-NMR-spectra described in literature and showed to have \> 99% purity (Figure [1](#F1){ref-type="fig"}).

![**NMR spectra of native and chemically modified lipoteichoic acid (LTA) of LGG.A**) ^1^H-NMR spectrum (600 MHz) of a representative sample of purified LTA from LGG wild type. **B**) ^1^H-NMR spectrum of purified LTA chemically treated to mainly remove D-alanine esters by alkaline hydrolysis in Tris buffer in D~2~O pH 8.5 for 24 h at 25°C. **C**) ^1^H-NMR spectrum of purified LTA treated by alkaline hydrolysis in 0.1M NaOH for 2 h at 37°C for chemical deacylation. Signals at 3.67 ppm (in B) and 3.56 ppm (in C) are due to the Tris buffer.](1475-2859-11-161-1){#F1}

The molecular structure determined by ^1^H-NMR spectroscopy confirmed that isolated LTA from LGG wild-type cells consists of a glycerolphosphate backbone decorated with D-alanyl esters as unique detectable substituents. The D-alanine content was calculated based on the integral ratios to be 71.8% (see structural representation in Figure [2](#F2){ref-type="fig"} based on NMR spectra in Figure [1A](#F1){ref-type="fig"}). The chain length (n) of the glycerophosphate residues in LTA from LGG wild-type in the LTA sample analysed was around 30, based on the ^1^H NMR integrals of native LTA (Figure [1A](#F1){ref-type="fig"}). Some variation was noted between different biological samples with values ranging between n = 30 and n = 76 (a representative LTA sample is shown in Figure [1](#F1){ref-type="fig"}). Analysis of the glycolipid moiety reveals an average fatty acid chain of C14, with one double bond per fatty acid (two double bonds in every membrane anchor). The position of this double bond cannot be verified from these spectra and is therefore arbitrarily represented in Figure [2](#F2){ref-type="fig"}. LTA was also isolated from the *dltD* mutant of LGG, but NMR analysis showed too much impurities for detailed studies. As an alternative, purified LTA from LGG wild-type was chemically deacylated and dealanylated for determination of the structure-activity relationships by two different treatments (moderate and harsh alkaline hydrolysis) based on \[[@B20]\] and structural changes were confirmed by NMR. A moderate alkaline hydrolysis in Tris buffer pH 8.5 for 24 h at 25°C resulted in a drastic reduction of D-Ala ester substitutions. More specifically, a 71.8% to 15.6% reduction in the degree of substitution was noticed, based on the integration ratios of chemical shifts (δ)~H~ 5.35 ppm, 4.25 ppm and 1.65 ppm (see Figure [1B](#F1){ref-type="fig"}). Only a minor effect on the acyl chains of the LTA was observed. (Compare signals at (δ)~H~ 1.2 ppm - CH~2~ (γ-ω) of the fatty acid chains - with those at (δ)~H~ 0.8 ppm - the terminal CH~3~ - in Figures [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}). The harsh alkaline hydrolysis treatment with 0.1M NaOH for 2 h at 37°C significantly reduced the acyl chains (from 100% to 45%) and removed all D-Ala substitutions (Figure [1C](#F1){ref-type="fig"}). In the remaining part of the manuscript, these chemically modified LTAs will be termed as mLTA1 (isolated LTA treated by mild alkaline hydrolysis, which results in partial removal of D-Ala) and mLTA2 (isolated LTA treated by harsh alkaline hydrolysis, which completely removed D-Ala residues and resulted in partial acyl chain removal) respectively.

![**Schematic representation of the structure of native LTA from LGG wild-type determined by NMR spectroscopy analysis.**^1^H-NMR spectra were analysed and integrated with Topspin 2.0 software. Based on the integrations of the corresponding signals in the spectra, the chain length (n) of the polyglycerolphosphate backbone was estimated between 30 and 78 depending on a biological variability (4 samples were tested). The second carbon of every glycerol was substituted with X, being either a hydroxyl group or D-alanine. The two acyl chains of LTA each have one double bound (of which the exact position was not determined) and have a total length of approximately n=14 (based on \[[@B17]\] and confirmed by integration data from this study). For the biological LTA sample of LGG wild-type (see also Figure [1](#F1){ref-type="fig"}), N = 30, while X = 71.8% D-Ala. For mLTA1, X = 15.6% D-Ala, while X = 0% for mLTA2. For mLTA2, 55% of the acyl chains of LTA were found to be removed.](1475-2859-11-161-2){#F2}

NF-κB activation of LGG LTA by interaction with TLR2/6
------------------------------------------------------

Since LTA of several bacteria, such as *Staphylococcus aureus* and *L. plantarum*, has been demonstrated to be a ligand for TLR2 \[[@B14],[@B18]\], we first aimed to investigate the importance of this PRR for the signalling capacity of LTA of LGG. Hereto, we used HEK293T cells transiently expressing TLR2 and containing a NF-κB reporter plasmid. Surprisingly, expression of only TLR2 on the surface of the HEK293T cells was insufficient to mediate NF-κB activation by LTA of LGG (Figure [3](#F3){ref-type="fig"}).

![**TLR2 mediated signalling using HEK293T cells.** HEK293T cells were transfected with pUNO-hTLR2 (Invivogen) alone (TLR2 (−)) or together with the reporter plasmid pNiFty2-SEAP (Invivogen). Cells were transiently transfected with the corresponding plasmids as described in Material & Methods. 24h after transfection, cells were incubated with Pam2CSK4 (50 ng/ml) and LTA (10 μg/ml) for 24 hours. After incubation, NF-κB-induced SEAP activity was assessed using pNPP (p-nitrophynyl phosphate) and measuring the OD at 405nm. Data represent mean values ± SD. ^\*^p-value \< 0.05 compared to the negative control (TLR2(−)).](1475-2859-11-161-3){#F3}

In contrast, transfecting the HEK293T cells with the vector pDUO-hTLR6/TLR2 (Invivogen), resulting in expression of both TLR2 and TLR6, did significantly result in activation of the NF-κB reporter plasmid by 10 μg/ml LTA of LGG (Figure [4](#F4){ref-type="fig"}). Lower concentrations of 0.1 and 1 μg/ml did not induce secreted alkaline phosphatase (SEAP) above baseline levels, which is in line with the literature \[[@B21]\].

![**TLR2/6 mediated signalling using HEK293T cells.** HEK293T cells were transfected with pDUO-hTLR6/TLR2 (Invivogen) alone (TLR2/6 (−)) or together with the reporter plasmid pNiFty2-SEAP (Invivogen). Cells were transiently transfected with the respective plasmids as described in Methods. 24h after transfection, cells were incubated with Pam2CSK4 (50 ng/ml), LTA at 0.1, 1 and 10 μg/ml, mLTA1 (10 μg/ml), mLTA2 (10 μg/ml), LGG wild-type or *dltD* mutant cells (10^8^ CFU/ml) for 24 hours. After incubation, NF-κB-induced SEAP activity was assessed using pNPP (p-nitrophynyl phosphate) and measuring the OD at 405nm. Data represent mean values ± SD. ^\*^p-value \< 0.05 compared to the negative control (TLR2/6(−)).](1475-2859-11-161-4){#F4}

Importance of D-Ala substituents and acyl chains for TLR2/6 interaction by LGG LTA
----------------------------------------------------------------------------------

To establish a structure-activity relation of LTA of LGG in interaction with TLR2/6 as PRRs, experiments were performed with chemically deacylated and dealanylated LTA, i.e. mLTA1 and mLTA2 described above. Partially modified mLTA1 showed a similar signalling through TLR2/TLR6 as native LTA, while the more severely deacetylated and fully dealanylated mLTA2 proved ineffective in activating NF-κB (Figure [4](#F4){ref-type="fig"}).

Interestingly, a similar trend was observed when investigating the interaction capacity of whole UV-irradiated bacterial cells. While LGG wild-type was able to significantly activate NF-κB signalling via TLR2/6 interaction, the *dltD* mutant, having modified LTA \[[@B17]\], significantly induced less NF-κB (Figure [4](#F4){ref-type="fig"}).

IL-8 mRNA induction in intestinal epithelial cells by LGG LTA
-------------------------------------------------------------

Subsequently, we investigated the structure-activity relationship of LGG LTA in intestinal epithelial cells. Native LTA of LGG was previously shown to induce IL-8 mRNA in Caco-2 epithelial cells with a maximum activity around 10 μg/ml \[[@B22]\]. To investigate the role of the D-Ala substituents and the lipid part of LTA, mLTA1 and mLTA2 were used as described above. Both treatments resulted in a significant reduction in IL-8 mRNA induction, highlighting the importance of both the acyl groups and the D-Ala ester substituents of LTA for this activity (Figure [5A](#F5){ref-type="fig"}).

![**Interleukin-8 mRNA response of human Caco-2 cells to 1.5 h stimulation with various compounds.** Cells were stimulated with LGG LTA, mLTA1 and mLTA2 (10 μg/ml) (panel **A**) or with live LGG WT or the *dltD* mutant strain (panel **B**) for 1.5 hours. DMEM medium served as a negative control. Bacteria were administered at 10^7^ CFUs per ml. The results shown are representative for at least three independent experiments. Data represent mean values ± SD. ^\*^p-value \< 0.05 compared to the negative control (DMEM).](1475-2859-11-161-5){#F5}

In addition, we investigated the IL-8 mRNA induction capacity of LGG wild-type versus the *dltD* mutant. Compared to the negative control (DMEM), LGG wild-type significantly induced IL-8 mRNA, while the *dltD* mutant did not (Figure [5B](#F5){ref-type="fig"}). Blocking experiments with anti-LTR2 antibodies confirmed the importance of TLR2 for IL-8 mRNA induction by LGG wild-type (data not shown).

Discussion
==========

In this study, we aimed to investigate the importance of the LTA structure of LGG in mediating pro-inflammatory effects in dedicated cell models. Hereto, we used highly purified native and chemically modified LTA of LGG. First, we showed that LGG LTA is able to activate NF-κB in HEK293T cells via interaction with the PRR couple TLR2/6. Importantly, this activity was not observed when the HEK293T cells were only transfected with TLR2, highlighting the importance of TLR6 as coreceptor for LGG LTA. This is in agreement with the PRRs of LTA molecules of *S. aureus* and group B streptococci, which were shown to interact with TLR2/6 and not TLR1/6 \[[@B23]\]*.* For lactobacilli, the pro-inflammatory potential of LTA of *L. plantarum* NCBIM8826 was shown to be TLR2 dependent using bone marrow cells isolated from TLR2^+/+^ and TLR2^−/−^ mice \[[@B18]\]. Experiments with LTA from *L. casei* and *L. fermentum* using HEK293T cells revealed that CD14 could only activate NF-κB when cotransfected with TLR2. Taken together, it seems that the pro-inflammatory character of LTA from different species is TLR2 dependent but needs coreceptors, such as TLR6 and/or CD14.

When we compared the activity of native with chemically modified LTA confirmed by NMR spectroscopy, i.e. mLTA1 (mild alkaline hydrolysis - partial removal of D-Ala) and mLTA2 (harsh alkaline hydrolysis - complete removal of D-Ala and partial acyl chain removal), D-alanylation does not appear to be crucial for TLR2/6 interaction. While a harsh chemical deacylation of 55% (mLTA2) completely abolished the NF-κB induction by LGG LTA, a severe chemical dealanylation of 72% (mLTA1) did not significantly affect this signalling. Our data confirm the essential role of the lipid part of LTA, as has been previously shown for other ligands in interaction with TLR2 \[[@B24]\]. Yet, they also suggest a modulatory role for the D-Ala substituents in signalling. This highlights the importance of structure-activity data for LTA of different species.

Subsequent experiments with the Caco-2 intestinal epithelial cells, a cell line that has been extensively used as a model for the intestinal barrier \[[@B25]\], indicate that LTA of LGG can indeed induce an inflammatory response, in this case a concentration-dependent increase in mRNA expression of the chemokine IL-8. Chemical dealanylation (mLTA1) was sufficient to significantly reduce IL-8 mRNA induction. These experiments suggest that the D-Ala substituents of LGG LTA are more important for IL-8 mRNA induction in Caco-2 cells than for NF-kB induction in HEK293T cells. This would be in agreement with the fact that full IL-8 mRNA induction in differentiated Caco-2 epithelial cells requires the interaction of LGG LTA with TLR2/6 and additional coreceptors interacting with the D-Ala substituents, which are not present in the less differentiated HEK293T cells. Furthermore, we could show in this study that the *dltD* mutant of LGG shows a significantly reduced pro-inflammatory capacity compared to wild-type, as assessed by TLR2/6-dependent NF-kB induction in HEK293T cells and IL-8 mRNA induction by Caco-2 cells. This is in agreement with its observed reduced pro-inflammatory and even enhanced anti-inflammatory and enhanced epithelial repair activity *in vivo*\[[@B16]\].

Due to pleiotropic effects of the *dltD* mutation, we were unable to isolate LTA from the *dltD* mutant that was sufficiently pure for immunomodulation experiments. Therefore, we are currently unable to answer the question as to whether the specific LTA structure of the *dltD* mutant as described in Perea Vélez *et al.*\[[@B17]\], results in this reduced pro-inflammatory capacity. Nevertheless, the data for NMR characterized chemical dealanylated and deacylated LGG LTA confirm the importance of both structural features for its pro-inflammatory potential.

In conclusion, we have convincingly shown in this study that LTA of LGG is an important MAMP of LGG for interaction with TLR2/6. Moreover, LTA is an important factor in the induction of pro-inflammatory markers in the Caco-2 intestinal epithelial cell line. This activity of LTA needs to be taken into account when selecting the optimal application for LGG. For example, in a disease situation like active colitis, extra pro-inflammatory stimulation by LTA of probiotic bacteria would be undesirable. Although human application of genetically modified probiotics such as the *dltD* mutant of LGG is not an option, experiments with mutants and isolated compounds such as shown in this study highlight the importance of tailored applications. Screening or searching for probiotic strains with less pro-inflammatory LTA seems to be important for specific disease conditions with a general pro-inflammatory profile. On the other hand, for application of probiotic strains or purified LTA as adjuvants, more immunostimulatory characteristics could be more appropriate.

Methods
=======

Bacterial strains, media and growth conditions
----------------------------------------------

LGG and the *dltD* mutant (CMPG5540) were grown at 37°C in MRS medium (Difco) or AOAC medium (Difco) under static conditions.

LTA purification
----------------

Overnight LGG wild-type cell cultures were centrifuged for 30 min (4000 rpm, 4°C) and the white cell pellets were stored at −80°C. Highly purified LTA was isolated according to Morath *et al.*\[[@B26]\]. In order to release LTA from the peptidoglycan matrix, the cells were disrupted mechanically by sonication. Hence, a defrosted aliquot of bacteria (ca. 40 g) was suspended in 80 mL of citrate buffer (0.1 M, pH = 4). The milky solution was placed in an ice bath and 4 subsequent sonication steps were accomplished (duty cycle = 50%, output \> 50). To remove the contaminant lipophilic cell molecules from the LTA containing buffer solution, an extraction step with n-butanone was applied as described by Morath *et al.*\[[@B20]\], as the classical extraction method with phenol-water at 68°C and subsequent dialysis was previously shown to partly desintegrate structure of the purified LTA.

The purification of LTA was accomplished by hydrophobic interaction chromatography (HIC) on an octyl-Sepharose CL-4B (GE Healthcare) packed in XK 16/60 column (GE Healthcare) using a linear gradient from 15% to 60% n-propanol in ammonium acetate (0.1 M, pH = 4.75). Hence, the lyophilized sample was suspended in 35 ml of chromatography start buffer (15% n-propanol in 0.1 M ammonium acetate, pH = 4.75) and centrifuged for 1 h (15000 rpm, 4°C). After sterilization by membrane filtration (0.2 μm), the supernatant was subjected to HIC.

The LTA containing fractions were identified by their phosphate content using the phosphomolybdenum test \[[@B27]\]. Therefore, to 50 μL of each fraction 200 μL of an acid solution (2 M H~2~SO~4~/0.44 M HClO~4~) was added and the samples were incubated at 140°C for 2 h. Next, a reduction solution (ammonium heptamolybdate solution: ascorbine solution 9:1) was added to each sample. After 2 h of incubation at 50°C, 200 μL of each sample was transferred into a microtiter plate. Absorption was measured at 700 nm. The phosphate content was determined by comparison to a P standard.

The LTA-containing fractions were collected and lyophilized after freezing with liquid N~2~. The lyophilized fractions were suspended in ca. 10 mL milli-Q water and lyophilized once more.

Dealanylation of LTA was achieved by increasing the pH of the water phase after butanol extraction, and by stirring at pH ≈ 8.5 with Tris buffer at RT (25°C) for 24 h as described previously \[[@B20]\]. Deacylation of LTA was performed at 37°C for 2 h with 0.1M NaOH \[[@B26]\]. LPS contamination was assessed with that LAL assay (\<0.1 EU/ml; QCL-1000; Lonza).

LTA structure analysis
----------------------

Proton (^1^H) Nuclear magnetic resonance (NMR) spectra of the LTA samples were measured in 5 mm Norell tubes on a Bruker Avance II-plus 600 MHz spectrometer equipped with a TXI-5 probe at 298 K. Spectra were obtained using D~2~O as a solvent, with the residual solvent peak at 4.7 ppm. The data acquisition, post-processing and integrations were performed in Bruker Topspin 2.1 and 2.0. The length of the polyglycerophosphate backbone, the percentage of substitution of the second position of every glycerol with D-alanine, and the average chain length of the fatty acids in the membrane anchor were calculated using the appropriate integrated signals of the proton NMR spectra.

Immunomodulation assay with Caco-2 cells
----------------------------------------

Caco-2 cells were purchased from ATCC (Rockville, MD). Cells were routinely grown in 75-cm^2^ culture flasks under conditions of 37°C, 5% CO~2~, and 90% relative humidity. Dulbecco modified Eagle medium (DMEM)/F-12 (GibcoBRL) (1:1) supplemented with 10% fetal bovine serum (FBS; HyClone) was used as the culture medium. Cells were passaged every 3 days (at 70 to 80% confluency) at a split ratio of 1 to 7. Epithelial cells were plated at a density of 10^4^ cells/cm^2^ in 12-well plates (Cellstar). Confluency was reached within 3 to 4 days after seeding, and monolayers were used for the experiments 5--7 days after seeding. Caco-2 cells were incubated with LGG wild type, the *dltD* mutant (10^7^ CFU/ml) or LTA (at different concentrations) for 1.5 hours.

Analysis of cytokine mRNA levels by qRT-PCR
-------------------------------------------

To determine cytokine mRNA levels by qRT-PCR, commercially available methodologies for performing total cellular RNA extraction (High pure RNA isolation kit, Roche), reverse transcription (SuperScript III first-strand Synthesis system, Invitrogen) and real-time DNA amplification (TaqMan Universal PCR Master Mix, Applied Biosystems) were applied. For real-time quantitative PCR (RT-qPCR) amplification, the StepOnePlus Real Time PCR system (Applied Biosystems, Lennik, Belgium) was used. All primers and probes were designed based on published sequences and chemically synthesized by Eurogentec (Seraing, Belgium). Purified plasmid DNA specific for each targeted cytokine gene served as cDNA plasmid standards and was used to quantify the respective cytokine in the test samples. The relative abundance of each mRNA species was measured by qPCR using 45 amplification cycles, with each cycle consisting of a denaturation step (94°C for 15 s) and a 1 min combined annealing-extension step (60°C). qPCR data are presented as a ratio of the mRNA level for cytokine genes over that of the housekeeping gene peptidylprolyl isomerase A (PPIA), one of the most stably expressed genes in Caco-2 cells \[[@B28]\].

Transient transfection of HEK293T cells and measurement of alkaline phosphatase activity
----------------------------------------------------------------------------------------

HEK293T cells (ATCC, Manassas, VA, USA) were cultured in DMEM/F-12 (GibcoBRL) (1:1) supplemented with 6% fetal bovine serum (FBS; HyClone) and 50μg/mL gentamycin in 5% CO~2~, 37°C, RH 90%. The cells (0.5 x 10^6^ cells/mL) were seeded in 12-well plates. These cells were then transfected with either pNiFty-SEAP alone or in combination with pUNO1-hTLR2 or pDUO-hTLR6/TLR2 (Invivogen Inc., San Diego, USA) using branched polyethylenimine (MW \~ 25.000). Transfected cells were cultured for 24 h and then incubated with Pam2CSK4 (positive control from Invivogen), LTA, UV inactivated LGG or the *dltD* mutant for an additional 24 h. All experiments were performed at least two times. After incubation, NF-κB-induced secreted alkaline phosphatase (SEAP) activity was assessed using pNPP (p-nitrophynyl phosphate) at 1 mg/mL and measuring the OD at 405 nm.

Statistics
----------

Values are presented as mean ± SD. Difference were evaluated by the unpaired Student's t test. A value of p ≤ 0.05 was considered to be significant.

Competing interests
===================

The authors declare that they have no competing interests.

Authors' contributions
======================

IJJC and MES designed and performed part of the experiments, analysed the data and wrote the paper. TLAV performed the immunomodulation experiments with Caco-2 cells. MD performed the NMR analysis of the LTA. MD and BS participated in the analysis of the NMR data and editing of the paper. SCJDK, JV and SL participated in the design and coordination of the experiments, analysis of the data and writing of the manuscript. All authors read and approved the final manuscript.

Acknowledgements
================

At the time of experiments, SL was a postdoctoral researcher of the fund for scientific research (FWO) of Flanders, Belgium and MES and IJJC held a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT--Vlaanderen). MD acknowledges FWO (Grant 1.1.955.10N). Funding of this project at the K.U.Leuven was in part through the Fund for Scientific Research, Flanders (FWO) (Grant G.0236.07) and in part through TEKES (Finnish Funding Agency for Technology and Innovation) (Grant 201/08). The authors would like to especially thank A. Demuynck for providing us the highly purified LTA.


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Q:

Is Romeo and Juliet a tragedy?

A friend of mine introduced me this riddle in a game some time ago in prose and I decided to try to make it in verse. It's a well known one. I hope it's a bit easier than the original because I was allowed some probing questions.

I enter the bedroom and immediately see
Romeo and Juliet dead, lying on the floor
Water and shattered glass all around me
What else am I supposed to look for?
Evidence and a culprit, my partner agrees
Everything clean but the garden backdoor
Some wet footprints towards the tree
There, staring at us, the perpetrator!
The crime wasn't that hard to foresee
The predator has done similar before
Poor couple, drowned to some degree
A bit confusing that sounds in the report
My dear and old friends ceased to be
An aquarium was their doom and my sore
Black story... What just happened here?
In just one phrase, simple, no more.

A:

What happened was

They were fish

I enter the bedroom and immediately see
Romeo and Juliet dead, lying on the floor

Their tank broke.

Water and shattered glass all around me
What else am I supposed to look for?

The tank was made out of glass.

Evidence and a culprit, my partner agrees
Everything clean but the garden backdoor

Yes.

Some wet footprints towards the tree
There, staring at us, the perpetrator!

Maybe a cat?

The crime wasn't that hard to foresee
The predator has done similar before

Yeah.

Poor couple, drowned to some degree
A bit confusing that sounds in the report

Drowned of air.

My dear and old friends ceased to be
An aquarium was their doom and my sore

Yeah.

Black story... What just happened here?
In just one phrase, simple, no more.

Fish.


1. Introduction {#sec1}
===============

Diabetes mellitus (DM) is a chronic disease accompanied by a series of metabolic disorders due to insulin deficiency or impaired insulin action. The prevalence of DM is increasing globally. According to the latest data from the International Diabetes Federation (IDF), 382 million people are living with diabetes in 2013, and this figure will rise up to 592 million by 2035 \[[@B1]\]. Type 2 diabetes mellitus (T2DM) is the most common form, which accounts for around 90% of all diabetes worldwide. It is characterized by hyperglycemia and dyslipidaemia, due to a dysfunction of insulin to activate signaling pathways (insulin resistance) in metabolic target tissues \[[@B2], [@B3]\]. T2DM may result in severe complications, including renal failure, blindness, slow wound healings, and cardiovascular diseases \[[@B4]\].

Protein tyrosine phosphatases (PTPs), the regulators of tyrosine phosphorylation dependent cellular events, play an important role in numerous critical physiological processes and metabolism \[[@B5]\]. Malfunction of PTPs activity has significant implications in many human diseases \[[@B5]\]. PTP1B is the first member of PTPs superfamily isolated from human placenta and characterized as a \~50 kDa protein (435 amino acid residues), consisting of an N-terminal catalytic domain and a C-terminal segment. PTP1B is a ubiquitously expressed nonreceptor tyrosine phosphatase and contains a highly conserved catalytic motif, in which Cys215 and Arg221 are crucial for its phosphatase catalytic activity \[[@B6], [@B7]\].

PTP1B is a key negative regulator of insulin signaling pathway, which blocks the insulin-stimulated tyrosine phosphorylation of the IR and thereby the insulin receptor substrate-1 (IRS-1) \[[@B8], [@B9]\]. PTP1B-null mice displayed enhanced tyrosine phosphorylation of IR and IRS-1 in muscle and liver as a consequence of increased systemic insulin sensitivity. Moreover, these PTP1B-null mice were viable and resistant to T2DM and obesity when fed with a high fat diet \[[@B10], [@B11]\]. On the basis of these findings, screening inhibitors of PTP1B has provided a definitive strategy in the therapy of T2DM and obesity. Antisense oligonucleotides of PTP1B (depletion of PTP1B expression) and BEOV (bis-ligand oxovanadium (IV) compound, a PTP1B inhibitor) have already entered phase II clinical trials \[[@B12], [@B13]\].

Magnolia bark is a traditional Chinese medicine, known under the name of*houpu* (*Magnolia officinalis* Rehder & E. H. Wilson). It is also a drug-food resource identified by the Ministry of Health of the People\'s Republic of China. The principal substantial compounds in magnolia bark are phenolic compounds and terpenoids. Magnolia bark has been used for years to treat a variety of disorders including anxiety, nervous disturbances, "stagnation of qi" (low energy), asthma, and digestive problems \[[@B14]\]. In the present study, we prepared*Magnolia officinalis* extract (ME) from dried root barks of*Magnolia officinalis*. We found that ME contains potent inhibitors of PTP1B and displays high selectivity towards PTPs. Meanwhile, ME can activate insulin signaling pathway in 3T3-L1 adipocytes and C2C12 myotubes. More importantly, it can dramatically lower the blood glucose levels in db/db mice. The data highlighted the inhibitory potential of ME targeted on PTP1B as anti-T2DM drug discovery.

2. Materials and Methods {#sec2}
========================

2.1. Materials and Chemicals {#sec2.1}
----------------------------

All the dried magnolia barks were purchased from the Jilin Grand Pharmacy and were identified by Academy of Traditional Chinese Medicine and Chinese Medicinal Materials of Jilin Province, China, as*Magnolia officinalis* Rehder & E. H. Wilson. Recombinant proteins containing the catalytic domains of protein tyrosine phosphatase 1B (PTP1B), T cell protein tyrosine phosphatase (TCPTP), Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 (SHP1), Src homology 2 (SH2) domain-containing tyrosine phosphatase 2 (SHP2), and hematopoietic protein tyrosine phosphatase (HePTP) were purified as previously described \[[@B15]--[@B18]\]. Bovine serum albumin (BSA), Tris, p-nitrophenyl phosphate (pNPP), and PVDF membranes were purchased from Milipore. 3T3-L1 preadipocytes and C2C12 myoblasts were obtained from KeyGEN Biotech, and all cell culture reagents were from GIBCO. Antiphosphotyrosine (sc-7020), anti-GLUT1 (sc-7903), anti-GLUT4 (sc-1608), and anti-phospho-ERK1/2 (sc-7383) antibodies were purchased from Santa Cruz. Anti-IR*β* (\#3025) and anti-phospho-IR*β* (\#3021) were purchased from Cell Signaling Technology. Anti-ERK1/2 (bs-0022R) and HRP-conjugated secondary antibodies were purchased from Beijing Biosynthesis Biotechnology. Anti-*β*-actin (TA-09) was purchased from ZSGB-BIO. The necessary apparatus for SDS-PAGE and Western blot were bought from Bio-Rad.

2.2. Preparation of ME {#sec2.2}
----------------------

ME was extracted from the dried root barks of*Magnolia officinalis*. Briefly, the dried root barks of*Magnolia officinalis* were extracted three times through condensate reflux with 95% methanol (*W* : *V* = 1 kg : 10 L) in 55°C water bath, then filtered, and concentrated to obtain the methanol extract. The methanol extract was dissolved in distilled water (*W* : *V* = 1 : 1) and extracted three times with n-hexane at a volume ratio of 1 : 1 and aqueous phase I was separated. The aqueous phase I was extracted three times with dichloromethane at a volume ratio of 1 : 1 to obtain aqueous phase II. The aqueous phase II was extracted three times with ethyl acetate at a volume ratio of 1 : 1, and ethyl acetate layer was concentrated with a rotary evaporator in 50°C water bath to obtain ME.

2.3. PTP Inhibition Assays {#sec2.3}
--------------------------

The PTPs activities were measured by addition of 10 *μ*L of 100 mmol/L*p*NPP (as the substrate) into a buffer solution (pH 7.0) containing 50 mM MOPS, 100 mM NaCl, 1 mM ethylene diamine tetra-acetic acid (EDTA), 1 mM DL-dithiothreitol (DTT), 1 mg/mL BSA, and 50 *μ*g/mL recombinant PTP1B or other PTPs, along with or without different concentrations of ME diluted in dimethyl sulfoxide (DMSO). After incubation for 10 min at 37°C, the reactions were terminated with 0.1 M NaHCO~3~, and the amount of the product,*p*-nitrophenol (*p*NP), was measured by UV absorbance at a wavelength of 405 nm. The inhibitory potency of inhibitors was evaluated by IC~50~ values.

2.4. Determination of Inhibition Kinetics {#sec2.4}
-----------------------------------------

Substrate (*p*NPP) with different concentrations was added separately to the reaction mixtures containing various concentrations of ME. The absorbance at 405 nm was measured to determine the amount of the produced*p*-nitrophenol. The inhibiting kinetic analysis was carried out according to the Lineweaver-Burk plot, 1/*v* versus 1/\[*S*\].

2.5. Cell Culture and Differentiation {#sec2.5}
-------------------------------------

3T3-L1 preadipocytes and C2C12 myoblasts were cultured in Dulbecco\'s Modified Eagle\'s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) at 37°C in humidified atmosphere with 5% CO~2~. For differentiation, 3T3-L1 preadipocytes were grown to confluence and further cultured for an additional 2 days. Differentiation was initiated by culturing in DMEM with 10% FBS, 1% PS, 0.2 nM insulin, 0.25 *μ*M dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for 2 days. Then, the medium was changed to DMEM with 10% FBS, 1% PS, 0.2 nM insulin for another 2 days. Finally, the medium was changed to DMEM with 10% FBS and 1% PS for full maturation into adipocytes. The differentiation of 3T3-L1 preadipocytes was detected by oil red O staining \[[@B19]\]. C2C12 myoblasts were stimulated to differentiate into myotubes according to a standard method \[[@B20]\]. Briefly, C2C12 myoblasts were cultured to 75% confluence in DMEM with 10% FBS, 1% PS. Then, cells were cultured in differentiation medium (DMEM supplemented with 2% horse serum and 1% PS) for 4--6 days to mature myotubes.

2.6. Western Blot {#sec2.6}
-----------------

The tyrosine phosphorylation of cellular proteins was detected in mature 3T3-L1 adipocytes and C2C12 myotubes. Cells were stimulated with ME at various concentrations for 30 min. The effects of ME on insulin signaling were also investigated in both 3T3-L1 adipocytes and C2C12 myotubes. After serum-free starvation for 4 h, cells were incubated with vehicle or ME (dissolved in DMSO) at various concentrations for 30 min and then stimulated with vehicle or 10 nM insulin (dissolved in sterile water with pH 4.0) for 5 min. All the stimulations were stopped by ice-cold PBS. Then the stimulated cells were lysed in ice-cold whole cell extraction buffer (WCEB) containing 25 mM *β*-glycerophosphate (pH 7.3), 5 mM EDTA, 2 mM EGTA, 5 mM *β*-mercaptoethanol, 1% Triton X-100, 0.1 M NaCl, and a protease inhibitor mixture (Roche Applied Science), and the lysates were centrifuged at 12,000 g for 15 min at 4°C. The supernatants were collected individually and the protein concentrations were determined by use of Bradford methods. To investigate the GLUT4 translocation, cells were incubated with ME and then insulin as above. The cell membrane fractions were harvested according to membrane protein extraction kit (BestBio, China). The proteins were separated on a 10% SDS polyacrylamide gel and electrotransferred to polyvinylidene fluoride (PVDF) membrane. Western blot analysis was performed with antibodies against actin, phosphotyrosine, phospho-IR*β*, anti-IR*β*, phospho-ERK1/2, ERK1/2, GLUT1, and GLUT4. Proteins were visualized using the enhanced chemiluminescence (ECL) method and quantified by densitometry scanning with Quantity One software.

2.7. Animal Experiments {#sec2.7}
-----------------------

Twenty SPF grade db/db mice (from the Model Animal Research Center of Nanjing University), male, 8--10 weeks of age, were selected for the studies. The mice were randomly divided into two groups with 10 mice in each group. Group 1: diabetic control mice treated through oral gavage with 0.9% saline (vehicle). Group 2: diabetic mice treated through oral gavage with 0.5 g/kg dose of ME (0.1 mL/10 g body weight) once a day for 4 weeks. All animal trial procedures instituted by the Ethical Committee for the Experimental Use of Animals and for Drug Safety Evaluation in Jilin University were followed. All mice were housed 5 in a cage and maintained at ambient temperature of 22 ± 2°C in a 12:12 h light/dark cycle with free access to food and water. During the treatment period, body weight and blood glucose of 16 h fasted mice were monitored every week.

2.8. Acute Toxicity Assay {#sec2.8}
-------------------------

Acute toxicity of ME was determined by use of Chinese Kunming mice (Mus musculus). All animal trial procedures instituted by the Ethical Committee for the Experimental Use of Animals and for Drug Safety Evaluation in Jilin University were followed. Fifty Chinese Kunming mice of both sexes, 8 weeks, 20--22 g weight (from the Experimental Animal Center of Jilin University), were randomized into five groups with 5 males and 5 females in each group and orally treated with ME once in dose of 3.2, 2.56, 2.048, 1.638, and 1.311 g/kg, respectively. The occurrence of massive toxic reactions and/or death was observed during the first 30 min periodically during the first 24 hours after administration of treatments and then daily for the following 14 days. At the end of the observational period, necropsy was performed on each animal to observe macroscopic reactions in brain, heart, kidneys, liver, spleen, stomach, large and small intestine, and lungs. The acute toxicity of ME was preliminarily evaluated by LD~50~ value (the drug dose leading to 50% lethality).

2.9. Statistical Analysis {#sec2.9}
-------------------------

Data are presented as mean ± SEM. Experiments were performed at least three times. Data were analyzed by Student\'s *t*-test and one-way ANOVA by use of Graph Pad Prism 5 software. Statistical significance was assigned if *P* \< 0.05.

3. Results {#sec3}
==========

3.1. ME Exhibited Potent PTP1B Inhibitory Activity {#sec3.1}
--------------------------------------------------

The inhibitory potency of ME on the PTP1B was evaluated according to the concentration-dependent inhibition curves as shown in [Figure 1](#fig1){ref-type="fig"}. ME was identified as a PTP1B inhibitor, with an IC~50~ of 55.96 *μ*g/mL. Our results also demonstrated selectivity of ME towards PTPs ([Table 1](#tab1){ref-type="table"}). We determined the content of magnolol and honokiol in ME by using HPLC-MS/MS. The content of magnolol and honokiol in ME is about 50% and 5%, respectively (Figure S1 available online in Supplementary Material at <http://dx.doi.org/10.1155/2015/139451>).

3.2. ME Inhibited PTP1B in a Competitive Manner {#sec3.2}
-----------------------------------------------

In order to further determine the inhibition mode of the ME on PTP1B, Lineweaver-Burk analysis was conducted. As shown in [Figure 2](#fig2){ref-type="fig"}, a common intercept of four Lineweaver-Burk lines on the *y*-axis can be obtained as ME concentration increases, suggesting that ME was a typical competitive inhibitor against PTP1B. The inhibitor constant (Ki) was a complex dissociation constant of enzyme with inhibitor. From [Figure 2](#fig2){ref-type="fig"}, −1/Ki value was determined from the *x*-axis intercept, and thus Ki was calculated to be 49 *μ*g/mL.

3.3. ME Enhanced Tyrosine Phosphorylation of Cellular Proteins {#sec3.3}
--------------------------------------------------------------

According to our knowledge, PTP1B is involved in some critical regulatory steps of signal transduction by dephosphorylation of signaling molecules; therefore, ME presumably increases protein tyrosine phosphorylation by the inhibition of PTP1B. Due to the limitation of the direct method used in detecting PTP1B enzyme activity in cells, we investigated tyrosine phosphorylation of cellular proteins to confirm whether ME has the inhibitory effect on PTP1B. Western blot analysis showed that ME enhances tyrosine phosphorylation of cellular proteins in a dose-dependent manner in 3T3-L1 adipocytes ([Figure 3(a)](#fig3){ref-type="fig"}). To confirm our results found in 3T3-L1 adipocytes, we performed the experiment in C2C12 myotubes. As indicated, the tyrosine phosphorylation of cellular proteins has been largely increased in the ME-treated C2C12 myotubes ([Figure 3(b)](#fig3){ref-type="fig"}). Therefore, it is acceptable to draw the conclusion that the inhibition of PTP1B is at least partly accounted for by the enhanced tyrosine phosphorylation.

3.4. ME Enhanced Insulin Signaling Pathway {#sec3.4}
------------------------------------------

Considering that PTP1B directly dephosphorylates IR and IRS-1, thereby negatively regulating insulin signaling pathway, we further explored whether ME could affect insulin signaling pathway.

Our study indicated that, in 3T3-L1 adipocytes, ME enhanced the insulin-induced tyrosine phosphorylation levels of IR*β* without altering the total protein levels of IR*β* in a dose-dependent manner ([Figure 4(a)](#fig4){ref-type="fig"}). These results were confirmed in C2C12 myotubes. As indicated, the insulin-stimulated phosphorylation levels of IR*β* were obviously increased in the ME-treated C2C12 myotubes ([Figure 4(b)](#fig4){ref-type="fig"}). Based on the findings that ME sensitizes insulin signaling, we next detected the effects of ME on downstream insulin signaling pathway (ERK pathway). As shown in Figures [4(c)](#fig4){ref-type="fig"} and [4(d)](#fig4){ref-type="fig"}, ME also enhanced insulin-induced the phosphorylation levels of ERK in a dose-dependent manner in 3T3-L1 adipocytes, as well as in C2C12 myotubes. We also analyzed the effects of ME on Akt in 3T3-L1 adipocytes and C2C12 myotubes, with similar results to that of IR*β* and ERK1/2 (Figure S2). Moreover, we found that ME alone cannot activate insulin pathway in the absence of insulin (Figure S3). Our findings suggested that improvement of insulin sensitivity and activation of the downstream signaling pathway by ME may be mainly attributed to the inhibition of PTP1B.

3.5. ME Promoted GLUT4 Translocation {#sec3.5}
------------------------------------

Another downstream consequence of insulin signaling is GLUT4 cell membrane translocation. Upon the demonstration that ME can activate insulin-induced ERK pathway, we investigated whether ME is also able to promote GLUT4 translocation. Compared to untreated cells, after stimulation of insulin or insulin plus various concentrations of ME, GLUT4 were aggregated at the cell membrane fractions ([Figure 5(a)](#fig5){ref-type="fig"}). The similar results were confirmed in C2C12 myotubes ([Figure 5(b)](#fig5){ref-type="fig"}). These results suggested that ME has a potential hypoglycemic function*in vivo*.

3.6. ME Decreased Blood Glucose Level in db/db Mice {#sec3.6}
---------------------------------------------------

After the confirmation of the finding that ME enhanced insulin signaling pathway and promoted GLUT4 translocation in cells, we went on to challenge these promising findings with*in vivo* situation. The db/db mice with the genetic mutation of leptin receptors suffer insulin resistance and severe hyperglycemia and thus have been widely used as a model of T2DM. As shown in [Figure 6](#fig6){ref-type="fig"}, the blood glucose levels were dramatically decreased after treatment with ME at 0.5 g/kg daily for 4 weeks in db/db mice compared to control group. The result fits well with*in vitro* and cell experiments outlining ME with its bioavailability as an efficient blood glucose lowering agent, though further*in vivo* experiments, particularly with lower administration doses and treatment time, are required to better estimate the therapeutic potential of ME. We also found that the weight gain of db/db mice during the period of intragastric administration with ME can be controlled effectively (Figure S4).

3.7. Acute Toxicity Trials of ME {#sec3.7}
--------------------------------

To explore the potential toxicity, we treated Chinese Kunming mice with different dose of ME ([Table 2](#tab2){ref-type="table"}). Lethality of mice in different groups orally treated with ME in dose of 3.200, 2.560, 2.048, 1.638, and 1.311 g/kg was 100%, 80%, 50%, 30%, and 0%, respectively. The occurrences of death were observed in 6 h after administration of treatments. Two mice died in the following day, while the surviving mice were viable over the following 14 days. Thus, the acute toxicity of ME is preliminarily evaluated as median lethal dose with 95% confidence (LD~50~ = 2.0 ± 0.2 g/kg) calculated according to the improved Bliss method. The necropsy was performed on each experimental mouse to observe macroscopic reactions in brain, heart, kidneys, liver, spleen, stomach, intestine, and lungs, and the results showed no lesions. These results indicated that ME has little toxicity and is well tolerated.

4. Discussion {#sec4}
=============

PTP1B has been identified as the main negative regulator of insulin signaling. Normally, insulin evokes a cascade of phosphorylation events, starting with the autophosphorylation of IR on multiple tyrosyl residues, which enhances IR kinase activity and leads to recruitment of IRS-1, followed by activation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB; also known as AKT), and finally glucose transporter 4 (GLUT4). Activated GLUT4 could translocate to membrane and thus improve the glucose uptake \[[@B21], [@B22]\]. Current evidence indicates that PTP1B could dephosphorylate IR with substrate specificity, inhibiting its kinase activity, and thus block insulin signaling transduction \[[@B23], [@B24]\]. Mice lacking PTP1B gene display improved insulin sensitivity with increased or prolonged tyrosine phosphorylations of IR and stay away from T2DM or obesity \[[@B10], [@B11]\]. PTP1B knockout mice do not have apparent disease phenotype and keep normal fetal survival rate compared with wild-type controls \[[@B10], [@B11]\]. Therefore, advisably inhibiting PTP1B activity is considered to evoke multiple series of physiological responses to resist T2DM and obesity via facilitating tyrosine phosphorylation of insulin signaling molecules. In fact, several studies have reported that oral administration of PTP1B inhibitor can decrease glucose in T2DM mice \[[@B25]--[@B28]\]. Antisense oligonucleotides of PTP1B (depletion of PTP1B expression) and BEOV (bis-ligand oxovanadium (IV) compound, a PTP1B inhibitor) have already entered phase II clinical trials \[[@B12], [@B13]\]. These studies indicate that PTP1B inhibitors may be promising candidates for novel antidiabetic drug development. In the present study, we revealed that ME potently inhibits PTP1B*in vitro* and greatly facilitates GLUT4 translocation from cytoplasm to cell membrane by activating insulin signaling pathway in both 3T3-L1 adipocytes and C2C12 myotubes. More importantly, we found that ME can lower blood glucose in db/db mice. Herein, we highlight ME\'s valuable potential in the development of drug against T2DM.

Although there has been growing concern on developing inhibitors of PTP1B, most of these efforts are in vain due to the challenges arising from the highly structural conservation of PTPs family and toxicity of inhibitor itself. PTP1B shares about 75% sequence identity in its core catalytic domain with TCPTP that is essential for normal hematopoiesis. TCPTP-null mice die as a result of hematopoietic defects within weeks of birth \[[@B29]\]. On the other hand, under physiological conditions, PTP1B modulates insulin signaling pathway in a reversible feedback manner, which warrants the orderly amplification and attenuation of insulin signal. However, most of the noncompetitive inhibitors act via oxidation of the catalytic Cys215 or by preventing the closure of the WPD loop, which are irreversible, consequently bringing toxicity*in vivo*. Our results showed that ME exhibits potent inhibition against PTP1B in a competitive manner and good selectivity towards PTPs*in vitro*. Meanwhile, oral LD~50~ value of ME was 2.0 ± 0.2 g/kg, suggesting the toxicity of ME is at low levels, which may be attributed to its inhibition type and selectivity. In addition, ME enhanced tyrosine phosphorylation of cellular proteins, indicating ME conquers its cell permeability and bioavailability restrictions.

Insulin signaling diverges mainly to the MAPK cascade, leading to the activation of gene expression and growth regulation, and the PI3K-AKT/PKB pathway which further regulates several different metabolic activities including glycogen synthesis and glucose uptake, as well as gene expression and survival \[[@B30]\]. The Western blot results revealed that ME not only augments the phosphorylation of ERK involved in the MAPK pathway though effects of ME on MAPK cascade should be further clarified in future studies, but also promotes GLUT4 translocation which is more relevant to the glucose lowering effects involved in the PI3K-AKT/PKB metabolic pathway. Thus the activation of insulin pathway by ME may be mainly attributed to its potent inhibition against PTP1B. However, considering ME contains a variety of ingredients and signal transduction is a complicated process, the hypoglycemic effect of ME via other mechanisms cannot be fully excluded.

As published, the antihyperglycemic effect of metformin resulted from the suppression of lipid oxidation and hepatic glucose production through AMPK \[[@B31]\]. Our findings verified that ME enhance insulin-stimulated tyrosine phosphorylation of IR*β*, and this effect was transduced downstream to yield a sustained increase in ERK phosphorylation and GLUT4 translocation, indicating that the hypoglycemic mechanism of ME is different from metformin. Interestingly, we also found that ME alone cannot activate insulin pathway (Figure S3). Actually, in the absence of insulin, the insulin pathway maintains a low level in cultured cells. Haque et al. reported that insulin is a protein hormone. It is hard to find some substance insulin-mimetic for its substitution \[[@B32]\]. Therefore, the current evidence revealed that ME functions as an insulin-sensitizer rather than an insulin-mimetic agent.

Magnolia bark is a highly aromatic herbal material from the cortex of*Magnolia officinalis* (Magnoliaceae family). As reported, magnolia bark exerts numerous and diverse pharmacological effects, including anti-inflammatory, antioxidant, antimicrobial, anxiolytic, and antiarrhythmic \[[@B14]\]. The principal substantial compounds in magnolia bark are different phenolic compounds and terpenoids. Several compounds had been identified, including gallic acid, sennosides A and B, hesperidin, naringin, syringin, and especially two neolignan compounds, magnolol and honokiol \[[@B14]\]. Previous report from Sohn et al. revealed magnolol is able to decrease the fasting blood glucose levels in GK rats and attributed the improvement of insulin resistance to chronic glycemic control \[[@B33]\]. In addition, Honokiol has been proved to be a partial nonadipogenic PPAR*γ* agonist*in vitro* which prevented hyperglycemia and weight gain in diabetic KKAy mice \[[@B34]\]. As is well-accepted, potential antidiabetic agents may act via different mechanisms. These significant findings shifted our research object to the magnolia bark, as the initial phase in series of related researches, with an attempt to reveal the inhibitory activity of ME against PTP1B and its potential antihyperglycemic mechanism due to the convenient purchase of magnolia bark from the drug store at a cheap price and easy preparation of ME in the laboratory. Through the experiment results, we are convinced of the effective inhibition of ME against PTP1B. Moreover, our results showed that the magnolol and honokiol are the major components in ME (Figure S1). Based on these findings, further studies on magnolol and honokiol have been carried out in the subsequent experiments to explore their inhibitory activity against PTP1B.

The db/db mice, generated by genetic mutation of leptin receptors resulting in overeating and subsequently obesity and diabetes, have been widely used as a model of T2DM. Here we demonstrated that oral treatment with ME resulted in a profound attenuation in hyperglycemia in db/db mice. The mechanism may be associated with improvement of insulin sensitivity by ME but more studies were needed. It should be mentioned that the db/db model may not fully replicate T2DM in human, although copy number variation at the leptin receptor gene locus is associated with metabolic traits and the risk of T2DM in human subjects \[[@B35]\]. The etiology of T2DM in human may be comprised of genetic and environmental factors. Therefore, further studies are required to validate antihyperglycemic effect of ME in other models of insulin resistance or obesity (e.g., the diet-induced obese model). In addition, oral LD~50~ value of ME indicated that ME is of low toxicity. Hence, ME is promising to benefit T2DM patients without adverse effects although its safety and efficient dosage remain to be validated. Our future research concern goes to the detection of the ME potentials on insulin signaling cascade in muscle, liver, and adipose tissue, and the effects on blood lipid profile (e.g., total cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride, and free fatty acids), along with the assessment of the cardiovascular effects of ME.

In conclusion, we demonstrated that ME contains potent inhibitors against PTP1B and displays good selectivity towards PTPs*in vitro*; meanwhile ME can efficiently enhance insulin-induced tyrosine phosphorylations of IR*β* in cell level. Furthermore, this effect is transduced downstream to yield a sustained increase in ERK phosphorylation and GLUT4 translocation, which provides a possible mechanism for its antihyperglycemic effects.

Supplementary Material {#supplementary-material-sec}
======================

######

The Supplementary Materials involve 4 figures of the quantitative analysis of magnolol and honokiol in ME by HPLC-MS/MS，Effects of ME on Akt，Effect of ME alone on IR, and The effect of ME on body weight of mice, thanks to the reviewer\'s constructive suggestion to further illustrate the components and functions of ME.

This work was supported financially by the National Natural Science Foundation of China (81102859) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (2013M541323).

DM:

:   Diabetes mellitus

T2DM:

:   Type 2 diabetes mellitus

PTPs:

:   Protein tyrosine phosphatases

PTP1B:

:   Protein tyrosine phosphatase 1B

IR*β*:

:   Insulin receptor *β*-subunit

ERK1/2:

:   Extracellular signal-regulated kinases 1/2

GLUT4:

:   Glucose transporter 4

ME:

:   *Magnolia officinalis* extract

IRS-1:

:   Insulin receptor substrate-1

TCPTP:

:   T cell protein tyrosine phosphatase

SHP1:

:   Src homology 2 (SH2) domain-containing tyrosine phosphatase 1

SHP2:

:   Src homology 2 (SH2) domain-containing tyrosine phosphatase 2

HePTP:

:   Hematopoietic protein tyrosine phosphatase

GLUT1:

:   Glucose transporter 1

PI3K:

:   Phosphatidylinositol 3-kinase

PKB:

:   Protein kinase B (also known as AKT)

AMPK:

:   AMP-activated protein kinase.

Highlights
==========

ME potently inhibits PTP1B*in vitro* and greatly facilitates GLUT4 translocation from cytoplasm to cell membrane by activating insulin signaling pathway in both 3T3-L1 adipocytes and C2C12 myotubes. More importantly, ME can lower blood glucose in db/db mice.

Conflict of Interests
=====================

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors\' Contribution
======================

Jing Sun and Yongsen Wang contributed equally to this work.

![Inhibitory potency of ME. Concentration-dependent inhibitory curve of PTP1B by ME.](BMRI2015-139451.001){#fig1}

![Inhibition kinetic analysis of ME against PTP1B. (a) Lineweaver-Burk plot of 1/*v* (min. *μ*M^−1^) versus 1/\[pNPP\] (mM^−1^) at various fixed concentrations of ME. (b) The inhibitor constant (Ki) of ME against PTP1B.](BMRI2015-139451.002){#fig2}

![Tyrosine phosphorylation of cellular proteins stimulated by ME. Differentiated 3T3-L1 adipocytes (a) and C2C12 myotubes (b) were stimulated with different doses of ME for 30 min. Equal amounts of total proteins were subjected to Western blot analysis with antibodies against phosphotyrosine (pY) and actin. Arrows indicate proteins whose phosphorylation is significantly enhanced by ME.](BMRI2015-139451.003){#fig3}

![Effects of ME on insulin signaling pathway. Differentiated 3T3-L1 adipocytes (a and c) and C2C12 myotubes (b and d) were starved for 4 h before stimulation. The cells were incubated with vehicle or ME at various concentrations for 30 min and then stimulated with vehicle or 10 nM insulin for 5 min. Tyrosine phosphorylations of IR and ERK were determined by Western blotting with anti-phospho-IR*β* and anti-phospho-ERK1/2 antibodies and were normalized with IR and ERK protein, respectively, which were then calculated as fold changes of insulin alone. Data are presented as mean ± SEM (*n* = 3). ^∗^ *P* \< 0.05, ^∗∗^ *P* \< 0.01, ^∗∗∗^ *P* \< 0.001 versus insulin alone.](BMRI2015-139451.004){#fig4}

![Effects of ME on GLUT4 translocation. Differentiated 3T3-L1 adipocytes (a) and C2C12 myotubes (b) were starved for 4 h before experiment. The cells were incubated with vehicle or ME at various concentrations for 30 min and then stimulated with vehicle or 10 nM insulin for 5 min. The cell membrane fractions were harvested. GLUT4 and GLUT1 were determined by Western blotting with anti-GLUT4 and anti-GLUT1 antibodies. Quantities of GLUT4 in cell membrane fractions were normalized against GLUT1 content and then calculated as fold changes of the insulin alone. Data are presented as mean ± SEM (*n* = 3). ^∗^ *P* \< 0.05, ^∗∗^ *P* \< 0.01, ^∗∗∗^ *P* \< 0.001 versus insulin alone.](BMRI2015-139451.005){#fig5}

![The effects of lowering the blood glucose of ME in db/db mice. Mice were orally treated with 0.9% saline alone (vehicle) and ME (0.5 g/kg) once a day for 4 weeks. Data are presented as mean ± SEM (*n* = 10). ^\#\#\#^ *P* \< 0.001 versus ME group (0 week), ^∗∗∗^ *P* \< 0.001 versus diabetic control group (4 weeks).](BMRI2015-139451.006){#fig6}

######

Selectivity of ME towards a panel of PTPs.

  ---------------------------
  PTPs    IC~50~ (*μ*g/mL)\
          ME
  ------- -------------------
  PTP1B   55.96 ± 1.14

  SHP1    501.94 ± 11.55

  SHP2    521.56 ± 15.76

  TCPTP   395.59 ± 9.76

  HePTP   677.88 ± 13.25
  ---------------------------

IC~50~ values of ME against PTPs including PTP1B, SHP1, SHP2, TCPTP, and HePTP were calculated. Data were expressed as mean ± standard error (*n* = 3).

######

Acute toxicity trials of ME (*n* = 10).

  Groups (ME g/kg)   Deaths   Lethality
  ------------------ -------- -----------
  3.200              10       100%
  2.560              8        80%
  2.048              5        50%
  1.638              3        30%
  1.311              0        0%

[^1]: Academic Editor: Julian Weghuber


/*
 * h323.c
 *
 * Copyright (C) 2015-20 ntop.org
 * Copyright (C) 2013 Remy Mudingay <mudingay@ill.fr>
 *
 */

#include "ndpi_protocol_ids.h"

#define NDPI_CURRENT_PROTO NDPI_PROTOCOL_H323

#include "ndpi_api.h"

struct tpkt {
  u_int8_t version, reserved;
  u_int16_t len;
};

void ndpi_search_h323(struct ndpi_detection_module_struct *ndpi_struct, struct ndpi_flow_struct *flow)
{
  struct ndpi_packet_struct *packet = &flow->packet;
  u_int16_t dport = 0, sport = 0;

  NDPI_LOG_DBG(ndpi_struct, "search H323\n");

  /*
    The TPKT protocol is used by ISO 8072 (on port 102)
    and H.323. So this check below is to avoid ambiguities
  */
  if((packet->tcp != NULL) && (packet->tcp->dest != ntohs(102))) {
    NDPI_LOG_DBG2(ndpi_struct, "calculated dport over tcp\n");

    /* H323  */
    if(packet->payload_packet_len > 4
       && (packet->payload[0] == 0x03)
       && (packet->payload[1] == 0x00)) {
      struct tpkt *t = (struct tpkt*)packet->payload;
      u_int16_t len = ntohs(t->len);

      if(packet->payload_packet_len == len) {
	/*
	  We need to check if this packet is in reality
	  a RDP (Remote Desktop) packet encapsulated on TPTK
	*/

	if(packet->payload[4] == (packet->payload_packet_len - sizeof(struct tpkt) - 1)) {
	  /* ISO 8073/X.224 */
	  if((packet->payload[5] == 0xE0 /* CC Connect Request */)
	     || (packet->payload[5] == 0xD0 /* CC Connect Confirm */)) {
	    NDPI_LOG_INFO(ndpi_struct, "found RDP\n");
	    ndpi_set_detected_protocol(ndpi_struct, flow, NDPI_PROTOCOL_RDP, NDPI_PROTOCOL_UNKNOWN);
	    return;
	  }
	}

	flow->l4.tcp.h323_valid_packets++;

	if(flow->l4.tcp.h323_valid_packets >= 2) {
	  NDPI_LOG_INFO(ndpi_struct, "found H323 broadcast\n");
	  ndpi_set_detected_protocol(ndpi_struct, flow, NDPI_PROTOCOL_H323, NDPI_PROTOCOL_UNKNOWN);
	}
      } else {
	/* This is not H.323 */
	NDPI_EXCLUDE_PROTO(ndpi_struct, flow);
	return;
      }
    }
  } else if(packet->udp != NULL) {
    sport = ntohs(packet->udp->source), dport = ntohs(packet->udp->dest);
    NDPI_LOG_DBG2(ndpi_struct, "calculated dport over udp\n");

    if(packet->payload_packet_len >= 6 && packet->payload[0] == 0x80 && packet->payload[1] == 0x08 &&
       (packet->payload[2] == 0xe7 || packet->payload[2] == 0x26) &&
       packet->payload[4] == 0x00 && packet->payload[5] == 0x00)
      {
	NDPI_LOG_INFO(ndpi_struct, "found H323 broadcast\n");
	ndpi_set_detected_protocol(ndpi_struct, flow, NDPI_PROTOCOL_H323, NDPI_PROTOCOL_UNKNOWN);
	return;
      }
    /* H323  */
    if(sport == 1719 || dport == 1719) {
      if((packet->payload_packet_len >= 5)
	 && (packet->payload[0] == 0x16)
	 && (packet->payload[1] == 0x80)
	 && (packet->payload[4] == 0x06)
	 && (packet->payload[5] == 0x00)) {
	NDPI_LOG_INFO(ndpi_struct, "found H323 broadcast\n");
	ndpi_set_detected_protocol(ndpi_struct, flow, NDPI_PROTOCOL_H323, NDPI_PROTOCOL_UNKNOWN);
	return;
      } else if(packet->payload_packet_len >= 20 && packet->payload_packet_len <= 117) {
	NDPI_LOG_INFO(ndpi_struct, "found H323 broadcast\n");
	ndpi_set_detected_protocol(ndpi_struct, flow, NDPI_PROTOCOL_H323, NDPI_PROTOCOL_UNKNOWN);
	return;
      } else {
	NDPI_EXCLUDE_PROTO(ndpi_struct, flow);
	return;
      }
    }
  }
}

void init_h323_dissector(struct ndpi_detection_module_struct *ndpi_struct, u_int32_t *id, NDPI_PROTOCOL_BITMASK *detection_bitmask)
{
  ndpi_set_bitmask_protocol_detection("H323", ndpi_struct, detection_bitmask, *id,
				      NDPI_PROTOCOL_H323,
				      ndpi_search_h323,
				      NDPI_SELECTION_BITMASK_PROTOCOL_TCP_OR_UDP_WITH_PAYLOAD,
				      SAVE_DETECTION_BITMASK_AS_UNKNOWN,
				      ADD_TO_DETECTION_BITMASK);

  *id += 1;
}


Immunohistochemical localization of some extracellular molecules and their integrin receptors in the rat Pacinian corpuscles.
The present results suggest that laminin-1 and 3 are localized in the specialized Schwann cells of Pacinian corpuscles, in spite of incomplete deposition of the basal lamina on the surface of their cytoplasmic processes. In addition, laminin-3 is concentrated and probably function as a stop protein not only in the neuromuscular junction, but also in the specialized Schwann cells enveloping the dendritic zone of the afferent axon. No significant changes of immunostaining for both laminins and their integrin receptors following denervation of Pacinian corpuscles indicate that their synthesis is independent to afferent axon as a prerequisite for successful reinnervation.


1. Field of the Invention
The present invention relates to a method and device for inspecting a touch panel, and more particularly, to a method and device for inspecting strips (or sensors or lines) of a touch panel.
2. Description of the Prior Art
Touch panel are widely used in various electronic devices. With the advance of technology and user demands, touch control has brought convenience into many devices, from small portable electronic devices to large display panel. However, during manufacturing of touch panels, connections of strips (or sensors) on the touch panel may fail (broken or have poor contact) due to various reasons, which adversely affects production cost and product functionality. In order to ensure quality of the products and to improve the manufacturing process, a method and device inspecting the connection status of the strips on a touch panel to determine if there is a broken strip as well as the accurate location on the strip is sought after.
In order to solve these problems, the present invention provides a method and device for inspecting strips of a touch panel, which readily determines if there is a broken strip on the touch panel and the accurate broken location on the strip to provide information for improving manufacturing processes and ensure quality of the products.


Q:

How do I safely unpack optionals (AlamoFire response object) in Swift

I am using the below code to download data from a server. According to Crashlytics, we see a crash occurred (EXC_BREAKPOINT) on the conditional evaluation (the 'if' statement). I suspect it is because the code unpacking the optional member "statusCode" - I am new to Swift (10 years doing Obj-C) - and I am not certain what the best, safest way is to unpack this variable without causing a crash.
Note that this app its using SwiftyJSON, though I do not think that is relevant.

    Alamofire.request(url).responseJSON { (response) in
        if (response.response?.statusCode)! >= 200 && (response.response?.statusCode)! < 300

A:

Alamofire.request(url).validate().responseJSON { response in
    switch response.result {
    case .success(let json):
        // do something with json
    case .failure(let error):
        // handle error
    }
}

The validate() method replaces your line checking the statusCode. It defaults to using acceptableStatusCodes which are 200..<300.
I think that's the best way to handle this specific case.
For more general cases, you should avoid force unwrapping. Unwrap the optional using guard or if let. The Swift docs explain that in detail.


# Copyright 1999-2016 Gentoo Foundation
# Distributed under the terms of the GNU General Public License v2

EAPI=6

PYTHON_COMPAT=( python2_7 )
PYTHON_REQ_USE="threads(+)"

inherit eutils gnome2-utils distutils-r1

DESCRIPTION="Python experimental psychology toolkit"
HOMEPAGE="http://www.psychopy.org/"
SRC_URI="https://github.com/psychopy/psychopy/archive/${PV}.tar.gz -> ${P}.tar.gz"

LICENSE="GPL-3+"
SLOT="0"
KEYWORDS="~amd64 ~x86"

DEPEND="
	dev-python/setuptools[${PYTHON_USEDEP}]
	"
RDEPEND="
	dev-python/configobj[${PYTHON_USEDEP}]
	dev-python/lxml[${PYTHON_USEDEP}]
	dev-python/matplotlib[${PYTHON_USEDEP}]
	dev-python/moviepy[${PYTHON_USEDEP}]
	dev-python/numpy[lapack,${PYTHON_USEDEP}]
	dev-python/pillow[${PYTHON_USEDEP}]
	dev-python/pygame[${PYTHON_USEDEP}]
	dev-python/pyglet[${PYTHON_USEDEP}]
	dev-python/pyopengl[${PYTHON_USEDEP}]
	dev-python/wxpython:*[${PYTHON_USEDEP}]
	media-libs/avbin-bin
	media-libs/opencv[python,${PYTHON_USEDEP}]
	dev-python/scipy[${PYTHON_USEDEP}]
	"

python_install_all() {
	distutils-r1_python_install_all
	newicon -s scalable psychopy/monitors/psychopy-icon.svg psychopy.svg
	make_desktop_entry psychopyApp.py PsychoPy psychopy
}

pkg_postinst() {
	gnome2_icon_cache_update
}
pkg_postrm() {
	gnome2_icon_cache_update
}


Chip Somodevilla/Getty ImagesWASHINGTON, DC - SEPTEMBER 21: House Judiciary Committee member Rep. Jason Chaffetz (R-UT) questions Internal Revenue Service Commissioner John Koskinen during a hearing in the Rayburn House Office Building on Capitol Hill September 21, 2016 in Washington, DC. Despite the lack of evidence against him, Koskinen is facing impeachment threats from conservatives in the House of Representatives for his role in the destruction of computer backups containing thousands of emails sought by Congress in its investigation of political targeting. (Photo by Chip Somodevilla/Getty Images)

Tonight, House Oversight Committee chair Rep. Jason Cheffetz (R, UT-03) wrote a letter to the FBI giving them one week to turn over “memoranda, notes, summaries, [and] recordings” of James Comey’s that may detail President Donald Trump’s attempt to end the FBI investigation of Michael Flynn.

As Chairman, Rep. Chaffetz has the power to subpoena the documents if they do not cooperate.

The letter follows a lurid New York Times report that says Trump asked fired FBI Director James Comey to end the investigation into former National Security Advisor Michael Flynn, saying “I hope you can let this go.”

The story, however, is a reported summary of a phone conversation conducted with FBI officials who have access to Comey’s memos. The New York Times has not acquired the memos themselves; only other FBI officials have seen them.

Comey allegedly wrote the memo following a conversation with Trump on the day after Flynn resigned. The memos say Trump told Comey:

“I hope you can see your way clear to letting this go, to letting Flynn go. He is a good guy. I hope you can let this go.”

“While the President has repeatedly expressed his view that General Flynn is a decent man who served and protected our country, the President has never asked Mr. Comey or anyone else to end any investigation, including any investigation involving General Flynn. The President has the utmost respect for our law enforcement agencies, and all investigations. This is not a truthful or accurate portrayal of the conversation between the President and Mr. Comey.”

Trump himself has not weighed in on the story to confirm or reverse the White House’s statement, as he has with this week’s revelation that he revealed sensitive intelligence to Russian diplomats.

Reaction to the New York Times report was swift, with Speaker of the House Paul Ryan and Republican members of Congress expressing unease and calling for Comey’s immediate testimony.

Speaker Ryan spox on Comey memo: "We need to have all the facts, & it is appropriate for the House Oversight Committee to request this memo"


La prima rassegna stampa del nuovo anno include un intervento di Roberto Calasso sul Corriere della Sera a proposito del modo in cui parliamo dell’Isis, le migliori innovazioni tecnologiche secondo Wired, e un’interessante intervista a Bret Easton Ellis su Huck.

Attualità, politica, esteri

The Obama Speeches – N+1

Mentre il mandato di Barack Obama volge al termine, la rivista letteraria analizza la sua carriera di “narrator in chief”.

Intellectuals for Trump – New Yorker

Chi è il gruppo di intellettuali che sta cercando di costruire un sistema di idee intorno a Trump, che però rifiuta qualunque ideologia.

Dobbiamo superare le reticenze sull’islam – Corriere della Sera

Un intervento di Roberto Calasso sul perché sbagliamo a non prestare attenzione alle parole dell’Isis, e sull’importanza di «studiare il nemico».

Media, giornali, tech

2017’s big ideas – part one: from driverless cars to interstellar travel – Guardian

Grandi nuove idee per il nuovo anno, raccolte da varie firme del giornale britannico, in vari campi che spaziano dalle neuroscienze al cibo, dai trasporti al mondo dell’arte.

2017 Is the Year That Twitter Learns to Thrive or Dies? – Wired

Nell’ultimo anno il social network non ha acquisito nuovi utenti (o meglio ne ha acquisiti pochi) e ha faticato a trovare un modello di business. Sarà l’anno della sua fine o della sua rinascita?

Cultura

A blunt conversation about life online with Bret Easton Ellis – Huck

Un’intervista a Bret Easton Ellis su internet, distrazione, morte del romanzo, la serie online da lui diretta e la tendenza a fingere che la realtà non esiste.

Bert Kaplan, l’uomo che archivia i sogni – Pagina99

Negli anni Cinquanta uno scienziato ha iniziato a registrare i pensieri notturni delle persone, a cominciare dai nativi americani. Oggi un libro racconta la sua storia.

Tv, cinema, pop

Generation X Lost Too Many Touchstones in 2016 – Vulture

David Bowie, Prince, Leonard Cohen, George Michael, e Carrie Fisher: cosa significa la scomparsa di tante figure di riferimento per la Generazione X.

Moda, viaggi, cibo, stili di vita

The Many Ways Society Makes a Man – National Geographic

Si parla molto di generi, e delle linee di separazione tra maschi e femmine. Un dato interessante è che ogni società ha un’idea diversa di cosa fa di un uomo un uomo.

Scienza

Rewriting the code of life – New Yorker

Dove sta andando la ricerca sull’editing del Dna, tecnica che potrebbe aiutare a combattere malattie gravi e cambiare il destino di alcune specie, in questo longread del New Yorker.

What not to eat – New York Times

Abbiamo passato decadi a preoccuparci dei grassi saturi, ma il vero nemico del genere umano è lo zucchero, sostiene Gary Taubes in un libro di cui si sta parlando molto.

Il video della settimana

Un ragazzo che soffre di acrofobia prova a superare la sua paura lanciandosi con un paracadute, in questo mini-documentario selezionato dallo staff di Vimeo tra i migliori del momento.

Alcuni contenuti o funzionalità qui non sono disponibili a causa delle tue preferenze sui cookie ! Ciò accade perché la funzionalità / i contenuti contrassegnati come “Vimeo framework” utilizzano i cookie che hai scelto di mantenere disabilitati. Per visualizzare questo contenuto o utilizzare questa funzionalità, abilitare i cookie: clicca qui per aprire le tue preferenze sui cookie.

La gallery della settimana

The Cut ha pubblicato una serie interessante di scatti da Cuba: le foto sono state caricate su Instagram e poi embeddate qui.


Common psychotic symptoms can be explained by the theory of ecological perception.
The symptoms of psychiatric illness are diverse, as are the causes of the conditions that cause them. Yet, regardless of the heterogeneity of cause and presentation, a great deal of symptoms can be explained by the failure of a single perceptual function--the reprocessing of ecological perception. It is a central tenet of the ecological theory of perception that we perceive opportunities to act. It has also been found that perception automatically causes actions and thoughts to occur unless this primary action pathway is inhibited. Inhibition allows perceptions to be reprocessed into more appropriate alternative actions and thoughts. Reprocessing of this kind takes place over the entire frontal lobe and it renders action optional. Choice about what action to take (if any) is the basis for the feeling of autonomy and ultimately for the sense-of-self. When thoughts and actions occur automatically (without choice) they appear to originate outside of the self, thereby providing prima facie evidence for some of the bizarre delusions that define schizophrenia such as delusional misidentification, delusions of control and Cotard's delusion. Automatic actions and thoughts are triggered by residual stimulation whenever reprocessing is insufficient to balance automatic excitatory cues (for whatever reason). These may not be noticed if they are neutral and therefore unimportant or where actions and thoughts have a positive bias and are desirable. Responses to negative stimulus, on the other hand, are always unwelcome, because the actions that are triggered will carry the negative bias. Automatic thoughts may include spontaneous positive feelings of love and joy, but automatic negative thoughts and visualisations are experienced as hallucinations. Not only do these feel like they emerge from elsewhere but they carry a negative bias (they are most commonly critical, rude and are irrationally paranoid). Automatic positive actions may include laughter and smiling and these are welcome. Automatic behaviours that carry a negative bias, however, are unwelcome and like hallucinations, occur without a sense of choice. These include crying, stereotypies, perseveration, ataxia, utilization and imitation behaviours and catatonia.


Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro.
Previous studies have shown that calcineurin activity plays a critical role in the myotoxic activity induced by crotoxin (CTX), a group II phospholipase A(2) (PLA(2)) with neurotoxic and myotoxic actions. In order to address whether calcineurin is also important for the activity of non-neurotoxic group II PLA(2) myotoxins we have compared the effects of calcineurin inhibition on the myotoxic capacity of CTX and the non-neurotoxic PLA(2)s, myotoxin II (Mt II) and myotoxin III (Mt III) from Bothrops asper venom. Rats were treated with cyclosporin A (CsA) or FK506, calcineurin inhibitors, and received an intramuscular injection of either CTX, Mt II or Mt III into the tibialis anterior. Animals were killed 24 h after injection of toxins. Tibialis anterior was removed and stored in liquid nitrogen. Myofibers in culture were also treated with CsA or FK506 and exposed to CTX, Mt II and Mt III. It was observed that, in contrast to CTX, CsA and FK506 do not attenuate myotoxic effects induced by both Mt II and Mt III in vivo and in vitro. The results of the present study suggest that calcineurin is not essential for the myotoxic activity of Mt II and Mt III, indicating that distinct intracellular pathways might be involved in myonecrosis induced by neurotoxic CTX and non-neurotoxic Bothrops sp. PLA(2) myotoxins. Alternatively, calcineurin dependent fast fiber type shift might render the muscle resistant to the action of CTX, without affecting its susceptibility to Bothrops sp. myotoxins.


Free Tone Body Wash Sample #free #samples

“Offer valid through 6/15/14 or while supplies last. Open to US legal residents, ages 18 and up. Limit one (1) offer per email account and per household. Offer can not be combined or redeemed for cash. Not transferable. VOID where prohibited by law. Not responsible for incomplete or incorrect submissions. Allow 6-8 weeks for delivery.”

Disclaimer: This is not a paid or sponsored post. I like to share offers/samples that I sign up for myself. I always check them out first and only post offers/samples that are legit or appear to be legit to the best of my ability. I cannot guarantee them as they do not come from me. Sign up at your own responsibility.


16-Channel Organic Electrochemical Transistor Array for In Vitro Conduction Mapping of Cardiac Action Potential.
16-Channel organic electrochemical transistor arrays (OECTs) are developed for mapping the propagation and studying the characteristics of action potentials of primary cardiomyocytes. The physiological activities of a rat cardiomyocyte monolayer during a long-term culturing is revealed by this biocompatible, low-cost, and high transconductance organic electronic device. OECT has great potential to be used in cardiac and neuronal drug screening.


Menu

loving food and words

Chocolate Mud Icecream

I’ve desperately wanted to learn how to make my own ice cream for years. A lack of an ice cream maker put that on hold for a while, though. I tried the (very good) method of buying top quality commercial ice cream and adding special ingredients for dinner parties, but it wasn’t quite the same as making from scratch. Luckily for me, last year for a (ahem) ‘special age’ birthday, my lovely parents-in-law gave me a KitchenAid mixer a gift. I’ll go into all the thousands of reasons I love my Kitchenaid another day, but I will say one of those reasons is ICE CREAM MAKER ATTACHMENT.

The ingredients

No, you don’t need a KitchenAid to make ice cream. You don’t even need an expensive ice cream maker. I’ve seen them on special for as little as $30. Keep an eye out at those sales. They’re a handy little toy to have in the kitchen. I will be adding a couple of posts in the near future that go into more detail so far as ice cream making is concerned, including recipes that don’t always need a specialty machine.

This recipe is a Mandy-altered version of one that came courtesy of KitchenAid. It’s delicious. And full-on. The richest, most decadent chocolate ice cream you’re ever likely to spoil yourself with. And it’s far easier to make than you might imagine…

The Mixture

What You Need:

600ml of thickened cream

300ml full-fat milk (sorry, but there’s just no point going skim when you’re doing this one…)

300g of best quality dark cooking chocolate, roughly chopped. Best quality means best quality. You need a minimum of 60% cocoa. Trust me on this. I’ve tried to cheat. It doesn’t work. At all.

2 teaspoons of vanilla extract. In a perfect world, this means pure vanilla extract. I didn’t have that this time, so used the artificial stuff.

4 egg yolks.

155 grams of caster sugar.

Churn it!

I’ve made this dozens of times now, and except for the time I used cheap chocolate buds, it’s never failed me. The one thing I always do before beginning is to have all my ingredients ready and lined up on the bench. That means sugar weighed, eggs separated.

Step One: Place cream, milk and chocolate into a large metal bowl over a saucepan containing simmering water. Be really careful not to let any water or steam come into contact with your chocolate or it will seize. Cook cream, milk and chocolate over a low heat, stirring occasionally until it comes together as a smooth mixture.

Step Two: I use a wire whisk on my Kitchenaid for this step, but you could use any electric mixer. Place the chocolate mixture into a large bowl and allow to cool for about 10 minutes. On a low-medium speed, begin to mix. Add the vanilla, egg yolks (one at a time) and sugar. Once all ingredients are added, mix for one further minute. Remove the mixture and place in the refrigerator to chill for around half an hour. I’ve been known to leave it at this step overnight.

Step Three: The churning bit. With most ice cream makers, there’s a special bowl that needs to be frozen to use. The principal is that they’re hollow and filled with a liquid that freezes solid. I keep mine in the freezer all the time it’s not in use. That way, it’s ready when I need it, rather than having to think 24 hours in advance. The most common mistake when making homemade ice cream is not having this bowl sufficiently frozen.

I try to make sure my chilled chocolate mixture is in a bowl that’s easy to pour from, just to try and cut down on the mess factor. Following your ice cream maker’s instructions, turn the machine on and slowly pour in the chocolate mix. Churn for around 25 or 30 minutes – obviously this will vary between machines. The KitchenAid starts to make a ‘clicking’ noise as the ice cream rises up the paddle, letting you know when it’s ready. At this point, your ice cream will be more like a soft serve than anything you can scoop, so it really needs to go back in the freezer – covered – for about two hours. For the recipe I’m making later in the week, I froze mine in a square slice tin.

Enjoy with strawberries, raspberries or simple whipped cream.

*It’s important to eat your chocolate ice cream within four days due to the raw egg factor.


Lets Get The Party Started

Lets say she’s just one of the 12,964,285 individuals that earned income in 2014 requiring them to lodge an individual tax return.

Her group, the 55.1% of Australia’s population (of 23,125,868) that self generated their income for that year.

Most interesting though if we remove the portion of the population that is not likely to be in employment and requiring the lodgement of an individual tax return – [Those 8,715.527 persons either under 17 or over 65] – then Ms. Avaridge now represents 88.3% the of the 14,410.341 of the 'working age group'.

Which still leaves of course, for one reason or another, 1,446,056 persons (11.7%) of the 'working age group' that didn't lodge an individual tax return in 2014.

Our nation is a corporation which each year incurs expenses, requiring income.

Because the days of piracy are gone and we can’t roam the high seas or land masses of the planet plundering the wealth of others and shipping it back in chests of booty and we, the nation, have given up owing businesses that returns profit to the nation, such as the Commonwealth Bank and telecommunications, we are left with having to generate this income solely by one method only – Taxing the locals

So just how much a year does Ms. Avaridgeearn?

Well her tax group (individuals) paid $166.2 billion (Yes that is correct 'billion') in Income tax that year, so her average share of Tax on that tax payer figure is $12,820.

Based on the 'personal' tax rates of 2014, paying $12,820 in tax, requires her to have an annual taxable income of $65,450.

Providing for an effective income tax rate of 19.6% of her income

After Income Tax, this leaves Justa with a disposable income of $52,630.

'Disposable' is used as a loose term because as we shall see Ms. Avaridge does not get to 'dispose' of this amount totally as she may wish.

So in the words of that famous commercial advertisements “- wait-we know you want more “. - More taxes that is.

Australians paid $52 billion in GST tax in 2014.

Now a GST tax, as you know, is applied to the purchase of goods and services BUT is passed down the line to be born by the end user - such as Ms. Avaridge.

A significant portion, say 90% of the burden for this $52 billion will most logically fall on individual Australians and 88.4% of those will be in the purchasing age group (17 to death) and because the working age group (14,410,341) are in the period of their life where they are most economically active they will shoulder the highest proportion. I have chosen 80% of the 88.4% of the 90% of the total

So Justa's 2014 share of GST Tax, on this bases, is $3,014.

After GST Tax this leaves Justa with a disposable income of $49,616.

But wait-we know you want more . - More taxes that is.

The government collected in 2014 a range of miscellaneous taxes placed on things like fringe benefits; alcohol; luxury cars; wine; and rent for petroleum products, which collectively add another $32.8 billion.

No doubt these cost initially placed on Industry will be passed down the line in the final cost of products and services charges, to be eventually paid for by the consumer.

At the end of that line is our unfortunate Ms. Avaridge.

So Justa's 2014 share of these taxes , on this bases, is$2,530.

After miscellaneous Tax this leaves Justa with a disposable income of $47,078.

But wait-we know you want more . - More taxes that is.

The government collected in 2014 $74.3 billion in Capital Gains taxes, This tax, which everyone knows, is placed on the sale of things sold for more value than what you paid for them. No that's not correct! - The money is fine, the definition is not!

Well! - Except of course, if the things you sold were things that you brought to sell as part of the business of buying and selling things - Well that’s not accurate either.- It gets pretty messy

Let's use an example:

if you’re a storekeeper and you buy packs of biscuits for $1.25 and sell them for $1.50 thus making $0.25 profit then, after deducting 'operating expenses', you will pay income tax on the remainder of the profit (taxable Income)

BUT and here is the crunch- Yep pun intended!

As a storekeeper if you buy shares in say a tech company for $1.25 and then sell them for $1.50 making $0.25 profit, in this scenario, you will be subjected to a capital gains tax on the transaction - go figure.

Now you know why there is a robust and growing part of the economy for tax lawyers and commercial accountants.

But hey -I've wandered far off topic, let’s get back to Ms. Avaridge's share of the $74.3 billion of Capital Gains tax collected in 2014.

Now in all fairness to reality, a whole host of people go on for a number, if not many, years without ever incurring capital gains tax, but when they do they usually are quite large values in nature.

For example a person with a rented property may sell the property some 10/15 years after purchasing it and may make a gain in the value of the property of say $200,000.

Unfortunately for that individual that gain will fall within the year of sale and be added on to the person’s income for that year to determine the tax.

So how, you say, does the tax department know how much tax is pertinent to a capital gains?

Well the devil is in the detail. If you have filled out a tax return and most of you haven’t, (at least 75% use tax agents to do their returns) you will have noticed that in the process, there is the requirement to detail any capital gains events over a certain value that has occurred in that financial year.

I suspect this is where they get their data from.

But hang on we still haven’t worked out how much is Ms. Avaridge's average, annual, share?

Well for my purposes and this has to be a personal view, to find the average I’ve taken the total tax paying entities for 2014:

12,964,285;

Individuals;

762,287;

Companies;

381,273;

Super funds;

335,579;

Trusts;

75,557;

Partnerships;

17,575,500

TOTAL

and divided that into the $74.3 billion

So Justa's 2014 share of this tax , on this bases, is$4,227.

After Capital Gains Tax this leaves Justa with a disposable income of $42,859.

This figure of course, is of absolutely no use whatsoever except to demonstrate the concept of how ubiquitous and pervasive taxes are in populating the daily living expenses of an individual.

NOTE: Australia’s gross domestic product (GDP) in 2014 was $1.57 trillion. The total revenue collected by the Federal government in taxation for that year was $337.3 billion or 20.8% of GDP- Ms. Avaridge contribution from her annual personal production (APP) at this stage is at 34.5% of her GPI (Gross Personal Income).

But wait-we know you want more . - More taxes that is.

State Taxes
Now that we have done with the Federal taxes let’s now look at State taxes and what Ms. Avaridge will need to fork out from her ever diminishing disposable income.

To do this however we will need to induct Justa into one of the states or territories of Australia.

Given that I live in Queensland she may as well come and reside here, in the now hotter part of the country.

State taxes are a little bit easier to avoid as long as you want to live in hibernation in a cave, rely on foot-power or just a horse for transportation and a have monk like attitude towards possession of earthly goods.

For Queensland this is where this state derives its taxation money

*

Payroll tax

*

Land tax

*

Other immovable property taxes

*

Stamp duties

*

Financial institutions taxes

*

Gambling tax

*

Taxes on insurance

*

Motor vehicle taxes

*

Franchise taxes

*

Misc taxes; fees and fines.

I know what you’re about to say, a lot of these look like consumption taxes. Which is what of course they are.

But wasn't, I hear you say, the Australian community promised in 1997 that all State's consumption taxes would be eliminated with the introduction of the new federal GST?

YES! of course-But they weren't eliminated and why?

Because the people that manage state governments are just people. They have the same uncontrollable urges that are found across the whole spectrum of humans, of which the most commonly demonstrated is the urge to acquire as much as you can whenever the opportunity presents.

So the States gladly accepted the GST dollars from the Federal government but proceeded merrily on their way and held on to a number of their consumption taxes (which they had promised to forego) but of course under the Australian Constitution of 1901 the Federal government and the community for that matter, were powerless to do anything about it.

So the introduction of GST was just a sham or more precisely put - a tax scam.

Any rate let’s see what Ms. Avaridge might be up for, from her disposable income.

The Queensland Government was not helpful in providing Revenue figures for the financial year 2014. The only figures I could find online indicated $12.1 billion in taxes for the FY 2014 and that year had a population 4,737,700 of which 2,582,085 or 54.5% lodged individuals income tax returns.

So about 2.6 million commercially active adults.

However

Staying with my thesis that all taxes other than 'income' levied on businesses are treated by business as a cost of production and factored into the final sale price of any product or service these taxes will filter down to the community individuals.

You only have to listen to any good or service provider's organisation that is called to explain an unexpected price rise (in excess of inflation) and you will be presented with an explanation that the root cause is a consequence of legislative impositions (including taxation) on their business

If you need any further evidence, just have the government put up excise duty and check out the price rise in a bottle of beer.

In the final analysis businesses rarely get left with the tax cost of doing business.

Individuals in the community are at the end of the line, and just like the trickle-down economy, the trickle-down taxation affect is that Ms. Avaridge will end up footing her share for most of this $12.1 billion bill as well.

But how much is an interesting question and your guess might be just as useful as mine but I am saying that individuals will pick up at least 80% of the states revenue raising.

For this exercise I am saying that individuals of the working age group will pick up 75% of that that 80%.

So Justa's 2014 share of these taxes , on this bases, is$2,554.

After State taxes this leaves Justa with a disposable income of Just $40,249.

But wait-surely you want more . - More taxes that is.

Well sadly yes there is one lot to come - local government

Australian local governments raised approximately $11 billion in 2014 in land associated taxes but the good news for Ms. Avaridge is that again, if she doesn't own or rent property then she can totally escape local government taxes.

But let’s assume that Justa is not going to live on the bank of the creek under a sheet of iron or plastic tarp.

There are 14 million properties in Australia paying $11 billion in local governments taxes

So Justa's 2014 share of these taxes , on this bases, is$810.

After Local taxes this leaves Justa with a FINAL disposable income of her initial gross income of $65,450 of Just $39,433

That is just 60.2% of GPI (Gross Personal Income) left for the non tax components of -