Bortezomib (Velcade) is approved by the FDA for IV or SC injection in select patients with multiple myeloma or mantle cell lymphoma. The SC route functions as an alternative to IV administration for patients with poor IV access. Learn about effective strategies used to reduce injection site reactions that can occur with SC delivery. The approval for myeloma was based on a phase III international randomized noninferiority trial evaluating the safety and efficacy of bortezomib administered either IV or SC using established doses . In addition, the incidence of peripheral neuropathy (PN), a common dose-limiting toxicity for bortezomib, was reduced with SC administration compared to the IV route: 38% vs. 58%, all grades; 6% vs. 16% for grade 3 or higher . It is important to note, however, that the reconstitution of SC bortezomib is different from that for the IV administration .These data have recently been updated, confirming sustained efficacy and safety at 10 months of follow-up in the same study population; see Table 1 . The incidence of PN has been shown to be reduced further in regimens using the weekly dosing schedule for bortezomib either as a single agent or in combination with other agents . No other significant changes in the adverse event profile for bortezomib were noted in the updated analysis .The SC route offers the ability to deliver medication into the adipose tissue with more rapid absorption than oral administration and a longer duration of drug activity than intramuscular administration, and it functions as an alternative to IV administration for patients with poor IV access. To be effective, medications administered using the SC route must be delivered into the subcutaneous adipose tissue and must demonstrate consistent pharmacokinetics.The most common medication delivered through the SC route is insulin, thus its administration techniques have been adopted from that clinical experience. Two recent literature reviews evaluating SC injection technique for the administration of insulin confirm limited evidence-based guidelines . Many of the papers included in these reviews focused on patient self-injection techniques in adolescents and children. The key recommendations offered in these reviews include the continuation of accepted techniques for site preparation using an antiseptic agent and rotation of SC injection sites .Key recommendations to ensure delivery of the medication into adipose tissue and not muscle include site selection based on skin thickness and subcutaneous fat, appropriate skin fold technique prior to injection, injection angle, and needle length. The thickness of the skin in adults is between 1.9 and 2.4 mm, with little variation in different body locations or among individuals of varying race, sex, or body mass index . There are, however, differences in the thickness of subcutaneous tissue by site and sex, with women having greater SC thickness than men, SC thickness being greatest in the abdomen, and SC thickness being reduced in lean individuals .Commonly available needle sizes for insulin administration are 4 mm (5/32"), 5 mm (3/16"), 8 mm (5/16"), and 12.7 mm (1/2"). Needles that are between 4 and 6 mm have been proven effective in adults of all body sizes, allowing penetration of the skin into the adipose tissue and avoiding intramuscular penetration . Subcutaneous injections using these needles should be given at a 90° angle. Subcutaneous injections given using needles > 6 mm should be administered using a 45° angle to avoid intramuscular penetration, which is associated with different pharmacokinetics.Unlike insulin, which is self-administered by patients, the majority of chemotherapeutic agents are administered by registered nurses. Injection site reactions have been reported in clinical trials and postmarketing experiences for chemotherapeutic agents administered using the SC route . Although injection site reactions are rarely severe, they may be associated with discomfort, body image concerns due to visible skin changes, and emotional distress for the patient. Patients experiencing injection site reactions may be reluctant to continue treatment using the SC route in the presence of such reactions . Experiences gained from the SC administration of 5-azacitidine, a hypomethylating agent used to treat myelodysplastic syndromes, have provided insight into strategies to reduce local injection site reactions in patients receiving SC chemotherapeutic agents. Use of the "air sandwich" technique has been effective in limiting the severity of injection site reactions during azacitidine administration ; see Table 2 for details.Injection site reactions are common but generally mild with SC administration of bortezomib. The most common finding is a 2-cm to 3-cm area of hyperpigmentation at the injection site with or without pruritus, which is self-limiting and resolves over a period of days to weeks . More severe reactions have been reported in clinical practice, including areas of hyperpigmentation exceeding 10 cm, variable degrees of induration, and in some cases scab formation at the injection site . Treatment for injection site reactions may include the administration of oral antihistamines. Topical application of cool compresses, antihistamines, or corticosteroids is not recommended within 4 hours of drug administration, to avoid any changes in pharmacokinetics. After 4 hours, any topical treatment should be applied gently to avoid unnecessary friction to the site.Clinical trials using the SC route for other chemotherapeutic agents are in process. One agent administered via the SC route, omacetaxine mepesuccinate (Synribo), was approved by the FDA on October 26, 2012, for the treatment of adult patients with chronic or accelerated phase Philadelphia-positive chronic myeloid leukemia with resistance and/or intolerance to two or more tyrosine kinase inhibitors. Adopting a protocol for SC administration of chemotherapeutic agents that reduces the incidence and severity of injection site reactions, while ensuring delivery of the drug into the adipose tissue, will facilitate continuation of treatment using the SC route and its associated benefits: treatment efficacy, a reduced incidence of PN in the case of bortezomib, and limited time spent by patients in the clinical setting. Proper injection site protocols should include guidelines for site selection, needle size, volume of medication per site, patient education for postinjection care, and guidelines for proper documentation.


Extinction is a fundamental form of memory updating in which one learns to stop expecting an event that no longer occurs. This learning ensues when one experiences a change in environmental contingencies, that is, when an expected outcome fails to occur (simple extinction), or when a novel inflated expectation of a double outcome (overexpectation) is in conflict with the real outcome, and is a process that has been linked to amygdala function. Here, we show that in rats, the same neuronal population in the amygdala central nucleus updates reward expectancies and behaviour in both types of extinction, and neural changes in one paradigm are reflected in the other. This work may have implications for the management of addiction and anxiety disorders that require treatments based on the outcome omission, and disorders such as obesity that could use overexpectation, but not omission strategies. There are two forms of extinction learning, which are vital for adaptive behaviour: simple extinction, when an expected outcome fails to occur, and overexpectation, when an exaggerated expectation is in conflict with the actual outcome. Iordanova et al. show that both forms of extinction learning have a common neural substrate in the amygdala. The accurate representation of predictive relationships between events in the environment is critical for adaptive behaviour. In an ever-changing world, the brain must constantly update its predictions, so that costly resources are no longer spent in attempts to acquire previously offered but now unavailable rewards. Such a reduction in the predictive potency of a cue can be achieved by holding the expectation constant and manipulating the delivery of the outcome—that is, simple extinction2Omission and overexpectation represent two distinct conditions that promote extinction learning and are underscored by the same theoretical framework2323To shed light on this issue, we recorded single-unit activity from the amygdala central nucleus (CN) in rats during a single task in which expectations were violated either by omitting expected rewards (simple extinction)911The behavioural design used in this study is illustrated in As the overexpectation condition necessarily required the compounding of two previously reinforced cues, it also created the possibility that behavioural and neural responding would be influenced by the compound novelty or the integration of audio–visual information. Omission and control conditions that also use novel audio–visual compounds control for the influence of these extraneous variables. This optimizes the direct comparison of responding  between the three compounds during the critical compound probe phase.To confirm that the overexpectation and omission conditions resulted in reduction in the association between the target cues and reward, behavioural and neural responding was examined during non-reinforced presentation of the overexpectation and omission cues on test. The data presented below were obtained from 9 rats  that exhibited behavioural evidence of overexpectation and extinction during this test. The 6 rats  that failed to show these behavioural effects, also did not exhibit the neural profile described below see .Conditioned responding to the cues during the pre-conditioning and conditioning phases developed as expected, with high levels of magazine entries to the reinforced cues but not the non-reinforced  cue by the end of each phase .The critical phase of the experiment, the compound phase, followed conditioning and consisted of the following three conditions: overexpectation, omission and control. The first day of compound training, the compound probe phase, was of primary interest from both a behavioural and neural perspective. This probe session presented the greatest mismatch between real and expected outcomes for the overexpectation and for the omission compounds. Therefore, it provided the best opportunity to examine how such a mismatch, formally referred to as negative prediction error, altered behavioural and neural responding in the two extinction (overexpectation and omission) and the control compounds. It also offered insight into how the combined presentation of two previously conditioned cues summed to influence behavioural and neural responding in the overexpectation condition.post hoc comparison test for each block of four trials , and during the subsequent days of compound training . The high level of responding to the reinforced compounds was in stark contrast to that seen to the non-reinforced omission compound, which started high and similar to the control compound , but quickly declined across trials  and remained low on subsequent compound training days . Linear regression analyses supported these results: the omission compound =13.1, P<0.05), but not the other two compounds  showed a linear trend across trials. Our data confirm a well-established finding, namely that conditioned responding to previously reinforced stimuli declines when these stimuli are no longer reinforcedBehavioural responding to the reinforced compounds, that is, overexpectation and control, was similarly high across trials of the compound probe session  but not during the second half  of the compound probe session, the latter result being consistent with updated expectations.Notably, the overexpectation compound resulted in a heightened expectation of reward. Although the rats did not increase responding during presentation of the overexpectation compound when levels were already high, they did respond more during (1 s) and some time after (30 s) food pellet delivery compared with the control compound , as if cq=1.2, P>0.05) and lower compared with the control cue . Notably, the overexpectation effect observed earlier during the post-reward period in the compound probe . In other words, the greater the overexpectation of reward during the first half of the compound probe, the lower the expectation of reward to the overexpectation cue on test 4 days later.To verify that the omission and overexpectation training reduced the expectation of reward to the omission and overexpectation cues but not to the control cue, rats were tested following the end of the fourth  day of compound training. The test consisted of the non-reinforced presentation of each of the three auditory cues intermixed with reinforced presentations of the previously trained visual cue. In line with predictions, nd probe  predictend probe . That isCN neurons did not signal the discrepancy between the expected and actual rewards in overexpectation and omission  during the compound probe phase see .P<0.03). Although this population of neurons fired to reward, it also fired during the first 9 s of the compound cues that preceded reward delivery reflecting reward expectancies. This is depicted on P<0.05), a main effect of trials =6.6, P<0.05) and an interaction =2.2, P<0.05). Although the expectation of reward to the omission and control compounds was similar at the start of the compound probe session, it diverged by the end of this session such that the omission compound signalled fewer or no rewards compared with the control compound. This was reflected in both the behavioural responses (see earlier) and neural responses. Compared with the control compound, neural firing in response to the omission compound was similar during the early trials  of the compound probe day. When presenting two previously conditioned cues in compound, an overexpectation of reward was generated. In other words, more rewards were expected during the overexpectation compound compared with the control compound. Neural firing to the overexpectation compound reflected the following: compared with the control compound, neural firing to the overexpectation compound was greater during the early trials  and equivalent during the late trials (q<1) of the compound probe. A linear regression analysis confirmed the change in neural firing to the overexpectation and omission compounds, which showed a decline across trials =3.8, P=0.05; omission: F=8.0, P<0.05), but not for the control compound =1.6, P>0.05). See Neural analyses were performed on the trials of the compound probe day, that is, the first day of compound training. In all, 87 units were recorded from the CN during this session , and 65 P<0.05) and omission =4.1, P<0.05) compounds but not for the control compound =−0.1, P>0.05). In addition, the overexpectation and omission compound distributions each differed from the control compound distribution (overexpectation versus control: t(64)=2.5, P<0.05; omission versus control: t(64)=3.5, P<0.05). These results show that the reward-responsive population represented a somewhat homogeneous set of cells, which showed a decline in firing across trials to the overexpectation and omission compounds, but not the control compound.These findings were also reflected at the level of individual neurons. We computed an index of change for each cell by taking the cue-evoked neural firing to each compound at the start  minus that at the end  of the compound probe session. We obtained a distribution of positive indices , reflectpost hoc comparison test, q=2.8, P<0.05) and omission cues  showed equivalent levels of firing to each other  but lower levels of firing compared with the control cue (P<0.05), and the omission and the control cues =−2.4, P<0.05) during the initial trials of test  on test. Neural firing in these neurons reflected the differences in reward expectancy associated with the individual auditory cues, such that the overexpectation  recorded on the compound probe day (reported above) showed a decline in neural firing to both the overexpectation and omission compounds (min t(898)=2.0, P<0.0455), a number greater than expected by chance , but neither of which correlated with the control compound . Shuffling among the 16 trials . The regression coefficient confirmed this relationship ). Similarly, neural firing during the early trials of the Omission compound reflected the greatest expectation of reward for the omission condition, and this neural firing predicted the decline in behavioural responding across time during the omission compound , also confirmed by the regression coefficient ). Critically, the common population of cells uniquely predicted the relationship between neural firing and behaviour. The population of cells  see n=16 cells, r2=0.03, P=0.66, β=0.54, confidence interval  see Given that the behavioural and neural responding reported here are in line with theoretical stipulations of learningells see  that unir2=0.78, P<0.05, β=2.89, confidence interval ). This was not the case for the common population: neural summation did not predict behavioural summation  see Although neural summation at time of the reward period in overexpectation-unique population did not predict the decline in behaviour, it predicted behavioural summation  and overexpectation, we show that CN cells represent expected outcomes resulting from explicitly trained or flexible integration of associative relationships, update outcome expectation real-time as a result of changing contingencies, and do so in a correlated manner between the two paradigms. As a result these findings extend prior work using temporary or permanent cell-silencing techniques that have implicated the CN in learning as a result of outcome omission8Before delving into the functional role of the CN in simple extinction (omission) and overexpectation as revealed by the present data, a number of important alternate accounts for the increase in cell firing to the overexpectation compound can be excluded. The presentation of an auditory and visual compound during the compound phase presents both a novel as well as a multisensory stimulus. Critically, the novelty, multisensory and integrative nature of the overexpectation compound matched those in the omission and control compounds, thus making these constructs an unlikely explanation for the higher level of cell firing in overexpectation compared with the other two compounds. In addition, the behavioural and neural data obtained from the overexpectation compound do not represent greater certainty of the arrival of two pellets following overexpectation compared with the other two compounds. If so, then this certainty would be matched by the delivered outcome and should not result in a lower level of responding on test to the overexpectation cue compared with the control cue.Of course, brain regions other than the CN have been shown to be involved in learning under conditions of reward omission or overexpectation. Areas upstream such as the orbitofrontal cortex (OFC) and the basolateral amygdala (ABL) have been implicated in these processes. For example, cell firing in both OFC and ABL tends to track learning, increasing when cues are paired with reward and decreasing when they are unrewarded16171819201791714The finding that a common population of CN cells processes the decline in reward expectancy in omission and overexpectation supports the idea that the CN is a site for convergence of information when fewer-than-expected rewards are delivered at both a structural and single-unit level. Notably, changes in neural firing in this common population across learning in omission predicted similar changes in overexpectation. Thus, despite the large procedural differences between the two behavioural paradigms, cells in the CN reflected associative updating in those paradigms in a coordinated manner. It must be noted that since the omission and overexpectation compounds did not signal the same outcome , the correlation in the change in neural firing across time cannot reflect the absolute value of outcome expectancy given the compounds, but instead some other information. That information is likely to be the reduction in the reward expectancy suffered by both compounds. If this reduction represents inhibitory learning, then it must be specific to cues that suffer loss in previously established associations, as recent work shows that the CN is not necessary when a novel cue is established as a conditioned inhibitor, that is, a signal for the omission of an outcomeIn addition, the present data provide another important insight that it is the common population (not the unique populations) of cells that track the decline in reward expectation and behaviour. In other words, both learning and changes in behaviour that result from omission of an expected reward or the inflation of an expected reward expectancy have a common neural locus. This carries the implication that disruption in one of these processes, such as overexpectation, may be predictive of a general inability to learn from situations when fewer-than-expected rewards are delivered, such as in omission. Indeed work on addiction is suggestive of this; rats with history of exposure to cocaine show disruption in learning from overexpectationIt is not to say of course that neural  setting cannot be ignored. Indeed, the delivery of fewer-than-expected rewards in simple extinction (omission) and overexpectation can be said to activate an aversive motivational state123738407Twelve naive male Lister Hooded rats were used in this experiment weighing between 343 and 425 g. Rats were obtained from Charles River Laboratories . All experimental procedures were in accordance with University of Maryland and National Institute of Drug Abuse guidelines for animal testing.Immediately before and during surgery, anaesthesia was induced and maintained using an isoflurane and oxygen mix. Rats were placed in a stereotaxic apparatus (Kopf Instruments) and burr holes were drilled in the skull directly above the area of implantation. Two sixteen-channel bundles of chronic drivable electrodes were placed bilaterally at 2.3 mm posterior and 4 mm laterally to bregma. The electrodes were lowered 7 mm below the surface of the brain at the site of implantation. Rats were allowed minimum 14 days (14–20 days) for recovery from surgery before the start of the behavioural and recording procedures.Electrodes were built in-house using formvar-insulated nichrome wire . The day before surgery, the bundle of wires were freshly cut to extend ∼1 mm below the cannula holding them together and were electroplated with platinum  at an impedance of 300 K Ohms.The behavioural apparatus, where behavioural training and recording took place, consisted of aluminium chambers of black sloping walls narrowing to a 12 × 12 in floor area. The chamber floor consisted of a black plastic over a wire mesh, under which was an aluminium tray. The chamber was equipped with a recessed food cup at the center of the left sidewall, as well as two speakers, a mechanical clicker and two visual stimuli (Coulbourn Instruments) located on the same wall. A pellet dispenser was mounted on the outside of the wall and attached to the food cup, where the pellets were delivered. Head entries into the food cup were monitored by a photobeam.Three auditory and two visual stimuli were used in this experiment. The auditory cues were a 75 dB tone, 72 dB white noise and a mechanical clicker pulsing at a rate of 4 Hz (Coulbourn Instruments). The visual cues included one flashing light (light pulsing at a rate of 4 Hz) and one steady light. All cues lasted 10 s. The reward consisted of two grape-flavoured sucrose pellets.Rats were food deprived to 85% of their body weight before the start of behavioural training. Before surgery, all rats received magazine training and 5 days of pre-conditioning. Magazine training consisted of placement into the behavioural chambers with 20 sucrose pellets made available in the magazine. The session lasted 30 min and two sucrose pellets were delivered with an average intertrial interval (ITI) of 4 min (2.5–5.5 min). During the subsequent 4 days, all rats received pairings between each of three auditory cues  with two sucrose pellets as well as discriminative conditioning with two visual cues , one of which was reinforced with two sucrose pellets, while the other was not. Sucrose pellet delivery occurred at the 9th second of cue presentation and took a second to complete. The average ITI during Pavlovian training was 90 s (60–120 s). The purpose of this pre-training was to ensure that all rats were able to perform in the task and were able to acquire the visual discrimination. Once rats had recovered from surgery, a conditioning phase commenced and lasted 5 days. Training was identical to that described for the pre-surgery period. On day 6, compound training began which consisted of the presentation of each of the auditory cues in compound with a visual cue. For the overexpectation condition, one of the auditory cues  was presented simultaneously with the previously reinforced visual cue . For the omission condition, one of the remaining auditory cues  was presented in compound with the previously non-reinforced visual cue . For the control condition, the third auditory cue  was presented together with the previously non-reinforced light . The role of each auditory cue and each visual cue was counterbalanced. Both the overexpectation and control compounds were each reinforced with two sucrose pellets in a manner identical to that described earlier whereas the omission compound was not reinforced. Compound training consisted of a single 2 h session comprised of 16 presentations of each of the 3 compounds, 16 reinforced presentations of the visual cue that was previously paired with sucrose pellets and 16 non-reinforced presentations of the visual cues that was not previously paired with reward. For the compound training phase, the overexpectation compound was intermixed with the reinforced visual cue in blocks of 32 trials, while the omission and control compounds were both intermixed with the non-reinforced visual cue in two blocks of 24 trials each. These blocks of training were delivered sequentially while the rats remained in the chambers. Stimuli duration, reward delivery and the ITIs were identical to that of conditioning. Compound training lasted 4 days. To confirm that compound training was effective, a test was performed immediately following the last training session on day 4. This test consisted of four non-reinforced presentations of each of the auditory cues among pairings of the previously reinforced visual cue with sucrose pellets. All behavioural training occurred in the same context as that used for magazine training. Behavioural data were recorded by Coulbourn Instruments software and analysed with Matlab.z-scores relative to baseline firing. The mean and standard deviation of the z-score normalization were taken from the first 5 s of the pre-CS period (10 s long) for the corresponding trial and cue.Analogue signals of the neural data were collected using several identical Plexon MAP systems and sorted online using Sort Client. The neural signal was amplified, filtered and waveforms with 2.5:1 signal-to-noise ratio showing stable activity (no drifting or disappearance of waveforms across the session on the PCA scatter plot with time as factor) were selected for subsequent analyses. The units were subsequently verified using a template-matching algorithm through Offline Sorter, Plexon Inc., and only units that showed stable firing across the day's behavioural session were used for further analyses. Unit time stamps and event markers were extracted with Neuroexplorer, verified and further analysed with Matlab. Time bins of 20 ms were constructed and a rectangular smoothing function using a 100 ms window was applied to the neural data across the duration of each training session. Neural data were normalized on a trial-by-trial basis to obtain t-test. This additional and prolonged period was necessary due to the nature of the reward (pellets), allowing for delivery, obtaining and consuming the food reward. Subsequent analysis of variance, Tukey's post hoc comparison tests or Student's t-tests were used to further examine the change in neural firing of these reward-responsive cells. These analyses focused on the reward expectation period, that is, first 9 s of compound cue. The reward expectation period was defined as the first 9 s of the 10 s cue per compound presentation. The last second of the cue per compound was not included in the reward expectation analyses period as it consisted of reward delivery.Subsequently, cells were screened for excitatory reward responses. Reward responses were defined as an increase in neural firing during the 6 s reward period , from the 5 s pre-compound baseline using a Student's Programming code and data reported in this manuscript are available on request from the authors.How to cite this article: Iordanova, M. D. et al. Neural correlates of two different types of extinction learning in the amygdala central nucleus. Nat. Commun. 7:12330 doi: 10.1038/ncomms12330 (2016).Supplementary Figures 1-5 and Supplementary Notes 1-5


Peptides released from enzymatic hydrolysis of certain proteins could exert immumodulatory effects on disorders such as food allergy. Peptide-based vaccines, corresponding to T cell epitopes of particular allergens, may retain immunogenicity, while being of insufficient length to cross-link allergen specific IgE on the surface of effector cells to elicit an allergic response. Ovalbumin (OVA) hydrolysates are a rich source of biologically bioactive peptides, some of which may present stimulatory activities on immune functions. On the other hand, cell cultures derived from mouse models of food allergy have proved very helpful in the evaluation of the allergenic and immunomodulatory abilities of hydrolysates and pure peptides. The aim of this study was to evaluate OVA hydrolysates as allergenic immune modulators by using splenocyte cultures of OVA-sensitized mice. The final goal is the development of new forms of immunotherapy, safer and more effective than those using whole allergens.OVA was hydrolyzed with commercial enzymes  and their products size-fractionated (3 kDa and 10 kDa). The hydrolysates and their fractions were characterized by SDS-PAGE, RP-HPLC and HPLC-MS/MS, and their IgE-binding properties were evaluated by inhibition ELISA using sera of egg-allergic patients. Balb/c mice were sensitized to OVA by the oral administration of the protein with cholera toxin. At the end point, mice splenocytes were isolated and cultured in presence of the hydrolysates and their fractions alone, or together with the sensitizing protein. The immunomodulatory effect of the hydrolysates was based on the cytokine profile, as determined by ELISA.In splenocyte cultures incubated in presence of the hydrolysates and their fractions, a marker down-regulation of Th2-biased cytokines  was observed. When samples were stimulated with OVA, the 10 kDa-fractions showed a more pronounced reduction of Th2 cytokines, as compared with the parent hydrolysates. The levels of Th1-biased cytokines (IFN-γ and TNF-α) increased in the presence of the hydrolysates with Alcalase and Neutrase. The hydrolysates produced with Neutrase significantly increased the levels of regulatory IL-10, even in the presence of the allergen.Hydrolysates of OVA with Neutrase exhibited an effective immunomodulatory effect in the cytokine profile of OVA-sensitized mice splenocytes. This might represent a novel immunotherapeutical approach in the treatment of egg allergy.


These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role.Transient receptor potential (TRP) channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca Drosophila melanogaster. A Drosophila mutant was isolated that showed a transient electrical light response in electroretinographic recordings upon application of a prolonged light stimulus -5-nitro-3-hexenamide. When the membrane-impermeable agent 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) was administered, no rise of intracellular Ca2+ levels could be observed. However, providing DTNB from the intracellular side resulted in increased intracellular Ca2+ levels. Consistently, extracellular application of the membrane-permeable methanethiosulfonate derivative 2-amino ethyl methane thiosulfonate hydrobromide (MTSEA) but not of membrane-impermeable (2- (trimethyl ammonium) ethyl) methane thiosulonate bromide (MTSET) resulted in TRPC5 activation. The authors concluded that the cysteine residues that are modified by TRPC5 agonists are located at the intracellular side. To identify these residues, every single cysteine residue was exchanged by serine and the resulting mutant TRPC5 channels were tested for agonist sensitivity. As a result, mutation of Cys553 and Cys558 led to a drastic reduction of TRPC5 activity upon stimulation by agonists. These two residues are located N-terminal of the pore-forming loop and are conserved in other TRP channels of the TRPC and TRPV subfamilies. Interestingly, sensitivity towards protons and temperature was increased in TRPV1 upon application of SNAP [2+ influx induced by NO donors was suppressed by expression of a dominant negative TRPC5 or by downregulation of TRPC5 via a siRNA approach [2+ influx in this system. Since ATP had been shown to activate endothelial NO synthases (eNOS) via G protein-coupled receptor stimulation, the authors tried to reproduce this pathway in the endothelial cells. Application of ATP led to an increase in intracellular Ca2+ levels that was blocked by eNOS-targeted siRNA as well as by suppressors of S-nitrosylation. In aggregation, these results provide compelling evidence that S-nitrosylation is a post-translational modification regulating the activity of TRP channels in the context of NO signaling.One such covalent modification is protein S-nitrosylation that conveys redox-based signals of the cell. The signaling molecule, nitric oxide (NO), is covalently attached to the thiol group of a cysteine residue within the target protein. Since cellular Caed by NO . By hete of SNAP . To inve of SNAP ,63. Natiapproach  showing 12,14-prostaglandin J2 (15d-PGJ2), hydrogen peroxide (H2O2), and protons (H+) [TRPA1 is expressed in the termini of nociceptive sensory neurons ,65,66 anons (H+) . Site-diin vivo [Wang and colleagues identified four different disulfide bonds that are formed between five different cysteine residues in vivo . These d2 sensor [2+ levels upon hyperoxia and hypoxia in cells that expressed TRPA1. TRPA1 exhibited hydroxylation at Pro394 that was removed upon hypoxia which led to channel activation. Current responses to hyperoxia were drastically reduced when Cys633 or Cys856 residues of TRPA1 were mutated. The authors proposed that under normoxia, TRPA1 is hydroxylated at Pro394 within the N-terminal ankyrin repeat by prolyl hydroxylases (PHDs) inactivating the channel. Under hypoxia, decreased O2 concentrations lower the activity of PHDs leading to elevated levels of active TRPA1 that is not modified at Pro394. Under hyperoxia, O2 directly oxidizes Cys633 or Cys856 or both. Oxidation overrides the inactivation by hydroxylation at Pro394 and activates the channel.Takahashi and colleagues uncovered a role of TRPA1 as an O2 sensor . When thDrosophila, TRPA1 is expressed in gustatory chemosensors to prevent the fly from taking up toxic electrophiles. The discovery of Drosophila TRPA1 and its role as a receptor of toxic electrophile compounds points to a common ancestral TRPA1 chemosensor for pungent substances. Thus, the detection of toxic electrophiles via covalent cysteine modification of TRPA1, which has the important biological function of prevention of intoxication, may be conserved from insects to man [In s to man .Collectively, current data point to a complex regulation of the TRPA1 channel by a large panel of substances some of which covalently bind to different cysteine residues. Formation of intramolecular disulfide bonds seems to influence TRPA1 conformation and might regulate the accessibility of other cysteine residues. In some cases, activation of TRPA1 rather depends on the chemical nature of agonists than on their three dimensional structure.−/−Trpv1 and −/−Trpa1 mice. Application of allicin induced action potentials and activated currents in DRG neurons from both −/−Trpv1 mice expressing TRPA1 and −/−Trpa1 mice expressing TRPV1. In a behavioral assay, the authors injected allicin into the paws of the −/−Trpv1 and −/−Trpa1 mice and their wild type littermates and determined the time the animals spent licking the injected paw. As a result, both −/−Trpv1 and −/−Trpa1 mice injected with allicin spent drastically more time licking their paws then control-injected animals indicating that TRPV1 or TRPA1 alone are able to elicit this behavior. Interestingly, the allicin-injected wild type littermates spent more time licking their paws than the single knockout mutants pointing to a synergistic effect of TRPV1 and TRPA1 in this scenario. In transfected HEK 293 cells expressing rat TRPV1, extracts from onion and garlic as well as allicin also induced currents that were reversed by DTT. Currents provoked by capsaicin, which does not modify cysteine residues, were not reversed by DTT. Pretreatment with allicin led to elevated capsaicin-induced currents. The cystein-reactive compound MTSEA elicited electrical responses in DRG neurons as well as in TRPV1-expressing HEK 293 cells. Pretreatment with MTSEA as well as MTSET led to elevated capsaicin-induced currents. Moreover, MTSEA resulted in an increased open probability of TRPV1 indicating that cysteine modification alone is sufficient to activate the channel. To identify cysteine residues that mediate sensitivity to cysteine-reactive compounds, Salazar and co-workers individually mutated the 18 cysteine residues of the rat TRPV1 primary sequence. Mutation of a single residue, Cys157 of rat TRPV1 (Cys158 in human TRPV1), residing in the predicted intracellular N-terminal region, resulted in insensitivity of the channel to MTSEA and garlic and onion extracts. Response to capsaicin, however, was not impaired. To provide further evidence that Cys157 is the only residue that is needed to confer TRPV1 sensitivity to cystein-reactive compounds, the authors generated a cysteineless TRPV1 mutant as well as a cysteineless TRPV1 in which only Cys157 was present. The cysteineless TRPV1 channel was functional and could still be activated by capsaicin but not by MTSEA or by onion and garlic extracts. In contrast, the cysteinless TRPV1 harboring Cys157 recovered activation by MTSEA and onion and garlic extracts that could be reversed by DTT. These experiments provide strong evidence for Cys157 as the only residue that confers sensitivity of TRPV1 to cysteine-reactive compounds.Besides TRPC5 and TRPA1, TRPV1 has been reported to undergo covalent cysteine modifications mediated by pungent compounds and by oxidative stress ,31. When2O2) that mimics oxidative injuries, Chuang and Lin observed a slow but robust increase of the currents elicited by capsaicin [2O2-mediated oxidation of cysteine residues confer TRPV1 sensitization. To investigate the sidedness of cysteines involved in this process, the authors used membrane-impermeable dithio-bis-nitrobenzoic acid (DTNB) and membrane-permeable phenylarsine oxide (PAO) that both link adjacent thiol groups. Extracellular application of PAO but not DTNB elicited a TRPV1 response indicating the modulation-sensitive cysteines reside within the cell. These cysteine residues were then identified in a reversion mutagenesis approach using chicken TRPV1. At first, all cysteine residues were mutated and then single residues were reverted to Cys and the resulting TRPV1 channels were tested. As a result, Cys772 and Cys783 single revertants were identified as being amenable to PAO sensitization. Since these single revertants harbor only one Cys residue, the authors concluded that these Cys residues form disulfide bonds between different TRPV1 subunits. To identify Cys residues that form intra-subunit disulfide bonds, sets of Cys residues were reverted. As a result, a double revertant harboring Cys393 and Cys397 exhibited PAO sensitization. Co-expression of Cys393 and Cys397 single revertants did not restore PAO sensitivity suggesting intra-subunit formation of a disulfide bond. Notably, two Cys residues near the N-terminus and one Cys residue in the vicinity of the C-terminus mediated PAO-induced suppression of basal activity of chicken TRPV1. These residues are absent from mammalian TRPV1. Three of the four Cys residues that were found to mediate oxidative sensitization in chicken TRPV1 are conserved in mammalian TRPV1 . Since Cys158 (Cys157 in rat TRPV1) had been reported to be covalently modified by allicin [2O2 and PAO treatment. Triple mutants harboring mutations in Cys158, Cys387, and Cys767 or in Cys158, Cys391, and Cys767 were resistant to PAO. The authors concluded that upon oxidative treatment, Cys387 and Cys391 form an intra-subunit disulfide bond while Cys158 or Cys767 form disulfide bonds between different channel subunits. Upon prolonged or repeated exposure to capsaicin, sensory neurons become unresponsive to capsaicin due to a reduction of the Ca2+ influx [2O2 treatment showed drastic synergism when applied in conjunction with phorbol di-butyrate that promotes phosphorylation of TRPV1 and low pH. Taken together, these results demonstrate that oxidative stress is a major signal to modulate TRPV1 channel activity that can override desensitization and augment previous stimuli.Treating human TRPV1-expressing HEK293 cells with hydrogen peroxide  and protein tyrosine phosphatases (PTPs). Whereas protein kinases preferentially phosphorylate their target proteins at certain residues that are flanked by a consensus sequence, protein phosphatases are believed to operate in a more unspecific manner. The phosphorylation level of a certain residue of a certain protein at a given time and physiological condition is the result of the activities of kinases and phosphatases that phosphorylate or dephosphorylate this residue. Thus, kinases and phosphatases have to be tightly regulated in order to generate certain phosphorylation patterns.in vivo [The members of the canonical TRP receptor subfamily, TRPC3, TRPC6, and TRPC7, are activated by the permeant diacyl glycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and this activation is reversed by the PKC activator 12-myristate 13-acetate (PMA) ,79,80,81in vivo . This phin vivo . Mutatioin vivo .Zhu and co-workers observed activation of mouse TRPC5 by carbachol that activates muscarinic receptors . HoweverAkin to TRPC3 and TRPC5, TRPC6 is inhibited by PKC-mediated phosphorylation  but phosin vitro kinase assay using PKCε.Besides N-glycosylation and covalent modifications of cysteine residues, TRPV1 undergoes phosphorylation. Cesare and McNaughton observed that the heat-activated current in small cultured dorsal root ganglion neurons was sensitized by application of bradykinin. The sensitization by bradykinin was mimicked by activators of protein kinase C and prolonged by phosphatase inhibitors . AlthougBecause sensitization of TRPV1 by PKA had been proposed , Rathee 2+ influx into the nerve cell is the major outcome of TRPV1 stimulation, Docherty and co-workers tested the possibility of a Ca2+-mediated desensitization of TRPV1 [2+ concentrations were lowered. Moreover, desensitization was inhibited by a Ca2+ chelator and by a specific inhibitor of the Ca2+/calmodulin-dependent protein phosphatase 2B . Therefore, they proposed that the capsaicin-induced rise of intracellular Ca2+ levels activates phosphatase 2B that in turn dephosphorylates TRPV1 to promote desensitization. Testing a panel of kinases, Jung and colleagues found that only calmodulin-dependent kinase II (CaMKII) was able to render TRPV1 susceptible for activation by capsaicin after previous desensitization [Since Caof TRPV1 . Using rtization . The autJin and co-workers examined the role of the cellular tyrosine kinase c-Src in the modulation of rat TRPV1 . They ob2+ level in the nociceptive cell.In general, phosphorylation sensitizes TRPV1 for activating stimuli whereas dephosphorylation renders it less susceptible for activation. Multiple kinases that are activated through different signaling pathways phosphorylate TRPV1 at different sites that exert different functions. Therefore, TRPV1 is not only activated by diverse ligands and heat but is also regulated through inputs from diverse signaling pathways. It is therefore believed that TRPV1 serves as a signal integrator combining different input signals that ultimately result in a certain Ca2+ transients that were observed in HEK293 cells expressing wild type TRPV4. In aggregation, the authors identified a single phosphorylation site of TRPV4 that is phosphorylated by a Src family tyrosine kinase under hypotonicity which is a prerequisite for gating of the channel [The TRPV4 channel is activated by hypotonicity and is believed to function as an osmosensor in vertebrates ,54. Beca channel .per se. These results suggest that autophosphorylation of the TRPM7 C-terminus promotes substrate recognition rather than kinase activity of TRPM7 [TRPM7 is ubiquitously expressed and interestingly, in conjunction to its function as a cation channel, displays serine/threonine kinase activity and autophosphorylation ,91. TRPMof TRPM7 .Drosophila phototransduction cascade is located within a specialized compartment of the photoreceptor cells in the compound eye, the rhabdomere. The rhabdomere is built by finger-shaped evaginations of the apical membrane, known as microvilli. Proteins of the phototransduction cascade are located within or at the cytoplasmic surface of the rhabdomeric membrane. A subset of these proteins is tethered by neither inactivation nor after potential (INAD) scaffolding protein. Interaction between INAD and its binding partners is mediated by PDZ domains . INAD harbors five of these domains that consist of approximately 90 amino acids [Drosophila, the TRP channel serves as an anchor that attaches INAD and the other INAD binding partners to the rhabdomeric membrane. In addition to TRP, a second light-activated channel, TRP-like (TRPL) is also present in the rhabdomeric membrane, but is probably not attached to the INAD signaling complex [cis-3-hydroxy retinal that is thereby isomerized to all-trans-3-hydroxy retinal [2), a building block of the rhabdomeric membrane, to yield soluble inositol-1,4,5-trisphosphate, diacyl glycerol (DAG) that stays in the membrane, and protons. It has been shown that the decrease of PIP2 and the increase of protons are necessary for finally activating the ion channels TRP and TRPL [2 results in a considerable change of the curvature of the rhabdomeric membrane as manifested by a light-triggered contraction of the entire rhabdomere that may open TRP and TRPL channels mechanically [The no acids . Throughno acids . Bindingno acids ,101,102. complex . The pho retinal . This isand TRPL . Recent anically . Howeverin vitro kinase assays using immunoprecipitated signaling complexes and radioactively labeled ATP, ePKC was shown to phosphorylate INAD as well as TRP [in vitro assays may not reproduce all aspects of the physiological conditions in the cell. The first identification of an in vivo phosphorylation site of the Drosophila TRP ion channel was reported by Popescu and colleagues [pkc null mutant fly, the respective phosphopeptides could not be observed. The authors concluded that Ser982 is a phosphorylation site that is phosphorylated by ePKC in vivo. A transgenic fly that expressed a modified TRP channel in which the Ser982 phosphorylation site was ablated by exchange to Ala, displayed a prolonged deactivation of the photoresponse. However, this phenotype only became evident upon application of a very bright light stimulus. Notably, the epkc null mutant P209inaC displayed a prolonged deactivation of the photoresponse as well, although it did so under every light intensity tested.Since ePKC is a member of the INAD signaling complex, it was speculated that this protein kinase might phosphorylate other members of this complex. Indeed, by l as TRP ,107,108.lleagues  who unamDrosophila mutants of the phototransduction cascade. Flies were light- or dark-adapted and fly heads were subjected to Western blot analyses using the phosphospecific antibodies. We observed strong phosphorylation of Ser936 in dark adapted wild type flies but no phosphorylation in light-adapted wild type flies. Conversely, we found weak phosphorylation of Thr849 and Thr864 in dark adapted wild type flies and strong phosphorylation in light-adapted wild flies. These results were in accordance with our data obtained by LC-MS/MS. Additionally, we observed strong phosphorylation of Ser936 and weak phosphorylation of Thr849 and Thr864, regardless of the light conditions in mutants of the phototransduction cascade that exhibit impaired vision. In contrast, a mutant expressing a constitutively active TRP channel exhibited weak phosphorylation of Ser936 and strong phosphorylation of Thr849 and Thr864. These data indicate that in vivo, TRP dephosphorylation at Ser936 and phosphorylation at Thr849 and Thr864 depends on the phototransduction cascade but activation of the TRP channel is sufficient to trigger this process.To identify TRP phosphorylation sites with a possible role in the regulation of vision, we analyzed TRP from light- and dark-adapted flies ,43. Usinepkc null mutants. Interestingly, we also found diminished phosphorylation in light-adapted pkc53e mutants suggesting that these two protein kinases C synergistically phosphorylate TRP at Thr849. Light-adapted rolled and snf1a mutants displayed significantly elevated phosphorylation levels of Thr849. Rolled is a mitogen-activated protein kinase that has been implicated in eye development [snf1a gene encodes an AMP-activated protein kinase. AMP-activated protein kinases have been associated to the cellular energy pathway [ck1a, licorne, tao-1, and mppe mutant flies. Neither of these mutations resulted a complete dephosphorylation of Thr864 showing that neither of these enzymes exclusively controls TRP phosphorylation at Thr864. Notably, MPPE is a metallophosphoesterase that mediates the activation of α-Man II that deglycosylates rhodopsin 1 in the process of its maturation [To identify kinases and phosphatases of Thr849 and Thr864, we conducted a candidate screen using available mutants of kinases and phosphatases that are expressed in the eye. We found that Thr849 phosphorylation was compromised in light-adapted elopment ,111. The pathway . Thr864 turation . Drosophila TRP channel permanently resides within the rhabdomeric membrane, TRPL undergoes a light-dependent translocation from the rhabdomeric membrane to a storage compartment of yet elusive nature within the cell body [Drosophila and is believed to function in long term light adaptation. Using mass spectrometry, we identified nine phosphorylated serine and threonine residues of the TRPL channel [trpl-eGFP fusion constructs. The mutated channels formed multimeres with wild type TRPL and produced electrophysiological responses when expressed in trpl;trp double mutant background indistinguishable from responses produced by wild-type TRPL. These findings indicated that TRPL channels devoid of their C-terminal phosphorylation sites form fully functional channels and they argue against a role of TRPL phosphorylation for channel gating or regulation of its biophysical properties. Since TRPL undergoes light-dependent translocation, we analyzed the subcellular localization of the phosphorylation-deficient as well as the phosphomimetic TRPL-eGFP fusion proteins by water immersion microscopy (see While the ell body . Light-d channel  (Figure Using immunocytochemistry, the eGFP signal of mutated TRPL in the dark was observed in restricted regions outside of the rhabdomere and differed from the diffuse eGFP signal observed in light-adapted flies expressing either the mutated or the native TRPL-eGFP channel. This result suggests that TRPL is localized in another subcellular compartment in dark-adapted TRPL8x-eGFP mutants that may be involved in degradation of TRPL. Unexpectedly, mutation of phosphorylation sites to Asp resulted in a similar phenotype as mutation to Ala comprising defects in TRPL localization and stabilization in the dark. This finding may be explained either by assuming that in this context, Asp did not mimic phosphate groups or that a specific pattern of TRPL phosphorylation—that changes upon exposure of the flies to light or darkness—is required for correct localization and stability of the channel. Taken together, our findings indicate that phosphorylation of TRPL at C-terminal residues seems to be required for retaining TRPL at its physiological site of action and thereby preventing degradation.Drosophila TRP channel is still elusive. Generation of transgenic flies expressing modified TRP channels in which the phosphorylation sites are modified will help to shed light on this question. Furthermore, even if the physiological function of a certain post-translational modification is known, it is oftentimes not clear how that physiological function is achieved mechanistically. The mechanistic understanding of how post-translational modifications exert their physiological functions will be a major challenge in this research field.The body of data about post-translational modifications of TRP channels is growing rapidly. However, some important questions remain unresolved. For many modifications it has not been thoroughly evaluated what fraction of the total amount of channels is modified at a given site. In the case of TRP phosphorylation, this fraction may range from a few percent to almost complete phosphorylation of all channel molecules. With regard to that, it would be important to find out whether a certain post-translational modification triggers a physiological response when present at a single subunit of a channel tetramer or only when this post-translational modification occurs at all four subunits at once. TRP regulation by modification of cysteine residues seems to be complex in some cases involving intra- and inter-subunit disulfide bonds that might influence the accessibility of cysteines by cysteine-modifying channel activators. In future work, the conditions under which certain disulfide bonds are formed have to be determined in greater detail and the physiological consequences have to be examined carefully. The physiological role of the massive C-terminal phosphorylation of the


Class II phosphoinositide 3-kinase α-isoform (PI3K-C2α) is involved in regulating KCl-induced vascular smooth muscle contraction. The current study was to investigate the effects of sevoflurane (SEVO) and isoflurane (ISO) on KCl-elicited PI3KC2α mediated vasoconstriction in rat aortic smooth muscle.Isometric force, in the absence or presence of SEVO or ISO , PI3K inhibitor LY294002, Rho kinase inhibitor Y27632, and membrane translocation of PI3K-p85, PI3K-C2α, Rho kinase (Rock II), or phosphorylation of MYPT1/Thr853, MYPT1/Thr696, CPI-17/Thr38 and MLC in response to KCl (60 mM) was measured by using isometric force transducer and western blotting analysis, respectively.p <0.05, p < 0.01, respectively). Not only Y27632 (1 uM), but also LY294002 (1 mM), inhibited KCl-induced Rock-II membrane translocation (p < 0.01). SEVO and ISO inhibited KCl-stimulated MLC phosphorylation, PI3K-C2α and Rock-II,not PI3K p85 membrane translocation in a concentration-dependent manner in rat aorta. Both SEVO and ISO suppressed the MYPT1/Thr853, not MYPT1/Thr696 and CPI-17/Thr38, MLC phosphorylation in response to KCl.KCl elicited a rapid and sustained contraction of rat aortic smooth muscle that was inhibited by both SEVO and ISO in a concentration-dependent manner, and also suppressed by LY294002 (1 mM) and Y27632 (1 uM). LY294002 (1 mM) and Y27632 (1 uM) also inhibited KCl-induced MLC phosphorylation. LY294002 (1 mM) inhibited KCl-induced PI3K-p85, PI3K-C2α membrane translocation in response to KCl (PI3K-C2α mediates part of SEVO and ISO-mediated vasodilation in rat aorta. The cellular mechanisms underlying the inhibitory effect of volatile anesthetics might be mediated by KCl/PI3K-C2α/Rho kinase/MYPT1/MLC pathway. Membrane depolarization by KCl increases Ca2+ influx via voltage-dependent Ca2+ channel receptors on VSM cells, leading to an increase in cytosolic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i activates Ca2+-calmodulin-dependent myosin light chain kinase, resulting in MLC phosphorylation and vascular contraction i in VSM [2+-induced Ca2+ sensitization in regulating VSM contraction. The current study involved investigating the effects of volatile anesthetic modulation of PI3K subunit and Rho kinase activity on vasoconstriction in response to KCl in rat isolated aortic smooth muscle.We previously demonstrated that SEVO inhibited GTPγ S-stimulated Rho-Rho kinase-mediated vasoconstriction in isolated rat aortic VSM . We alsoi in VSM . We recei in VSM . HoweverThis protocol was approved by Shandong University Animal Care and Use Committee and Qilu Hospital of Shandong University Medical Ethics Committee (No. KYLL-2013-51). Aortic rings and strips were prepared according to previous protocols –18. Male2, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 24.8; and dextrose 10) at 37 °C and gassed with a mixture of 95 % (v/v) O2 and 5 % (v/v) CO2 with a fresh gas flow of 2 L/min. After 60 min equilibration, with the bathing fluid replaced every 20 min, the aortic rings were incubated with KCl (60 mM) to access their overall contractile responsiveness. Removal of the endothelium was confirmed with 3 × 10−7 M phenylephrine pre-contracted vessels that showed a lack of relaxation in the presence of 10−5 M acetylcholine. To examine the effect of anesthetics on KCl-induced contraction, six aortic rings from each individual rat (n = 7) were randomly exposed to 0, 1, 2, and 3 minimum alveolar concentration (MAC) of each anesthetic, 1 mM LY294002 (PI3K inhibitor), or 1 μM Y27632 (Rho kinase inhibitor) for 15 min before the addition of KCl  was used as an agonist according to previous results . KCl-elin = 8–12) and 0.19, 0.39, and 0.56 mM for gas concentrations of 1.2 % (1 MAC), 2.3 % (2 MAC), and 3.5 % (3 MAC) ISO, respectively (n = 8–12), after 15 min of equilibration.SEVO or ISO was delivered into the gas mixture with a fresh gas flow of 2 L/min via a calibrated agent-specific vaporizer  to aerate the KBS with equivalent human MAC. The concentration of the resulting gas mixture was monitored and adjusted using an Atom 303 anesthetic agent monitor . Our previous data using the same experimental system indicated that the concentrations of SEVO or ISO in KBS measured by gas chromatography  were 0.17, 0.28, and 0.41 mM for gas concentrations of 1.7 % (1 MAC), 3.4 % (2 MAC), and 5.1 % (3 MAC) SEVO, respectively  were bathed in oxygenated KBS and were equilibrated for 60 min before the start of the experiment. One strip was obtained from each animal.Previous data demonstrated that KCl (60 mM) induced a rapid increase in MLC phosphorylation and reached peak levels approximately 5 min after exposure to KCl . TherefoIn each experiment, proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis 12 % or 5 %) and transferred to nitrocellulose membranes. Membranes were incubated with anti-MLC, anti-CPI-17, anti-MYPT1/Thr696, anti-MYPT1/Thr853 , and antiphospho-MLC, antiphospho-CPI-17, antiphospho-MYPT1/Thr696, and antiphospho-MYPT1/Thr853 antibodies  for 2 h, followed by incubation with a horseradish peroxidase-conjugated antibody (1:2000) for 1 h [ % or 5 %The densities of immunoreactive bands were detected using chemiluminescence  and were assessed with image analysis software . Bands with molecular weights of 17 kDa, 20 kDa, and 110 kDa were identified as CPI-17, MLC, and MYPT1, respectively. The ratios of phosphorylated to total CPI-17, MLC, and MYPT1 were used as an indicator of activation of each enzyme and expressed as the percentage relative to the baseline control level (referred to as 100 %).For enzyme membrane translocation, PI3K and Rho kinase (Rock II) activity were measured using western blotting analysis as described previously . Briefly2, 100 mM NaCl, 2 mM EDTA, 1 mM 4-(2-aminoethyl) benzonesulfonyl fluoride, 20 μg/mL leupeptin, 20 μg/mL aprotinin, and 1 mM Na3VO4. Homogenates were centrifuged at 13,000 g for 15 min at 4 °C, and the supernatants were collected and centrifuged at 100,000 g for 30 min at 4 °C again. The supernatant (cytosolic fraction) was removed, and the pellet (membrane fraction) was re-suspended using the same lysis buffer. The protein concentrations of each fraction were determined using the bicinchoninic acid method [Frozen aortas were cut into small pieces and were homogenized in ice-cold lysis buffer containing 1 mM Tris/HCl, pH 7.5, 5.5 mM MgCld method .Equal amounts of total protein (30 μg) were used for every sample in each experiment. Proteins were separated by 7.5 % or 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to a nitrocellulose membrane. The membrane was treated with anti-PI3K-p85α, anti-PI3K-C2α, and anti-Rock-II antibodies  for 4 h, followed by incubation with a horseradish peroxidase-conjugated antibody (1:2000) for 1 h. Immunoreactive bands were detected using chemiluminescence  and were assessed with image analysis software . The amount of PI3K-p85α, PI3K-C2α, and Rock-II on the membrane was expressed as a percentage of the total value .SEVO and ISO were purchased from Dainabot Company Limited . Polyclonal antibodies against phospho-MLC (Thr18/Ser19), phospho-MYPT1 (Thr853), phospho-MYPT1 (Thr696), phospho-CPI-17 (Thr38), MLC2 (FL-172), MYPT1 (H-130), CPI-17 (H60), PI3K-p85α, and Rock-II, and the secondary antibody labeled with horseradish peroxidase (HRP) were all obtained from Santa Cruz Biotechnology, Inc. . PI3K-C2α was purchased from Abcam, Inc. . LY294002 was provided from Merck-Calbiochem Biosciences . Y27632 was purchased from Calbiochem-Novabiochem Corporation . All other reagents for experiments and western blotting were of analytical grade.n) refers to the number of rats from which the aortas were harvested. Data are presented as the mean ± standard deviation and were analyzed using the Dunnett test after analysis of variance for multiple comparison of isometric tension or protein phosphorylation. A p-value < 0.05 was considered significant.Data analyses were performed using the software program StatMate . The sample size    inhibited KCl-induced PI3K-p85 and PI3K-C2α membrane translocation in response to KCl (01) Fig. .2+-induced Ca2+ sensitization induced by volatile anesthetics contributes to its vasodilatory effect.The current study demonstrated that both SEVO and ISO inhibit KCl-induced MLC phosphorylation and vasoconstriction by modulating PI3K-C2α and Rho kinase activity in the rat aorta. These findings suggest that the inhibition of Ca2+ sensitivity. Because specific PI3K-C2α-inhibitors do not exist, it remains unclear whether the reduced MLC phosphorylation and vasoconstriction depends on class II PI3K-C2α-inhibition or inhibition of class I PI3K.Several studies demonstrate that LY294002 and Y27632 inhibit KCl-induced vasoconstriction by suppressing MYPT1 and CPI-17 phosphorylation in rat or rabbit aortic smooth muscle , 11. LY22+-induced Ca2+ sensitization [2+/PI3K-C2α/Rho kinase/MYPT1/Thr853 pathway.Accumulating evidence demonstrates that PI3K-C2α and Rho kinase, or PKC, MYPT1/Thr853, and CPI-17 are involved in vasoconstriction by regulation of Catization , 23–25. tization , 27. Taktization . It is p2+ sensitivity by regulating Rho kinase and PKC activity [2+ changes in the diabetic rat aorta [2+ sensitization, not Ca2+-dependent mechanisms, is involved in modulating vascular tension by SEV.Several reports also showed that SEVO decreased myofilament Caactivity , 17, 18.at aorta . These d2+ sensitivity mechanisms is mainly mediated by increasing Ca2+ influx via voltage-dependent calcium channels [2+-dependent pathway and not through the Ca2+ sensitivity pathway. In contrast to the above results, we recently demonstrated that ISO inhibits Ang II-induced vasoconstriction through regulating the phosphorylation of the MLCP subunit MYPT1/Thr853 [2+-induced Ca2+ sensitization pathway was also involved in the regulation of vasoconstriction by ISO.Evidence also demonstrates that ISO inhibition of agonist-induced contraction of Cachannels , 30. We channels . These d1/Thr853 . Further2+-induced Ca2+ sensitization through modulating PI3K-C2α, Rho kinase activity, and MYPT1/Thr853 phosphorylation in rat vascular smooth muscle.Overall, our data, in combination with other investigations, reveal that inhibition of KCl-elicited vasoconstriction by volatile anesthetics may involve the modulation of PI3K-C2α and/or Rho-Rho kinase activity, the upstream activators of MLCP , 11, 25.in vivo conditions. However, we used endothelium-denuded samples to avoid the effects of the endothelium. (2) Moreover, pre-arteriolar resistance arteries, not conduit arteries, mainly determine peripheral vascular resistance. Therefore, the current results obtained from endothelium-denuded aortas cannot be used to directly extrapolate to the in vivo condition. (3) Furthermore, the aortic rings are not necessarily reflective of the physiology of resistance arteries and thus may not be ideally suited to address issues related to the clinical hemodynamic effects of volatile anesthetics.However, this study has some limitations: (1) the aim of this study was to elucidate the cellular mechanisms responsible for anesthetic-induced inhibition of vasoconstriction. Therefore, we used endothelium-denuded aortic strips or rings for this study. Indeed, the endothelium plays an important role in regulating systemic vascular tone under In conclusion, our study demonstrates that both SEVO and ISO inhibit KCl-induced PI3K-C2α-participation, Rho kinase-mediated MLC phosphorylation, and vasoconstriction in rat aortic smooth muscle. The intracellular mechanisms of regulating VSM contraction of volatile anesthetics may be mediated by the KCl/PI3K-C2α/Rho kinase/MLCP/MLC pathway.Ang II, Angiotensin II; BCA, bicinchoninic acid; CaM, Ca2 + -calmodulin; CPI-17/Thr38, PKC-potentiated inhibitory protein/Thr38; GPCR, G protein-coupled receptors; ISO, isoflurane; MAC, minimum alveolar concentration; MLC, myosin light chain; MLCK, myosin light chain kinase; MLCP, MLC phosphatase; MYPT1/Thr696, myosin phosphatase target subunit/Thr696; MYPT1/Thr853, myosin phosphatase target subunit/Thr853; PI3K-C2α, Class II phosphoinositide 3-kinase α-isoform; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEVO, sevoflurane; VSM, vascular smooth muscle


Minimally invasive transforaminal lumbar interbody fusion (MI-TLIF) is increasingly popular for the surgical treatment of degenerative lumbar disc diseases. The constructs intended for segmental stability are varied in MI-TLIF. We adopted finite element (FE) analysis to compare the stability after different construct fixations using interbody cage with posterior pedicle screw-rod or pedicle screw-plate instrumentation system.A L3–S1 FE model was modified to simulate decompression and fusion at L4–L5 segment. Fixation modes included unilateral plate (UP), unilateral rod (UR), bilateral plate (BP), bilateral rod (BR) and UP+UR fixation. The inferior surface of the S1 vertebra remained immobilized throughout the load simulation, and a bending moment of 7.5 Nm with 400N pre-load was applied on the L3 vertebra to recreate flexion, extension, lateral bending, and axial rotation. Range of motion (ROM) and Von Mises stress were evaluated for intact and instrumentation models in all loading planes.All reconstructive conditions displayed decreased motion at L4–L5. The pedicle screw-plate system offered equal ROM to pedicle screw-rod system in unilateral or bilateral fixation modes respectively. Pedicle screw stresses for plate system were 2.2 times greater than those for rod system in left lateral bending under unilateral fixation. Stresses for plate were 3.1 times greater than those for rod in right axial rotation under bilateral fixation. Stresses on intervertebral graft for plate system were similar to rod system in unilateral and bilateral fixation modes respectively. Increased ROM and posterior instrumentation stresses were observed in all loading modes with unilateral fixation compared with bilateral fixation in both systems.Transforaminal lumbar interbody fusion augmentation with pedicle screw-plate system fixation increases fusion construct stability equally to the pedicle screw-rod system. Increased posterior instrumentation stresses are observed in all loading modes with plate fixation, and bilateral fixation could reduce stress concentration. Transforaminal lumbar interbody fusion (TLIF) is used to achieve a solid arthrodesis of spinal segments which sustains loading, while maintaining disc space height, preserving foraminal dimensions, and restoring sagittal plane alignment. Paraspinal muscle dissection and retraction in open TLIF remains a drawback, which would cause muscle denervation, atrophy and consequently persistent lower back pain . BecauseA finite element model has the advantages of easily modifying TLIF cage and posterior implant geometry to observe the altered load transfer on the individual motion segment, and to analyze the stress distribution on these constructs. A few studies have investigated the biomechanical performance of TLIF procedures with different instrumentation methods –11. DiffWe developed a three-dimensional nonlinear finite element model of the lumbosacral spine (L3-S1), which consisted of four vertebrae, three discs and associated spinal ligaments. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Third Military Medical University, Chongqing, China. After obtaining the informed consent of a volunteer, geometrical details of bony structures were obtained from 64 spiral computed tomography (CT) images of a 29-year-old healthy male (70kg and 175cm) without history of spine injury, osteoporosis and radiographic evidence of degenerative sign. The PC used in this study was Think Station D20 with Xeon X5650 CPU and 16 GB RAMThe CT scans of the L3-S1 were obtained at a 0.5-mm interval and 0.6-mm resolution using CT scanner . The image were imported into medical image-processing software  to reconstruct the geometric surface of the lumbosacral vertebrae. The surface model was then exported into Geomagic Studio 2012  to enhance the quality of the surface. We use the software to perform smoothing process, remove the high aspect ratio elements and repair gaps left in the surface model. The solid model was generated by SolidWorks 2012  from the geometric data. Eventually, it was imported to finite element software  for meshing and analyzing.The vertebrae were composed of a solid volume (cancellous bone) and a layer of shell  with a thickness of 0.5 mm . The intThe metal component and cage were made of Ti6Al4V and polyetheretherketone, respectively. The material properties and mesh type of lumbosacral components and implants were listed in The FE model was modified to simulate the MI-TLIF surgical procedure at the L4–L5 level via a transforaminal approach by the application of facetectomy and partial discectomy . The nucFive instrumentation models were established using the TLIF model : unilateThe insertion points of the ligaments and the interfaces of the intervertebral disc were attached onto the vertebrae, whereas the interaction between the facet joint was assigned with frictionless sliding contact formulation –16.The bTwo types of loading conditions corresponding to loads were used for model validation and model predictions. For four pure moments, the results of a range of motion were compared with those of previous in vitro studies by Dahl et al.  and SoriWe followed the convergence test method referred by Womack et al.  and creaThe intact model was used for the validation and the results of the range of motion were compared with those of previous in vitro studies by Dahl et al.  and SoriThe range of motion (ROM) of the L4-5 segment was reduced for all construction techniques compared with that in the intact condition . There wThe largest maximal stresses of pedicle screws were all detected in axial rotation among all the fixation models during all states of motion . For uniThe largest maximal stresses of longitudinal connector were all detected in axial rotation among all the fixation models during all states motion . For uniVon Mises stresses on the graft obtained for each construct were showed in The old plate system had been gradually replaced by the rod system . The pedAdditionally, the pedicle screw-rod system used in MI-TLIF is imperfect. The polyaxial pedicle screw was used in the rod system in MI-TLIF and its ball-in-cup structure significantly reduced the strength of the whole assembly compared with that of the monoaxial pedicle screw –28. ThisWe conducted the FE analysis to evaluate the stability of the five constructs under different loading conditions, assessing motion at the fixation level and estimating the stresses experienced by the posterior instrumentation and intervertebral graft. After performing simulations of the right side facetectomy and partial discectomy through a transforaminal approach, we found that all reconstructive configurations provided a substantial reduction in ROM compared with the intact lumbar spine. The pedicle screw-plate system fixation reduced ROM at the L4-L5 between 61%-88% compared with intact conditions in all loading modes, consistent with the results from previous studies –11. In cThe cross section area of the paired parallel bars on plate was designed to be the same with that of the single rod, so each bar with half of the rod cross section area suffered higher stresses than single rod under the same load. Biomechanical study indicated the decrease of the rotation torque in the round rod compared with the in the polygonal rod in cantilevered pedicel screw system . The plaIn this study, we also compared the fixation effect of the unilateral fixation and the bilateral fixation. Firstly, variation in ROM was noted with unilateral fixation when compared with bilateral fixation. Simultaneously, the stresses on intervertebral graft in unilateral fixation were larger compared with bilateral fixation. The distribution was eccentric under the axial force produced by flexion and pre-load modes. Moreover, the stresses on the posterior instrumentation were much larger on rod/plate and screws in unilateral fixation compared with the bilateral fixation. These results were consist with the previous studies about unilateral and bilateral posterior instrumentation in TLIF , 10. GoeThere are a few limitations to this study. The most commonly used pedicle screw in minimally invasive spine surgery is polyaxial designed with ball-in-cup construct. It is different from the monoaxial pedicle screw in the yield load of screw-rod assembly . We builFor single-level MI-TLIF procedures, posterior fixation using the new pedicle screw-plate system offers immediate stability and is equivalent to fixation using a pedicle screw-rod system, regardless of unilateral fixation mode or bilateral fixation mode. Moreover, augmentation with bilateral posterior fixation offers greater immediate stability compared with unilateral posterior fixation. Increased posterior instrumentation peak stresses are observed in all loading modes with plate fixation, and bilateral fixation could reduce peak stresses.S1 FigRed color region indicated the over 50.0 MPa Von Mises stresses distribution.(TIF)Click here for additional data file.S2 FigRed color region indicated the over 1.0 MPa Von Mises stresses distribution.(TIF)Click here for additional data file.S3 FigModel 1 with 102,455 meshes, Model 2 with 154,976 meshes, Model 3 with 200,581 meshes and Model 4 with 307,689 meshes.(TIF)Click here for additional data file.S4 FigThe difference between models was within 2.5%.(TIF)Click here for additional data file.S5 FigThe difference between models was within 1.15%.(TIF)Click here for additional data file.S6 FigThe difference between models was within 2.63%.(TIF)Click here for additional data file.


Cladiella australis. Our results indicated that 4-(phenylsulfanyl)butan-2-one could successfully inhibit pigment generation processes in mushroom tyrosinase platform assay, probably through the suppression of tyrosinase activity to be a non-competitive inhibitor of tyrosinase. In cell-based viability examinations, it demonstrated low cytotoxicity on melanoma cells and other normal human cells. It exhibited stronger inhibitions of melanin production and tyrosinase activity than arbutin or 1-phenyl-2-thiourea (PTU). Also, we discovered that 4-(phenylsulfanyl)butan-2-one reduces the protein expressions of melanin synthesis-related proteins, including the microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (Trp-1), dopachrome tautomerase , and glycoprotein 100 (GP100). In an in vivo zebrafish model, it presented a remarkable suppression in melanogenesis after 48 h. In summary, our in vitro and in vivo biological assays showed that 4-(phenylsulfanyl)butan-2-one possesses anti-melanogenic properties that are significant in medical cosmetology.In this study, we screened compounds with skin whitening properties and favorable safety profiles from a series of marine related natural products, which were isolated from Formosan soft coral  Cladiella australis show significant in vitro anti-inflammatory activity [2 hybridization) attached to sulfur. 4-(phenylsulfanyl)butan-2-one was found to be a safer and more effective ingredient than traditionally used agents for cosmetic and medical applications.Manufacturers claim that many of their cosmeceutical ingredients from natural products and chemically synthesized compounds have bio-functional properties. Marine natural resources have become a new inspiration of drug development research in recent years. Our group discovered and isolated several pure compounds from soft corals, which exhibit neuroprotective and anti-inflammatory activities ,2,3,4,5.activity . In thisin vitro and in vivo [In the cosmetic business, skin whitening is one of the most popular demands in Asia. Skin color is affected by melanin, which is produced in melanocytes in the basal layer of human epidermis. Melanin is formed through the activity of several melanogenesis-related proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, dopachrome tautomerase , tyrosinase-related protein-1 (Trp-1), and glycoprotein 100 (Gp100) ,8. MITF  in vivo ,13. Thus in vivo ,15,16.in vivo cytotoxic performance testing can be done through this embryonic sensitivity evaluation method.Zebrafish models have become a highly advantageous vertebrate animal system for high-throughput screening of melanogenic regulatory agents because zebrafish are small in size, inexpensive, easy to handle, short-lived, and have transparent embryos and physiological similarity to mammals . The zebin vivo pigmentation reversibly, and it maintained satisfactory treatment survival rates. Thus, this target compound could have significant application in the cosmetic field.In this work, we identified a sulfur-containing compound, 4-(phenylsulfanyl)butan-2-one, as a promising and potential skin whitening agent. In the mushroom model, it acted as a non-competitive tyrosinase inhibitor. In murine melanoma B16-F10 cells, we demonstrated that it suppressed melanin production, reduced the protein expression of melanosome maturation related proteins, and inhibited tyrosinase activity. Finally, in zebrafish we illustrated that it repressed in vitro. We searched for a new and efficient substance for hyper-pigmentation prevention and skin whitening and discovered that one compound, 4-(phenylsulfanyl)butan-2-one, significantly diminished mushroom tyrosinase activity . The Lineweaver–Burk equation plot butan-2-one for further studies. Initially, we measured mouse dermal melanoma B16-F10 cell viability via the Alamar Blue assay to investigate whether our compound causes cytotoxic damage (Preliminarily data from a number of c damage A. To be To clearly understand the inhibitory effect of 4-(phenylsulfanyl)butan-2 on melanogenesis, we assessed intracellular tyrosinase activity in B16-F10 cells. Cells were cultured in 10 and 50 µM (suitable concentrations as suggested by the cell viability assay) for two periods of time, 24 h and 48 h. After incubation, tyrosinase activities were suppressed to a greater extent than those of the vehicle control, 1,000 µM arbutin, and 300 µM PTU A. We stul-tyrosine. First, l-tyrosine is catalyzed to l-DOPA by tyrosinase, and l-DOPA is converted in to dopaquinone via the same enzyme. Dopaquinone, through catalysis by Trp-2, Trp-1, and tyrosinase, becomes the black or gray pigment, eumelanin; another bypass-path end product is the red or yellow pigment, pheomelanin [Melanin biosynthesis takes place in dermal melanocytes, and the substrate amino acid is omelanin . We revein vitro tyrosinase inhibition screening, we executed an in vivo zebrafish assay to examine the bioactivity of our target compound. All samples and doses being examined consisted of two embryos within one well (on a 96-well plate). The phenotypic effectiveness on zebrafish melanin was observed by analyzing the body pigment under a stereomicroscope at 28.5 °C butan-2-one condition treatments . Culturein vitro) was determined. Safe and effective skin lightening compounds are important for preventing hyper-pigmentation. Our target marine derivate compound exhibited diminished mushroom tyrosinase activity (in vivo examination models. We further investigated the mushroom tyrosinase inhibitory kinetic parameters of 4-(phenylsulfanyl)butan-2-one. According to the Lineweaver–Burk plot we obtained, the compound was a non-competitive inhibitor of the enzyme substrate.The most important pigment determining human skin color is melanin. Melanogenesis suppressors are useful as therapeutics for hyper-pigmentation and have application in the cosmetic industry ,24,25,26activity . Moreovel-DOPA as the substrate, and l-DOPA stimulates the mushroom tyrosinase but does not affect mammalian tyrosinase activities. In the second stage of testing, we determined whether cellular melanin genesis is suppressed in mammal B16-F10 cells. Using a microscope, we obtained cultured-cell photographs with the melanin accumulations in B16-F10 cells treated with various concentrations of 4-(phenylsulfanyl)butan-2-one. This implies that the compound is a stronger inhibitor than arbutin or PTU. In this mouse epidermal melanoma model, we found that the pigment concentration and tyrosinase activity were significantly reduced by the presence of 4-(phenylsulfanyl)butan-2-one in a dose-dependent manner. Since kojic acid and arbutin are weak epidermal de-pigment agents, and their use at high concentrations causes carcinogenic diseases, our compound was scrutinized for high skin-lightening effectiveness at low concentrations.Suppressed tyrosinase activity has been reported in numerous papers; however, most only tested this in a mushroom enzyme activity platform ,29,30,31In melanin biosynthesis, tyrosinase has a unique influence, but other proteins also contribute to this process, including MITF, Trp-1, Trp-2, and Gp100. DOPA-chrome is catalyzed to 5,6-dihydroxyindole-2-carboxylic acid by Trp-2, and the meddle-product is catalyzed to indole-5,6-quinone carboxylic acid, which is then used to synthesize eumelanin . In our in vitro screening using the mushroom model and mammal cell culture system, an in vivo zebrafish model was used as a more physiologically relevant platform to elucidate the mechanism for suppression [As a melanin synthesis regulatory agent is being developed, possible harmfulness and reliability must be put into scientists’ considerations, such as the toxicity, allergy, and sensitivity tests, even if most are not confirmed right now. When our dermal pigment inhibitory agent is being considered for potential cosmetic or therapeutic use in humans, the cytotoxic properties of 4-(phenylsulfanyl)butan-2-one become extremely important. There are several famous pigment production suppressors, including kojic acid, arbutin, PTU, and hydroquinone. They are being developed internationally as ingredients in cosmetics at the present time. However, these pigment synthesis suppressors may be tumorigenic at a high dose or with frequent usage ,36. Everpression ,38,39,40pression . 4-(PhenWe followed our published papers with minor modification to obtain the target compound . We founl-tyrosine (2 mM) in phosphate buffer (pH 6.8) was added at 37 °C for 30 min. Tyrosinase inhibitory activity was determined by the following equation:A is the optical density (OD490) without the testing substance; B is the OD490 without the testing substance, but with tyrosinase; C is the OD490 with the testing substance; and D is the OD490 with the testing substance, but without tyrosinase.Mushroom tyrosinase activity inhibition was determined spectrophotometrically , according to the method described previously with minor modifications . Evaluat2 atmosphere humidified incubator at 37 °C for 24 h. After 24 h incubation, 10% alamarBlue®  was aseptically added to measure cell viability, according to commercial kit protocols. The vehicle control group (without 4-(phenylsulfanyl)butan-2-one) was defined as 100%. The optical absorbance values (A) of the supernatants were quantified at 570 nm, and the cell viabilities were analyzed according to the following formula:The murine melanoma cells (B16-F10), human foreskin fibroblasts (Hs68), and human umbilical vein cell line (EA.hy926) were separately cultured on 96-well plates . B16-F105 per well) were placed in 24-well plates in 500 µL DMEM medium with 72×10−3 mg/mL l-tyrosine that acts as not only substrate but also regulator of melanogenesis [l-tyrosine as substrates mixed in 0.1 M phosphate buffer (pH 6.8). This reaction was then incubated at 37 °C for 3 h in the dark, and the absorbance at 475 nm was measured on a spectrophotometer. The vehicle control group tyrosinase activity (without 4-(phenylsulfanyl)butan-2-one) was defined as 100%.Tyrosinase activity was estimated by measuring the rate of dopachrome formation, based on the method described previously with minor modifications . B16-F10ogenesis ,44, and According to a previous method with minor modifications ,45, cellg for 20 min at −4 °C, and the supernatant concentration was determined using the DC protein assay kit . In total, 40 µg of protein was separated via 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membranes . The membranes were blocked with 5% non-fat skim milk in a tris-buffered saline (TBS)-Tween 20 solution. Tyrosinase , Trp-1 , Trp-2 , Gp100 , MITF  and β-actin  were all detected with a rabbit polyclonal anti-tyrosinase antibody, a rabbit polyclonal anti-Trp-1 antibody, a rabbit polyclonal anti-Trp-2 antibody, a goat monoclonal anti-Gp100 antibody, and a mouse monoclonal anti-β-actin antibody . The PVDF films were further incubated with horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized by enhanced chemiluminescence. All bound antibodies were then detected with a BioSpectrum® Imaging System .B16-F10 cells were treated with 4-(phenylsulfanyl)butan-2-one or a vehicle control for 24 h. Cells were washed twice with ice cold PBS buffer, harvested, and disrupted in lysis buffer  ,5. The cSynchronized embryos were collected and arrayed by pipette, three embryos per well, in a 96-well plate containing 200 µL embryo medium, according to a previous method . Test cot-test was used to determine the level of significance.Results are presented as a mean value of the data obtained from triplicate experiments. Student’s in vivo zebrafish model to determine melanin generation diminish by our compound. In B16-F10, Hs68 and EA.hy926 cell viability, and zebrafish models, it did not showed the cytotoxic, carcinogenic or teratogenic properties. We suggested that the 4-(phenylsulfanyl)butan-2-one is safe and efficacious to be developed into a skin whitening substance.In this study, we developed the anti-melanogenesis abilities of our target compound, 4-(phenylsulfanyl)butan-2-one. Via the mushroom tyrosinase inhibition test, we found that the compound had the tyrosinase inhibitory capacity, and the kinetic analysis showed that the suppressive mechanism was noncompetitive. In murine melanoma B16-F10 cells, 4-(phenylsulfanyl)butan-2-one also inhibited the cell-based tyrosinase activity, and decreased the melanin production. To confirm the pathway of melanogenesis inhibition, we used the western blotting to prove that MITF, tyrosinase, Trp-1 and Trp-2 were inhibited by 4-(phenylsulfanyl)butan-2-one, and the compound arrested the melanosome mature by Gp100 repression. Further, we executed the


FYN is a non-receptor tyrosine kinase belonging to the SRC family of kinases, which are frequently over-expressed in human cancers, and play key roles in cancer biology. SRC has long been recognized as an important oncogene, but little attention has been given to its other family members. In this report, we have studied the role of FYN in FLT3 signaling in respect to acute myeloid leukemia (AML). We observed that FYN displays a strong association with wild-type FLT3 as well as oncogenic FLT3-ITD and is dependent on the kinase activity of FLT3 and the SH2 domain of FYN. We identified multiple FYN binding sites in FLT3, which partially overlapped with SRC binding sites. To understand the role of FYN in FLT3 signaling, we generated FYN overexpressing cells. We observed that expression of FYN resulted in slightly enhanced phosphorylation of AKT, ERK1/2 and p38 in response to ligand stimulation. Furthermore, FYN expression led to a slight increase in FLT3-ITD-dependent cell proliferation, but potent enhancement of STAT5 phosphorylation as well as colony formation. We also observed that FYN expression is deregulated in AML patient samples and that higher expression of FYN, in combination with FLT3-ITD mutation, resulted in enrichment of the STAT5 signaling pathway and correlated with poor prognosis in AML. Taken together our data suggest that FYN cooperates with oncogenic FLT3-ITD in cellular transformation by selective activation of the STAT5 pathway. Therefore, inhibition of FYN, in combination with FLT3 inhibition, will most likely be beneficial for this group of AML patients. Expression of receptor tyrosine kinases has been shown to be deregulated in many cancers. Deregulation can be due to overexpression or through oncogenic mutations. A member of the PDGFR-family receptor tyrosine kinase , FLT3, has been shown to be deregulated in acute myeloid leukemia (AML) and in a small portion of acute lymphoblastic leukemia , 2. FLT3The downstream signaling of FLT3 is tightly controlled by FLT3 associating proteins. Activation of FLT3 is mediated through ligand binding and dimerization followed by phosphorylation of several tyrosine residues in the intracellular domain. Phosphorylation of tyrosine residues creates docking sites for the SH2 domain-containing proteins that further propagate or terminate signaling. For example association of non-receptor tyrosine kinases SRC and SYK, adaptor proteins GRB10 and SLAP resulted in enhancement of FLT3 signaling . In contThe proto-oncogene FYN is a member of the SRC family of protein tyrosine kinases (SFKs). The SFKs includes 11 non-receptor protein tyrosine kinases . All famIn this report we describe a novel role of FYN in FLT3 signaling. We show that FYN interacts with wild-type and oncogenic FLT3 through its SH2 domain in a FLT3 kinase-dependent manner. This association has no effect on FLT3 stability but overexpression of FYN in FLT3 expressing Ba/F3 cells slightly enhanced mitogenic signaling. Moreover, FYN expression significantly enhanced FLT3-ITD-mediated STAT5 phosphorylation as well as colony formation in semi-solid medium.P = 0.0096). FLT3-ITD is the most commonly mutated gene in AML and correlates with poor prognosis. We observed that patient displaying both high FYN expression and FLT3-ITD mutations further showed poor survival (P = 0.0026) in comparison to patient with low FYN expression , SRC , HCK 1919 and LYWe then aimed at identifying the binding sites in FLT3. Phosphorylation of FLT3 on tyrosine residues occurs at multiple sites in the intracellular domain. We used tyrosine-phosphorylated synthetic peptides corresponding to known or predicted tyrosine phosphorylation sites in FLT3 to identify candidate FYN interaction sites. We observed that FYN associates with FLT3 through binding to the FLT3-pY591, FLT3-pY599 and FLT3-pY955 residues Figure . These rFYN is phosphorylated on several tyrosine residues. We observed that wild-type FYN remains tyrosine phosphorylated in COS-1 cells and ligand stimulation slightly increased tyrosine phosphorylation of FYN Figure . HoweverAlthough we observed that FYN expression increased FLT3 tyrosine phosphorylation, we did not see any considerable increase in ubiquitylation of FLT3 in FYN transfected cells (data not shown). Furthermore, time-dependent stimulation with FL in the presence of cycloheximide, an inhibitor of protein synthesis, did not result in any significant difference in FLT3 degradation Figure .To understand the role of FYN in FLT3 signaling we used Ba/F3-FLT3-WT/empty vector and Ba/F3-FLT3-WT/FYN cell lines that overexpress FYN. Activation of wild-type FLT3 resulted in activation of the AKT, ERK and p38 signaling pathways. We observed that FYN expression slightly increased FL-induced AKT phosphorylation Figure  but did http://pantherdb.org) many of those genes are found to be involved in regulation of cell survival.Wild-type FLT3 and FLT3-ITD differ in their ability to activate downstream signaling. While wild-type FLT3 activates AKT, ERK and p38 signaling, FLT3-ITD activates in addition to AKT, ERK and p38 signaling also STAT5 signaling. Similar to what we saw with wild-type FLT3, we observed a slight increase in AKT phosphorylation in Ba/F3-FLT3-ITD/FYN cells Figure  but not Since we observed that FYN expression correlated with poor prognosis in AML patients carrying FLT3-ITD mutations and since FYN associates with both FLT3-WT and FLT3-ITD, we hypothesized that FYN may play a role FLT3-induced biological outcomes. We generated Ba/F3 cell lines expressing FLT3-WT or FLT3-ITD along with FYN or empty control vector. FYN expression slightly potentiated FLT3-ITD-induced cell proliferation Figure  and sligIn order to further understand FLT3-ITD induced AML pathogenesis, we defined the role of FYN in FLT3-ITD dependent AML. In a screen using mRNA data from AML patient samples we observed that among the 11 SFKs, FYN expression correlated with AML patient survival. Therefore, we aimed to understand the role of FYN in FLT3-ITD - dependent AML. We showed that FYN is involved in FLT3-ITD - induced transformation. FYN has previously been found to associate with other type III receptor tyrosine kinases such as KIT and PDGFRβ. FYN associates with KIT through the major SFKs binding site Y568 in KIT . A speciDue to high degree of structural similarity and abundant expression of many family members in most tissues, it still remains a challenge to study the roles of individual SFKs. We still do not have any highly specific SFK inhibitors. The major FYN interaction residues in FLT3, pY591 and pY599, are also well-known SRC binding sites , 21. AddThe exact mechanism by which FYN regulates STAT5 phosphorylation is not known. We have previously demonstrated that the scaffolding protein GAB2 is involved in regulation of STAT5 phosphorylation downstream of FLT3-ITD . PhosphoThe minimal differences in phosphorylation of AKT, ERK1/2 and p38 seen in FYN overexpressing cells is probably due to the fact that FLT3 downstream signaling is already saturated by endogenous expression of the predominant SFKs expressed in Ba/F3 cells, SRC and LYN, and probably the pY591 and pY599 sites are the major phosphorylation sites in FLT3 signaling that are required for activation of AKT, ERK1/2 and p38. FYN plays a unique role in STAT5 signaling since overexpression of FYN selectively elevated STAT5 phosphorylation as well as colony formation. In addition to the cell lines studied, we observed an enrichment of the STAT5 pathway in AML patients with higher FYN expression. Furthermore, AML patient carrying the FLT3-ITD mutation combined with higher FYN expression displayed relatively poor prognosis. Collectively we suggest that FYN by activating STAT5 signaling plays an important role in FLT3-ITD-mediated transformation in AML and therefore a combinatorial therapy targeting FYN in addition to FLT3-ITD would likely be beneficial for this group of patients.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14468).Data from AML patient samples were collected from the data set GSE14468 . This da2 at 37°C.The murine hematopoietic cell line Ba/F3 was cultured in RPMI-1640 media  supplemented with 10% heat-inactivated fetal bovine serum , 1% penicillin and streptomycin and 10 ng/ml murine interleukin 3 (IL3) as previously described . COS-1 cThe open reading frame (ORF) of human FYN (BC032496) was subcloned (BamHI-XhoI) into a modified pcDNA3.1 vector pcFLAG  expressiTo establish Ba/F3 cells stably expressing FLT3-WT and FLT3-ITD, EcoPack packaging cells were transfected with pMSCVpuro-FLT3-WT or pMSCVpuro-FLT3-ITD construct, and virus-containing supernatants were collected 72 h after transfection as described previously , 41. RetAfter 100 ng/ml FL-stimulation cells were washed once with cold PBS and lysed using Triton X-100 based lysis buffer. Immunoprecipitation and western blotting were performed following the previously describe methods . PhosphoApoptosis was measured using annexin V and 7-aminoactinomycin D (7-AAD) kit (BD biosciences) following the previously described method . For the