Human lung cancer is highly invasive and the most malignant among human tumors. Adenocarcinoma as a specific type of non-small cell lung cancer occurs with high frequency and is also highly resistant to radiation therapy. Thus, how to avoid radiation resistance and improve radiotherapy effectiveness is a crucial question. In the present study, human lung cancer A549 and H1299 cells were irradiated using γ-rays from a Co60 irradiator. Protein expression was detected by Western blotting. Cell cycle and apoptosis were measured by flow cytometry. Surviving fraction was determined by colony formation assay. γH2AX and 53BP1 foci formation were examined by fluorescence microscopy. In the results, we show that CHMP4C, a subunit of Endosomal sorting complex-III (ESCRT-III), is involved in radiation-induced cellular response. Radiation-induced Aurora B expression enhances CHMP4C phosphorylation in non-small cell lung cancer (NSCLC) cells, maintaining cell cycle check-point and cellular viability as well as resisting apoptosis. CHMP4C depletion enhances cellular sensitivity to radiation, delays S-phase of cell cycle and reduces ionizing radiation (IR)-induced γH2AX foci formation. We found that Aurora B targets CHMP4C and inhibition of Aurora B exhibits similar effects with silencing of CHMP4C in radioresistance. We also confirm that CHMP4C phosphorylation is elevated after IR both in p53-positive and-negative cells, indicating that the close correlation between CHMP4C and Aurora B signaling pathway in mediating radiation resistance is not p53 dependent. Together, our work establishes a new function of CHMP4C in radiation resistance, which will offer a potential strategy for non-small cell lung cancer by disrupting CHMP4C. Human lung cancer is highly invasive and most malignant among human tumors, which is classified into non-small cell lung cancer and small cell lung cancer. Non-small cell lung cancer accounts for about 85% of lung cancers and the current therapeutic strategy includes surgical resection together with radiation and/or chemo-treatment. Radiation resistance is the severe outcome of lung cancer radiotherapy and therefore poses a critical barrier for the radio-therapeutic effect. Hence, how to avoid the radiation resistance and to improve its treatment efficiency is a major challenge ,2.Ionizing radiation initiates DNA damage and arrests cell cycle progression, leading to cellular genomic instability and loss of genetic information. In response to the damage stress, the ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) are activated. ATM and ATR can also engage in cell division control via activating cyclins and cyclin-dependent kinases (Cdks) . A recenAurora B kinase is the key kinase of CPC  essential for the mitotic processes. Aurora B expression and activity are altered during the cell cycle and peaks at the G2-M transition. Aurora B is elevated in a variety of human tumors and its over-expression is susceptible to tumor formation and poor outcomes of non-small cell lung cancer radiotherapy, therefore indicating that Aurora B might be a drug target for lung cancer ,7,8. HowThat ESCRT  is required for the CPC -mediated cytokinetic abscission, during which CPC monitors the right abscission time of two daughter cells. ESCRT has six distinct complexes  engaging in multi-vesicular body formation, cytokinesis and HBV (Hepatitis B virus) budding ,10,11. EIn the study, we first demonstrate that the subunit of ESCRT-III CHMP4C is involved in cellular radiation reactions. Radiation enhances Aurora B expression and CHMP4C phosphorylation in NSCLC cells, collectively directing cell cycle check-point and promoting cell survival. CHMP4C silencing increases cellular sensitivity to radiation, hinders S-phase progression in cell cycle and diminishes ionizing radiation (IR)-triggered γH2AX foci formation. We show that CHMP4C is the major target of Aurora B and has a similar effect with Aurora B upon radiation. We further reveal that the phosphorylation level of CHMP4C is rising both in p53-positive and-negative cells, showing that the close correlation between CHMP4C and Aurora B signaling pathway in mediating radiation resistance is not p53 dependent. Overall, our work discovers a novel action of CHMP4C in radiation resistance, suggesting CHMP4C as a new drug target for non-small cell lung cancer treatments.To test if CHMP4C is involved in DNA damage response, cells were γ-irradiated with 2, 4, and 6 Gy and CHMP4C expression as well as phosphorylation was checked. We found that CHMP4C protein increases after irradiation (IR) and peaks at 24 h in A549 but not in H1299 cells A,B. HoweNext, we wondered if CHMP4C exerts its regulatory role in cell cycle progression. A549 and H1299 cells were depleted of CHMP4C for 24 h and the S phase delay was checked, indicating that CHMP4C could impact S phase exit A–D,G–J. In addition, CHMP4C knockdown has no influence on p21 expression E, and p2The above data indicate that CHMP4C inhibition can arrest S-phase of cell cycle. Colony formation assays were next employed to detect if CHMP4C acts in cell radioresistance. These assays showed that CHMP4C knockdown decreases cell survival compared to control with IR. Similarly, the result was also found in Aurora B clearing. Inversely, CHMP4C or Aurora B expression can increase cell growth upon irradiation A,B.To verify whether Aurora B regulates CHMP4C, we silenced or overexpressed the Aurora B with siRNA or Aurora B expression plasmids in A549 or H1299. CHMP4C knockdown has no effect on Aurora B protein level, whereas Aurora B silencing inhibits CHMP4C protein level indicating that Aurora B acts upstream of CHMP4C A. MoreovThe results of colony formation assay indicate that the surviving fraction of NSCLC cells was significantly attenuated after IR treatment of 4, 6 Gy. We next want to ascertain whether CHMP4C knockdown can enhance IR-induced apoptosis under three conditions in A549 cells: radiation only (6 Gy), CHMP4C knockdown, combined CHMP4C knockdown and IR (6 Gy). We found that combined CHMP4C silencing and irradiation produce more evident apoptosis than single treatment . Hence, Next, to analyze if CHMP4C is immediately involved in DNA damage signaling, we examined the γH2AX and 53BP1 foci assembly in A549 cells with or without CHMP4C silencing. The IR dose of 4 Gy was selected in this analysis according to the results of colony formation and apoptosis assay. As shown in 1 to S transition [Current investigations on CHMP4C mainly focus its function on endosome generation. However, whether and how it serves its part in cell cycle, viability and apoptosis under irradiation is not clear. It has been reported that p53 regulates transcription of CHMP4C in the autophagy and endosome production . p53 plaansition ,20,21. WFurther, our results also showed that CHMP4C can promote cell survival and cell viability upon IR, indicating that CHMP4C offer the radioresistance to the irradiated cells. We next studied if CHMP4C was connected with DNA repair, and found that CHMP4C inhibition repressed DNA repair by decreasing γH2AX and 53BP1 foci reacting to radiation stress. Hence, the novel functions of CHMP4C in cell cycle control and DNA repair not only enriches the content of the DNA damage pathway but also extends its specific role in cell regulation.p53 is often mutated in NSCLC and its mutation increases sensitivity to ionizing radiation in tumor cells . CHMP4C Several Aurora kinase inhibitors are tested in clinical trials . StudiesCHMP4C can check the cytokinetic abscission to prevent premature and accumulated DNA damage as an abscission timer. CHMP4C deficiency generates the defective abscission check, which makes injured cells quickly pass through M to S phase, leading to DNA repair disability and genomic instability. These data suggests that CHMP4C deficiency disorganizes the cell cycle and enhances cell sensitivity to radiation, thus providing a new combined strategy to diagnosis and treatment of the lung cancer.We propose that ATM is activated by Aurora B, followed by p53-mediated activation of CHMP4C. In turn, CHMP4C promotes cell viability and proliferation. However, there is another major tumor suppressor factor, the alternative reading frame (ARF), which is negatively regulated by ATM in a transcription-independent manner. Contrary to the function of CHMP4C on cell fate, inhibition of ATM enhances ARF levels and stimulates the tumor-suppressive effects of ARF in human oncogene-transformed and cancer cells . These f2 and treated with different radiation of 2, 4, and 6 Gy using Co60 γ-rays with a dose rate of 1 Gy/min in the Irradiation (IR) Center .The human NSCLC cell lines A549 (p53 wild type) and H1299 (p53 deficient)  were cultured in RPMI-1640 containing 10% fetal bovine serum , incubated in 37 °C humidified incubator with 5% COA549 and H1299 cells were grown at 80% confluence and then introduced with siRNAs against CHMP4C , Aurora B (Ambion), p53 and p21  as well as control siRNA (Ambion). CHMP4C, Aurora B, p53 and p21 siRNA sequence is respectively: 5′-CCUGCGUCUCUACAACUAU-3′, 5′-CAUGGAUCUGAACAAAAUATT-3′, 5′-CUACUUCCUGAAAACAACG-3′ and 5′-CCUCUGGCAUUAGAAUUAUTT-3′. siRNA tansfections were performed using Lipofectamine RNAi MAX reagent (Invitrogen). Twenty-four hours after transfection, the cells were irradiated and harvested.pcDNA3.1-CHMP4C or -Aurora B was contrasted and transfected into A549 or H1299 cells using Lipofectamine 3000 reagent (Invitrogen). Twenty-four hours later, cells were collected and performed for subsequent experiments as siRNAs treatment.Aurora B kinase inhibitor AZD1152-HQPA was dissolved in dimethyl sulphoxide (DMSO). The A549 and H1299 cells were treated with AZD1152-HQPA at the concentration of 10, 100 and 1000 nM. After 24 h of treatment, CHMP4C phosphorylation was examined by Western blot.g for 15 min at 4 °C and measured by BCA protein assay kit (Thermo Scientific Pierce). Equal amounts of protein were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and blotted on nitrocellulose membranes for Western blot analysis. The membranes were blocked in 5% nonfat milk and then incubated with the following primary antibodies: CHMP4C , phosphorylated (p)—CHMP4C , Aurora B (Abcam), p53 , p21  and β-actin . The CHMP4C antibody is diluted in 1:500, and the rest were used in 1:1000 dilutions. Membranes were washed in tris-buffered saline containing 0.5% tween-20 and then incubated with goat anti-rabbit lgG  or goat anti-mouse lgG  conjugated to horseradish peroxidase for 1 h at room temperature. The membranes were detected using Chemiluminescence liquid (Thermo Scientific Pierce) according to the manufacturer’s protocol and analyzed by the Image J software .The cells were harvested and lysed in RIPA lysis buffer . The protein was collected at 12,000× Ct−∆∆ method.Total RNA was extracted using SV total RNA isolation system kit  followed by reverse transcription using the GoScript reverse transcription system kit (Promega). The subsequent cDNA products were used as templates to perform the real-time PCR assays. The primers for the amplification of Aurora B or CHMP4C are as follows; Aurora B forward: TTTGAGATTGGGCGTCCTCT and reverse: CGCCCTCCTTCTCTATCTGG; CHMP4C forward: AGAAGCCCTGGAGAACTCAC and reverse: CTTGGGCAGTATCCTGTTGC. The β-actin was used as the internal control using primers forward: TGCCAGAAAACAAGATGAG and reverse: CACCTTCACCGTTCCAGTTT. PCR amplifications were performed in triplicate wells and each experiment was repeated for three times. The relative expression levels of genes were analyzed through the use of the 2Cells were harvested and treated with Vibrant Dyecycle green stain (Invitrogen) at 37 °C for 30 min in a dark place. Then, cell cycle was analyzed on a flow cytometer using 488-nm excitation and green emission.To analyze colony formation, Single cell was plated in the 6 cm dish at a density of 1000 cells/mL for 10 to 14 days to form spheres. Colonies were fixed with 4% paraformaldehyde for 20 min and then stained with 1% crystal violet for 15 min. Colonies were counted and surviving fraction was calculated as the mean number of colonies/(cells seeded × plating efficiency).6 cells/mL and stained with PE AnnexinV and 7-AAD (BD) for 15 min at room temperature in the dark. Samples were analyzed by flow cytometry.Cells were harvested and washed twice with cold PBS. Cells were resuspended in 1× binding buffer (BD) at a concentration of 1 × 10Cells were seeded on the glass sheets into 6-well plates. Cells were transfected with siRNAs and then exposed to 4 Gy of irradiation. 30 min later, cells were washed twice in PBS, and then incubated with anti-γH2AX , anti-53BP1 (Abcam) or anti-CHMP4C antibody at a dilution of 1:200 overnight at 4 °C. After washing twice in washing buffer (0.05% Tween-20 in PBS), the cells were incubated with goat anti-rabbit lgG  at 37 °C for 1 h. Cells were washed twice with washing buffer and once with PBS. The coverslips were then mounted with mounting medium for fluorescence with DAPI (Vectashield). The image of γH2AX and 53BP1 foci was detected using a Leica DMRXA fluorescent microscope . γH2AX and 53BP1 foci were counted in cells with more than five foci.t test using Microsoft Excel . p-value <0.05 indicates statistical significance.Data are presented as the mean ± S.E. of three independent experiments, and the statistics were analyzed by students’ in vivo, for example the function of CHMP4C in xenografts [in vivo evidence for the new action of CHMP4C.In conclusion, we identify that CHMP4C is a new radiation-inducible protein engaged in cell cycle and cell survival. We first confirm CHMP4C offers lung cancer cells radioresistance, which will provide a potential radio-therapeutic strategy for non-small cell lung cancer by disrupting CHMP4C expression. In future research, the role of CHMP4C combined with radiation nografts , will be


Using a large social media dataset and open-vocabulary methods from computational linguistics, we explored differences in language use across gender, affiliation, and assertiveness. In Study 1, we analyzed topics  across 10 million messages from over 52,000 Facebook users. Most language differed little across gender. However, topics most associated with self-identified female participants included friends, family, and social life, whereas topics most associated with self-identified male participants included swearing, anger, discussion of objects instead of people, and the use of argumentative language. In Study 2, we plotted male- and female-linked language topics along two interpersonal dimensions prevalent in gender research: affiliation and assertiveness. In a sample of over 15,000 Facebook users, we found substantial gender differences in the use of affiliative language and slight differences in assertive language. Language used more by self-identified females was interpersonally warmer, more compassionate, polite, and—contrary to previous findings—slightly more assertive in their language use, whereas language used more by self-identified males was colder, more hostile, and impersonal. Computational linguistic analysis combined with methods to automatically label topics offer means for testing psychological theories unobtrusively at large scale. How do women and men use words differently? While language use typically differs minimally across self-reported gender, statistical models can accurately classify an author’s gender affiliation with accuracies exceeding 90% , suggestThe study of gender differences in language has a long history that spans gender studies, psychology, linguistics, communication, and computational linguistics, among other fields. Investigating gender differences has been, at times, considered controversial , 3, althgender-linked language features, such as words, phrases, and sentence length, that are used consistently more by one gender than the other . In most studies, researchers have identified gender-linked features by comparing text samples from self-identified females and males, counting the frequencies of theoretically interesting features in each text , comparing average frequencies across gender, and then interpreting results in terms of psychological theory [Individual studies and meta-analytic reviews have found evidence for l theory , 10, 11.d| ≤ .20; [For example, a meta-analysis conducted by Newman et al.  compared| ≤ .20; ). Men us| ≤ .20; , 16. AcrMulac et al.  comparedLeaper and Ayres  summarizThe prevalence of affiliation/assertiveness in gender research has motivated inquiry into how these dimensions relate to the Big Five personality framework. Assertiveness was found to correlate with extraversion, particularly the activity and excitement-seeking facets, whereas affiliation is captured by empathy-related aspects of agreeableness , 22. Affclosed-vocabulary analyses. These methods define categories of words a priori, based on common psychological or linguistic functions determined by researchers. The most popular implementation of closed-vocabulary analysis in psychology is LIWC, which automatically counts words belonging to over 60 predefined categories, such as positive emotion , achievement , articles , and tentative words .Most work on language differences by gender, including those above, have relied on Closed-vocabulary methods depend on researchers at two levels: category definition and psychological labeling. Category definition refers to the creation of coherent groups of words, phrases, and other features . For example, word categories may be formed on the basis of a common syntactic function, such as first person singular words  or prepositions , or by semantic content .positive emotions word category indicates a speaker’s experience of positive emotions) [cognitive processes is associated with having had a self-transcendent experience of unity, but the words most frequent within that category  are likely references to a greater whole in this case, rather than indicators of a cognitive process [Psychological labeling refers to the process of inferring a category’s psychological meaning. Labeling is often done by the researcher or by trained raters and is often theory-driven. For example, Mulac  suggestsmotions) . Such ex process . Such diOpen-vocabulary methods of language analysis are newer within social science, but are common within computational linguistics and related disciplines . These mtopics. For example, one topic included the words “love”, “sister”, “friend”, “world”, “beautiful”, “precious”, and “sisters”, and a second topic included “government” “freedom”, “rights”, “country”, “thomas”, “political”, and “democracy”. These topics are generated in a data-driven, “bottom-up” way, as opposed to the theory-driven, “top-down” methods used in closed-vocabulary approaches.In a recent example, Schwartz et al.  applied Open-vocabulary methods may reveal new, unexpected patterns of gender similarities and differences. However, a challenge with language topics derived through open-vocabulary methods is how to infer their psychological meaning. Consider the two topics above: the first contains generally positive, relationship-related words, while the second appear to be words related to political discussions. The first topic has some salient social and emotional references, but the psychological meaning of the political topic is less clear. While we may have intuitions about the characteristics of the people who use each topic, the psychological meaning of a topic is not obvious. To this end, psychological theory can provide a framework for understanding and interpreting automatically derived topics.In two studies, we examined gender language differences through an open-vocabulary analysis of language. In Study 1, we generated thousands of topics, and compared their relative use in a sample of over 52,000 female and male participants. This identified hundreds of male and female-linked topics. In Study 2, we labeled these gender-linked topics by degree of assertiveness and affiliation in a sample of over 15,000 people, and compared the pattern of gender-linked language along these two dimensions. Most studies require human raters to manually sort topics as either assertive or affiliative, whereas our method did so automatically. Further, while we and others have previously correlated topics with personality, here we use the correlations as labels. This labeling method places our open-vocabulary findings into a broader psychological context and allows comparisons with previous findings in the literature.gender-linked topics—topics used consistently more by one gender. One advantage of using social media as a language corpus is that it constitutes naturally occurring language among friends, family, and acquaintances. Only later—with users’ permission—was this language retrieved for research purposes. This allowed us to study language use in the relatively naturalistic setting of an online social networking site.In Study 1, we used open-vocabulary methods to categorize a large set of language from social media into a smaller set of topics. By comparing the relative use of each topic across several thousand self-identified men and women, we identified status updates. These participants also agreed to written informed consent within the MyPersonality application. An archival dataset of over 10 million users, collected between 2007 and 2012 is available for research use  [Our language source was messages from Facebook, a popular social networking platform . Particiity.org) . We use Median = 23, SD = 9.3, interquartile range = 20 to 29). Between January 2009 and November 2011, our final sample wrote approximately 15.4 million status messages. The average user (mean) wrote 4,077 words across all status messages .Status updates are a primary form of communication on the Facebook platform. These messages are typically visible to all first-degree friends in one’s social network. Status updates allow users to instantly broadcast information about themselves, such as current moods, activities, reactions, and relationships, to their social network. We created our analytic sample by selecting users who granted the MyPersonality application access to their status messages, wrote at least 1,000 words across their status messages, provided both their gender and age, and indicated that they were between 16 and 64 years of age, resulting in a final sample size of 68,228 participants for studies 1 and 2. Within this sample, 52,401 participants  were included in study 1, while the remaining 15,827 were set aside for study 2. Participants in this latter group were selected because they had completed a 100-item personality measure, while participants included in study 1 had not. The average user age was 26.2 years old , and included more females than males (62.6% versus 50.9%). There were no significant differences in terms of age or the other personality characteristics.^-^”), non-standard punctuation , and unconventional spellings and acronyms .As topic-based linguistic analyses of gender differences have rarely been done, we used this open-vocabulary approach to generate insights that complement and go beyond prior closed-vocabulary analyses. Prior to identifying topics, we first identified single words within the language sample. Words were defined by an emoticon-aware tokenizer , which iAfter extracting and tokenizing words and other language features, we used topics, derived via an unsupervised algorithm, latent Dirichlet allocation (LDA) , to defiAs the number of topics needs to be pre-specified, we set the number of topics to 2,000 to balance breadth and semantic coherence, and to be consistent with the precedent we set by using this number in our prior work . The samA single topic consists of hundreds of words along with weights, but only a small handful of words have appreciable weights. We found that listing the most heavily weighted 5 to 10 words in order of decreasing weights is often sufficient to portray the semantic content captured by a given topic.p(word|user) is the user’s normalized use of a word and p(topic|word) is the probability of the topic given that same word (which is part of the output of the fitted LDA model).We then calculated the relative use of each topic for every user. Topic use for a given individual was defined as the probability of using a topic,d, the standardized difference in group means, and 95% confidence intervals.Lastly, we estimated the size of gender differences for all 1,281 topics using Cohen’s d|) across all 1,281 topics was 0.12. The full distribution of gender difference effect sizes is shown in d|) greater than 0.10; 250 had absolute effect sizes greater than 0.20. Only 5 topics reach the level of a “moderate” effect (|d| ≥ .5).We found hundreds of gender-linked language topics . The average absolute effect size , consistent with findings by Newman et al.  and MulaIn general, female-linked topics contained many more references to emotions than male-linked topics, replicating findings from earlier meta-analyses by Leaper and Ayres . One advOur method also replicated several findings of male-linked language. For example, male-linked language included swearing and references to sports and occupations . NotablThe pattern of topics can also be viewed in light of people-focused versus object-focused language, as others have suggested. Meta-analyses have found that men had much stronger interests and preferences for working with things relative to people, whereas women showed the opposite pattern , 47. LikAlthough several of our open-vocabulary findings converged with previous work, our method also generated hundreds of gender-linked topics that did not fit neatly into earlier frameworks. For example, several of the politically-related male-linked topics  are not easily categorized as objects- or people-oriented. In Study 2, we built on Study 1 by using a method to assign psychological labels to these topics and describe the pattern of gender differences along more psychologically meaningful dimensions relevant to the extant literature.Study 2 characterized gender-linked topics from Study 1 into meaningful psychological attributes. Our goal was to assess each topic according to dimensions that would be most relevant to past studies of gender language differences and also have broader psychological significance: affiliation and assertiveness.affiliation and assertiveness, are so common in language studies that Leaper and Ayres [Gender differences have often been characterized by at least one of two dimensions: (1) affiliation and interpersonal warmth versus impersonality and coldness, and (2) assertiveness and dominance versus indirectness and passivity. These two dimensions, which we call nd Ayres  organizend Ayres  summary nd Ayres . Others nd Ayres .The prominence of the dimensions of affiliation and assertiveness in language research follows a long history of describing interpersonal behavior and judgments along similar dichotomies: communion and agency , 54, lovDepue and Morrone-Strupinsky  describeAssertiveness reflects a tendency towards “dominance, ambition, mastery, and efficacy that is manifest in. . . interpersonal contexts” , p. 315)x, y) or polar coordinates . This space is often divided into distinct regions, each reflecting different interpersonal styles. The descriptive labels around the edge of the circumplex reflect the octants suggested by Wiggins [gregarious-talkative region, while highly assertive but highly unaffiliative behaviors fall within the arrogant-calculating region.Together, affiliation and assertiveness form the primary axes of the interpersonal circumplex , a rich  Wiggins . For exaTo determine the degree of affiliation and assertiveness of a given language feature, we considered the traits of the people who are most likely to use that language. That is, we reasoned that assertive language would be expressed disproportionately more often by people who scored high on measures of assertiveness. For example, if a language topic containing the words “family”, “friends”, “wonderful”, “blessed”, and “amazing” is used most frequently by people who are highly assertive and highly affiliative, then we label it as a highly assertive and highly affiliative language topic. Likewise, if the topic containing “computer”, “error”, “program”, “photoshop”, and “server” is used most by unassertive and unaffiliative people, then we label it as low on assertiveness and low on affiliation.To derive these labels, we examined correlations between topic use and self-reported personality measures in a sample of over 15,000 Facebook users (separate from the sample used in Study 1). These users completed measures of extraversion and agreeableness–the two Big Five domains most relevant to the interpersonal circumplex , 69, 70.We built on these findings by first calculating the correlations between each topic and facets of extraversion and agreeableness, and then rotating these see  above toMedian = 22, SD = 8.2, interquartile range = 20 to 27).Participants were users of MyPersonality who granted the application access to their status messages, wrote at least 1,000 words across their status messages, provided their gender and age, indicated that they were between 16 and 64 years of age, completed a 100-item personality measure, and were not a part of the Study 1 sample. Our resulting sample size was 15,827 individuals . The average participant’s age was 24.9  , 73. Thir = .24), we controlled for each trait when calculating correlations for every topic. We standardized topic use, extraversion, and agreeableness scores across users. Then, we regressed topic use on extraversion, agreeableness, gender, and age. The resulting regression coefficient for extraversion is equivalent to a Pearson correlation between the topic and extraversion, controlled for agreeableness, gender, and age, and the resulting regression coefficient for agreeableness is equivalent to a Pearson correlation between the topic and agreeableness, controlled for extraversion, gender, and ageTo determine the topic’s degree of affiliation and assertiveness, we first estimated each topic’s correlations with extraversion and agreeableness, controlling for age and gender. Controlling for age and gender ensured that our resulting labels did not merely reflect gender differences in underlying personality trait distributions. Because extraversion and agreeableness were correlated in our sample  coordinates, a process used in other studies utilizing circumplex models [xtopic, ytopic) are a topic’s loadings on affiliation and assertiveness, respectively, and rext and ragr are a topic’s correlations with extraversion and agreeableness, respectively. Thus, affiliation_topic and assertiveness_topic contain affiliation and assertiveness effect sizes for the given topic, which can be plotted within a two-dimensional plane where affiliation is the x-axis and assertiveness is the y-axis.Each topic’s correlations with extraversion and agreeableness were then used to create affiliation and assertiveness scores, which also determine its position in the interpersonal circumplex in Cartesian  of topics to their respective level of affiliation and assertiveness, and we calculated the Pearson correlation between ds  and each dimension. We also examined the language content of topics near the tails of each dimension to assess whether our automatic labels identified reasonably assertive and affiliative language, or their opposites (described above) deferential and cold-hearted, respectively.After labeling topics by affiliation and assertiveness, we analyzed the pattern of gender differences across each dimension. We first created scatterplots to compare the gender difference effect  across topics as estimated in the sample of 15,827 participants who also completed the 100-item personality questionnaire. We calculated these ds, too, and across both samples the ds were correlated at r = .98. We opted to use the ds from the Study 1’s sample of 52,401 participants due to the much larger sample size, but the pattern of results would not have meaningfully changed had we used gender difference ds from Study 2. Of the 1,281 topics, 905 met this criterion. We then used affiliation and assertiveness labels to place each gender-linked topic into the interpersonal circumplex, and explored the spatial distribution of these topics in two complementary ways. First, we visualized the pattern of differences by plotting topics in the circumplex and shading the corresponding points according to the size and direction of the gender difference. This visualization offers an overview of the differences across hundreds of topics.To focus specifically on patterns of gender-linked topics identified in Study 1, we limited our analysis to topics that had non-trivial gender differences, which we defined as those with |d of all gender-linked topics.Second, we compared the distributions of male- and female-linked topics within interpersonally distinct areas of the circumplex octants. Octants are formed around the primary and secondary axes, and we use the divisions and labels suggested by Wiggins . Within topics, not male and female participants). We visualized these summaries using iconic representations, which illustrate group differences within a circumplex space. To produce iconic representations, we calculated the mean angle or circular mean (θM) within each group of topics. A group’s circular mean describes its “predominate theme” or “center of gravity,” p. 417 [topic) of every topic asFinally, we summarized the central tendency and variability of each group of topics . We next used each θtopic to locate each topic along the circumference of the unit circle, or , asHere, θx and y, or xM and yM by averaging across the individual x’topic and y’topic, respectively. Each group’s circular mean, θM, is the angle corresponding to their respective xM and yM, orWe calculated each group’s mean n is the number of topics in each group. We then converted this to degrees and plotted ±1 unit of variance around each group mean. Finally, we estimated 95% confidence intervals around each group’s θM using the approximation suggested by Gurtman & Pincus (2003),To summarize the variability of each group of topics, we calculated the circular variance within each group asThe resulting iconic representations display the circular mean (as arrows), corresponding confidence intervals around the mean (as dark shading), and ±1 unit of variance around each mean (as light shading).Our method automatically labeled topics by assertiveness and affiliation, and patterns of male- and female-linked language reflected contrasting interpersonal styles. Highly affiliative language was used much more by female participants. Gender differences in the use of assertive language were less clear, though women used slightly more assertive language.The most affiliative topics  were cenaffiliationtopic) was positively correlated  to topic gender score (i.e. Cohen’s d between the topic and gender). Topic affiliation score (i.e. assertivenesstopic) was positively correlated with topic gender score , but examination of the scatterplot in Topic assertiveness score . Male-linked topics were largely in the less affiliative, colder half, but also spread more evenly in terms of assertiveness. Male-linked topics were both the most assertive (a swear word topic) and least assertive (topics with computer-related words).ds favored women; in the most and least assertive octants, mean and median ds favored men.Octant-level analyses of gender-linked topics and effect sizes were consistent with a pattern of greater affiliation across female-linked topics but greater variation in assertiveness across male-linked topics. The mean angles of gender-linked topics, displayed as arrows in not find clear gender differences in assertive language. Instead, we found that male participants were more likely to use language that was both highly assertive and colder , while women were more likely to use language that was highly assertive but also warmer . While average gender differences in assertive language were small, male-linked language in assertiveness was more variable. While some male-linked topics were cold and assertive, others were cold yet highly unassertive. These unassertive topics contained relatively neutral language about objects . Ultimately, the greatest distinction between female- and male-linked language was in terms of the level of affiliation and interpersonal warmth.Our labeling method automatically labeled affiliative and assertive topics of language. Affiliative, interpersonally warmer language was used more often by female participants. Contrary to past research  and popud = .40) and falls in the aloof-introverted octant. Neighbors of this topic in interpersonal space included topics about government and taxes, knives and stabbing, and death. While they are diverse in semantic content, they share the same aversive interpersonal style and are all potentially unsettling topics in an informal public social setting like Facebook. They were also used far more by male than female participants. On the other hand, the largest cluster of strongly female-linked topics in the gregarious-extraverted octant was loaded with positive evaluations and expressions about friends and families.Placing language into interpersonal space revealed similarities between topics that were not obvious from a direct analysis of their words. Consider the topic containing the words “opinion”, “opinions”, “logic”, “based”, “political”, and “fact”. This topic was among the most male-linked  was roughly an order of magnitude larger than the combined sample size across all studies included in Leaper and Ayres’ (2007) meta-analyses of gender language differences . Finally, effect sizes were small by conventional standards, although they were similar in size to other studies of language on social media. The large sample size provides power to detect small effects, but the practical meaning of small effects, especially in other contexts or for single users is unclear, and the results should be interpreted accordingly.Despite the limitations of social media samples, they allow researchers to study questions at on a much larger scale than is typically possible. The total sample size afforded by social media in our two studies (In a large study of gender and language, we found that men and women use language differently, with the greatest difference being in the degree of interpersonal warmth. The language most characteristic of self-identified females was warmer, friendlier, and focused on people, whereas self-identified males’ most characteristic language was more socially distant, disagreeable, and focused on objects. Contrary to expectations, women used slightly more assertive language than men. We found affiliative and assertive language through established assessments rather than human judgments, the latter of which are more prone to rater-bias. Our approach borrows equally from computational linguistics and psychological theory, and we propose that similar interdisciplinary approaches may be useful for seeing old psychological questions in a new light.


Aggregation of α-synuclein is believed to play an important role in Parkinson's disease and in other neurodegenerative maladies. Small molecule inhibitors of this process are among the most promising drug candidates for neurodegenerative diseases. Dendrimers have also been studied for anti-fibrillation applications but they can be difficult and expensive to synthetize. Here we show that RAFT polymerization can be used to produce a hyperbranched polyethylene glycol structure via a one-pot reaction. This polymer included a dopamine moiety, a known inhibitor of α-synuclein fibril formation. Dopamine within the polymer structure was capable of aggregation inhibition, although not to the same degree as free dopamine. This result opens up new avenues for the use of controlled radical polymerizations as a means of preparing hyperbranched polymers for anti-fibrillation activity, but shows that the incorporation of functional groups from known small molecules within polymers may alter their biological activity.  •Dopamine-containing hyperbranched polymer was synthesized by RAFT co-polymerization.•This polymer inhibited α-synuclein aggregation less effectively than dopamine.•It was also much less effective than dopamine in disaggregating α-synuclein fibrils.•The difference is likely due to loss of covalent dopamine–protein interactions. Autosomal dominant early-onset PD is induced as a result of six different missense mutations in the α-synuclein gene In recent years, polymers and nanoparticles have been explored, not only for the more established field of protein aggregate detection, diagnosis and destruction in vitro. We found that, similar to dopamine itself, DOPA-modified polymer interfered with the α-synuclein fibril formation promoting oligomer formation instead. However, effects of dopamine were significantly moderated by its incorporation into a polymer, and it was no longer capable of effectively disaggregating fibrils into oligomers. The ease at which the polymer structure and composition can be varied allows the potential to mechanistically study specific polymer/protein interactions in an attempt to find future therapeutic strategies for diseases associated with protein misfolding.In this study, we show the proof-of-principle, that RAFT polymerization can be used to produce a hyperbranched poly (ethylene glycol) (PEG) structure which contains a DOPA moiety hypothesized to provide anti-fibrillation properties. We aimed to investigate whether the inclusion of a DOPA molecule into a hyperbranched structure would reduce, enhance or have no effect on anti-fibrillation or aggregation behavior. For the synthesis of this functionalized polymer, a DOPA analogue with a methacrylamide group  was firstly synthesized and incorporated into a hyperbranched PEG via co-polymerization. This polymer, DOPA-PEG polymer (henceforth termed DP) was analyzed at different concentrations to assess its effect on α-synuclein aggregation/fibrillation 22.1-1), poly(ethylene glycol) diacrylate , 2,2-dimethoxy-2-phenylacetophenone and 1,10-Azobis-cyclohexane-carbonitrile (ACHN) were purchased from Sigma. Acetone 99.8+%, tetrahydrofuran, hexane 95%, dimethylformamide (DMF), ethyl acetate, hydrochloric acid, and methanol were purchased from Fisher Scientific. 4-Cyano-4-[(ethylsulfanylthiocarbonyl)sulfanyl] pentanoic acid was obtained as a kind gift from Dr. Hongyun Tai at Bangor University, UK. Other chemicals and supplies were from Sigma, Fisher or VWR.Dopamine hydrochloride, sodium bicarbonate, methacrylate anhydride, sodium borate, sodium hydroxide, magnesium sulfate, poly(ethylene glycol) methyletheracrylate .The dopamine methacrylamide (DMA) monomer was prepared via a previously reported protocol 2.3Protein aggregation in the automated format was carried out in a reaction volume of 0.1 mL in black, flat-bottomed 96-well plates in the presence of 5 μM thioflavin T (ThT). Two teflon balls  were placed into each well of a 96-well plate. The reaction mixture containing protein and ThT (320 μl) was split into three wells (100 μl into each well), the plates were covered by Mylar septum sheets (Thermo), and incubated at 40 °C with continuous orbital shaking at 280 rpm in an Infinite M200 Pro microplate reader . Reaction conditions: 0.25 mg/mL (17.4 μM) α-synuclein in 20 mM Hepes buffer (pH 7.5), 100 mM NaCl, 5 μM ThT and 50 μg/mL heparin. The kinetics was monitored by top reading of fluorescence intensity every 5 min using 444 nm excitation and 485 nm emission filters. Data from replicate wells were averaged before plotting fluorescence vs. time. The data were fit to a sigmoidal equation Eqn.  using Si−1), and tm is the midpoint of transition. The lag time (tl) of amyloid formation was calculated as tl = tm−2/k. Initiation rate was defined as the inverse of lag time.Here A is the initial level of ThT fluorescence, B is the difference between the final level of ThT fluorescence and its initial level, k is the rate constant of amyloid accumulation (hFibril disaggregation assays were conducted in a similar manner except preformed α-synuclein fibrils (0.05 mg/mL) were incubated  in the presence of variable concentrations of either dopamine or a dopamine-containing polymer (DP) in the plate reader with shaking, and ThT fluorescence was monitored. Fibril disaggregation was analyzed using the three parameter exponential decay equation using SigmaPlot .2.45 μl aliquot of the protein solution was adsorbed onto prewashed 200 mesh formvar/carbon-coated nickel grids for 5 min. The grid was washed with water (10 μl), stained with 2% uranyl acetate (10 μl) for 2 min and washed with water again. The samples were analyzed with a JEM 1400 transmission electron microscope (JEOL) operated at 80 kV.33.11H NMR results  as well as dopamine (0.5–10 mg/mL) and PEG 12 (1–20 mg/mL) on aggregation of α-synuclein. We used the aggregation conditions close to physiological that were previously shown to promote effective α-synuclein aggregation . Each experiment was run in triplicate, and independent experiments were performed at least 3 times for each data point.We found that DOPA-modified polymer inhibited formation of α-synuclein fibrils  A–E. A lDopamine itself was significantly more effective as an inhibitor of fibril formation. In the presence of dopamine the lag phase of α-synuclein aggregation increased and the ThT signal decreased significantly in the concentration-dependent manner. At dopamine concentrations of 10 mg/mL and higher, no increase in ThT fluorescence was observed indicating complete inhibition of fibril formation. Electron microscopy H confirmPEG 12 accelerated α-synuclein aggregation decreasing the lag phase of aggregation somewhat and significantly increasing the ThT fluorescence indicating higher yield of fibrils  A–E. And3.3In addition to interfering with formation of protein fibrils, some compounds are known to disaggregate the preformed fibrils. A peculiar shape of α-synuclein aggregation curves in the presence of dopamine B consistWe analyzed the morphology of α-synuclein aggregates by electron microscopy at different time points during aggregation in the presence of dopamine indicated by arrows in Since PD pathology is associated with dopaminergic neurons, interaction between α-synuclein and dopamine has been extensively investigated Overall, we synthesized a dopamine-containing hyperbranched polymer via RAFT co-polymerization with PEG monomers. We found that, similar to dopamine itself, the polymer interfered with the α-synuclein fibril formation promoting oligomer formation instead. However, effects of dopamine were significantly moderated by its incorporation into a polymer, and it was no longer capable of effectively disaggregating fibrils into oligomers. This is likely due to a specific mechanism of action of dopamine known to involve covalent interaction with the target proteins. The ease at which the polymer structure and composition can be varied allows the potential to mechanistically study specific polymer/protein interactions in an attempt to find future therapeutic strategies for diseases associated with protein misfolding.


Molecular functional imaging (MFI) has given a newer insight to the medical imaging and has diversified the role of imaging in the field of the translational cancer medicine and has an indispensible role to play in screening, early diagnosis, staging, predicting prognosis, therapy delivery, therapy monitoring and follow-up. Overall there has been a significant development in the field of molecular imaging and its utilisation in the perspective of the biomedical research which has led to better understanding of the signalling pathways in the tumourigenesis and novel drug discoveries.Review and Highlight the complementary role of these techniques in the detection and staging of tumours and which of these techniques is more adequate for clinical scenarios in oncology.• Cancer And Molecular Functional Imaging (MFI)• MFI Of Gene Expression, Receptors And Signalling Pathways• MFI of Multidrug-Resistance In Cancer• MFI Of Extracellular Matrix And Its Key Components• MFI Of Neoangiogenesis, Hypoxia And Metabolism• MFI And Small Animal Imagingst century and beyond.The future of molecular functional imaging in the coming era is its exploitation into understanding the gene expression profiling in-vivo and optimising the patient specific therapies using gene expression profiling. Quantitative molecular functional imaging, in conjunction with quantitative structural imaging, will be the future of 'personalised radiology,' 'personalised oncology,' 'personalised medicine,' and of oncologic research in the 21


Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis. A distinctive feature of tumor growth is uncontrolled cell proliferation that largely bypasses the process of programmed cell death, or apoptosis . As hypeXIAP is a member of the inhibitor of apoptosis (IAP) family of proteins that target caspases, the effector molecules of cell death . XIAP isPN1, known alternatively as SerpinE2 , is secrvia 1) an alteration in NF-κB signalling, lessening xiap transcription or 2) through a blockade of AKT signalling, preventing the stabilizing phosphorylation of XIAP at serine 87, therefore promoting the protein to degradation. Thus, the PN1-uPA regulatory axis may be capable of triggering apoptosis by modulating survival pathways and as a result the growth of prostate cancer cells.We have found that PN1-mediated inhibition of uPA and its signalling through the uPA receptor (uPAR), but not LRP-1, results in a downstream cascade of events leading to a cellular reduction in XIAP. This can occur either We show here and previously  that expHaving validated that increased levels of PN1 can enhance apoptosis, we sought to determine its effect on known cell death regulatory proteins. Lysates from PC3 cells transfected with an empty vector (Mock) or a PN1 expression vector were evaluated using arrays to detect 35 pro- and anti-apoptotic factors Figure . Of the These data Figure  suggeste3. The combinatorial effect of PN1 exposure and TRAIL (post-treatment) of PC3 xenografts resulted in slower growth compared to control  . These tumors were then treated with daily administration of TRAIL (40 mg/kg) intraperitoneally after tumors reached 100 mm) Figure . These dxiap RNA levels were determined using qRT-PCR at 24 h following transfection of increasing concentrations of the PN1 expression vector  and a cervical cancer cell line (SIHA) were tested. In all three cell lines, PN1 expression reduced xiap mRNA levels  in conjunction with a sandwich ELISA system for the detection of XIAP. Once more, inhibition of uPAR made a substantial impact on total XIAP protein while its levels were relatively static following LRP blockade Figure . We alsoxiap mRNA through the NF-κB pathway, we used an NF-κB inhibitor (BAY 11–7082). As expected, the inhibitor alone reduced xiap expression. That effect was amplified by expression of PN1 protein  alone or in combination with PN1 expression, reduced xiap expression . PN1 expression displayed a trend of decreasing in the presence of increasing p65 or XIAP  show an inverse correlation between PN1 expression and XIAP Figure . PN1 levOur data provides evidence that PN1 can regulate signalling by neutralising complexes uPA-uPAR and altering downstream pathways that contribute to the transcriptional activation and stabilization of the survival factor XIAP in prostate cancer cells. As a result, PN1 levels may affect the overall severity of prostate cancer. Therefore, induction of PN1-mediated signalling may be useful for enhancing XIAP-specific inhibition by small molecule compounds in cancer therapy.pn1−/− mice were a generous gift from MC Bouton . All knock out animals were compared against littermate controls. All animal experiments and protocols were reviewed and performed in accordance with UK Home Office and Oxford University regulations.pcDNA3-PN1 was a kind gift from Dr Peter Andreasen's lab . A range of point mutations were generated previously as described .Cancer cell lines PC3, SIHA, Jurkat and HL-60 cells were obtained from the American Type Culture Collection (ATCC) and regularly tested to ensure the absence of Mycoplasma contamination . New stock vials were thawed every 3–4 months and cell morphology regularly checked. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C with 5% CO2. PN1 expression vectors were transfected into cells using FuGENE 6 reagent (Roche). All transfection experiments performed for 24 h unless otherwise noted. Where indicated, inhibitors, recombinant proteins, or blocking antibodies were added to serum free medium during treatments: NF-κB inhibitor Bay11–7082 , PI3k/AKT inhibitor LY 294002 , AKT inhibitor MK-2206 (Santa Cruz), recombinant uPA (America Diagnostica), recombinant PN1 (R&D system), uPAR blocking antibody (R&D Systems), and LRP blocking antibody . Recombinant human TRAIL was generated as described . uPA actCell pellets were lysed in TNN buffer containing protease inhibitor cocktail . 50 μg of protein was loaded into 10% Bis-Tris gels (Invitrogen). Blots were probed with the indicated antibodies: anti-PN1, XIAP, DR5 (R&D Systems), beta-actin (Santa Cruz), p-AKT, PARP, all NF-κB molecules , and uPA (Abnova).All immunohistochemistry was performed on tissues harvested from wild type C57/B6 or pn1−/− animals on a B6 background. All harvested tissues were fixed for 24 hours in 4% paraformaldehyde and sliced at 10 μM thickness for staining. Briefly, microscope slide mounted tissues were hydrated and exposed to an antibody at a 1:50 dilution overnight following antigen retrieval . Pre-diluted Immpact secondary antibody (Vector Labs) was applied to the slides for 30 min and Immpact DAB (Vector Labs) was added to observe positive stain. Samples were evaluated microscopically and photographs were taken on a 20× objective.Apoptotic Protein arrays  were used to probe for alterations in protein amounts. PC3 cell lysates were prepared 24 h after transfection with PN1 expression vectors and 300 μg of lysate was diluted in blocking buffer. This solution was incubated with an apoptotic protein array containing 35 pro- or anti-apoptotic proteins overnight at 2–8°C. After 3× washing with PBS, reconstituted Detection Antibody Cocktail was added to the membrane and incubated for 1 hour. The membrane was then subjected to Streptavidin-HRP followed by a chemiluminescent reagent, per manufacturer's instructions.All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC  according to factory instructions. Briefly, 10 μg of cell lysate was combined with a provided XIAP capture antibody fused to the bottom of a 96 well plate overnight. Then a streptavidin-linked detection antibody was applied for 2 h before a substrate was added (20 min) for colorimetric detection at 450 nM using a Tecan m200 plate reader.RNA was extracted by Trizol and the RNA concentration was measured by a Nano-Drop 1000 spectrophotometer (Thermo). All qRT-PCR reaction mixtures were prepared using Superscript Platinum III one step kits with incorporated SYBR Green (Invitrogen). One step cDNA production and DNA amplification were performed on a Stratagene MX 3005P thermocycler. All amplified products normalized against GAPDH. Amplified products confirmed by electrophoresis using 1.5% high-resolution agarose gels.pn1,upa, lrp, upar, and siRNA negative control oligos were from Ambion. 10 nM of each siRNA was transfected into cells using the siPORTNeoFX transfection agent (Ambion). After 48 h, cells were washed and the media replaced with serum free media. Conditioned media was collected 24 h later.siRNA directed against 6 PC3 cells (suspended in 50 μl of serum free media and 50 μl of Matrigel supplemented with recombinant PN1 as indicated) were injected subcutaneously into the flanks of adult SCID mice as previously described. When distinct individual tumors reached 100 mm3, TRAIL/apo2 was administered at 40 mg/kg, daily by IP, in appropriate treatment groups. Tumor volumes were determined from calliper measurements of the tumor dimensions at the indicated times.Briefly, 1 × 10Human tissue microarrays  contained samples from 36 cases of prostate cancer with various Gleason scores and 12 examples of normal prostate tissue. TMA staining was assessed using a combination of intensity and percentage of positive stained cells, as previously described . The slip-value of < 0.05.All statistical measures were determined using the Prism 5 Graphpad software. Statistical significance was considered to be a


The use of baseline dopamine D1 and D2/3 receptor binding potential (BPND) as a proxy for dopaminergic tone is problematic because they reflect both endogenous dopamine levels (a change in radiotracer's apparent affinity) and receptor density. In this analysis of 31 healthy controls genotyped for the Val158Met polymorphism , we used amphetamine-induced displacement of [11C]FLB 457 as a direct measure of dopamine release. Our analysis failed to show a relationship between COMT genotype status and prefrontal cortical dopamine release. COMT genotype was also not predictive of baseline dopamine D2/3 receptor BPND.Basic investigations link a Val158Met polymorphism (rs4680) in the catechol-O-methyltransferase (COMT) gene to not only its enzymatic activity, but also to its dopaminergic tone in the prefrontal cortex. Previous PET studies have documented the relationship between COMT Val158Met polymorphism and D Higher D1 receptor BPND in the cortex of Val/Val subjects compared to Met carriers in this study was interpreted as a marker of decreased dopamine. Such an interpretation is consistent with the notion that COMT clears cortical dopamine ~40% more efficiently in individuals with the COMT Val compared to the Met allele [2/3 antagonist radiotracer [11C]FLB 457 and PET, Hirvonen and colleagues found no significant association between the Val158Met genotype and cortical D2/3 receptor BPND. The reason for the discrepant results observed with [11C]NNC 112 and [11C]FLB 457, which are both reportedly sensitive to chronic alterations in endogenous dopamine (despite the fact that they label different subtypes of the dopamine receptor), is unclear [11C]FLB 457 in the cortex makes its baseline BPND a less than ideal proxy for endogenous dopamine tone. It is also possible that Val158Met has an effect on D1 receptor BPND that is independent of endogenous dopamine levels, and this is not necessarily reflected in the D2/3 receptor BPND. The use of D1 and D2/3 receptor BPND as a proxy to interpret vivo dopamine levels in these prior studies complicates the interpretation of these data.Previous studies have demonstrated that the catabolic enzyme catechol-O-methyltransferase (COMT) plays a major role in regulating dopamine transmission in the prefrontal cortex, a region that is relatively devoid of dopamine reuptake transporters, compared to the striatum , 2. Humaal, BPND , 6. The t allele . However unclear . It is p11C]FLB 457 and PET have demonstrated the ability to measure dopamine transmission in the prefrontal cortex following an acute amphetamine challenge [11C]FLB 457 BPND in several cortical regions of interest including the dorsolateral prefrontal cortex, orbital frontal cortex, medial prefrontal cortex, and anterior cingulate cortex. This reduction in [11C]FLB 457 BPND following an amphetamine challenge has been shown to linearly correlate with increases in extracellular fluid dopamine as measured using microdialysis in non-human primates [11C]FLB 457 with PET provides an opportunity to test the relationship between dopamine transmission and COMT Val158Met genotype status. Here, we include data from a cohort of healthy controls who underwent [11C]FLB 457 PET scans before and after an acute oral amphetamine challenge (0.5 mg/kg). In this dataset, the relationship between COMT Val158Met genotype status and cortical [11C]FLB 457 BPND, as well as amphetamine-induced dopamine release , were examined. Based on the literature, we hypothesized that the COMT Val158Met genotype would be predictive of amphetamine-induced ΔBPND , but not baseline D2/3 receptor BPND in cortical regions.Recent studies with [hallenge –11. In hprimates . The abiThe Institutional Review Board and Radioactive Drug Research Committee of the University of Pittsburgh approved the study. All subjects provided written informed consent.11C]FLB 457 BPND and COMT Val158Met genotyping  were available for n = 31 healthy control  male or female aged 18–40 years; 2) no current or past DSM-IV axis I disorder as assessed by the Structured Clinical Interview for DSM-IV Axis I Disorders; 3) no current use of cocaine, opiates, cannabis, sedative-hypnotics, amphetamines, 3,4-methylenedioxy-N-methylamphetamine, or phencyclidine (as confirmed by urine drug screen at the study screening and PET scan day); 4) not currently pregnant or nursing; 5) no current or past chronic medical or neurological illnesses as assessed by a complete physical examination and laboratory examination; 6) no resting systolic blood pressure >140 mm Hg and no diastolic blood pressure >90 mm Hg; 7) no history of prior radioactivity exposure from nuclear medicine studies or occupation; 8) no metallic objects in the body that are contraindicated for MRI; 9) no more than one risk factor for coronary artery disease ; 10) no first-degree relative with a psychotic or mood disorder; and 11) not currently on any prescription or over-the-counter medications.11C]FLB 457 PET scan on the same day. In the remaining subjects, the baseline and post-amphetamine scans were acquired on different days, approximately a week apart. The details of this imaging protocol have been published [A magnetization prepared rapid gradient echo structural MRI scan was obtained using a Siemens 3 Tesla Trio scanner for determination of regions of interest. All but eight subjects underwent the baseline and post-amphetamine [ublished .11C]FLB 457 was synthesized using the methodology reported by Halldin, et al. [11C]FLB 457 and emission data was collected for 90 minutes. Arterial blood samples were collected to measure the plasma free fraction (fP) for [11C]FLB 457 and derive a metabolite corrected arterial input function for modeling using methods described previously [11C]FLB 457 was restricted to 0.6 μg [11C]FLB 457 scan was performed ~ 3 to 4.5 hours after the administration of 0.5 mg kg-1 of oral d-amphetamine. Amphetamine blood levels were measured before the post-amphetamine [11C]FLB 457 PET scan and were analyzed using methods previously described FLB 457 VT in the regions of interest (VT ROI) and cerebellum (VND) was performed using a two-tissue compartment kinetic analysis using the arterial input function as previously described [PET data were reconstructed and processed with the image analysis software MEDx  and SPM2  available to bind [11C]FLB 457 in vivo, KD is the in vivo equilibrium dissociation constant of [11C]FLB 457 (nmol L-1)PET outcome variables are defined in accordance to the consensus nomenclature for oligands . D2/3 reND (Δ BPND) was calculated as the difference between BPND measured in the post-amphetamine condition (BPND AMPH) and BPND measured in the baseline condition (BPND BASE), and expressed as a percentage of BPND BASEThe amphetamine-induced change in BP-1). PCR primers were designed around COMT rs4680 (G/A polymorphism) using PRIMER3 program (http://bioinfo.ut.ee/primer3-0.4.0/). The size of the amplicon is 348bp. The primer sequences are F- GGGCCTACTGTGGCTACTCA and R-CTTGGCAGTTTACCCAGAGC. PCR conditions were 94° for 10 minutes, followed by 35 cycles of 94° for 30 seconds, 54° for 30 seconds 72° for 30 seconds, and final extension at 72° for 7 minutes. PCR products were checked on 1.5% agarose gel. After gel checks, PCR products were treated with ExoSAP-IT and DNA sequencing was conducted using the BigDye Terminator V3.1 Cycle Sequencing Kit (Life Technologies). Sequencing traces were compiled on Sequencher V5.3 and two investigators read genotypes traces separately to confirm them. Discrepancies were resolved through re-analysis.DNA was extracted from frozen blood samples of subjects using the QIAMP DNA Blood Mini Kit. DNA was diluted to working stock (10ng ulND) were performed with paired t-tests. Prior to performing statistical tests, Box's M tests were performed on the dependent variables, BPND and ΔBPND, to evaluate the data, where p >0.05. Thus, genotype effect on baseline cortical BPND and Δ BPND was tested using a linear mixed model analysis with cortical regions of interest as a repeated measure and genotype as the fixed factor (IBM SPSS Statistics). Genotype, region, and genotype by region interaction were included in the model as explanatory variables. To control for the potential confounding effect of smoking status, its effect on the model was evaluated by including it as a factor following the primary analysis. A two-tailed probability value of p < 0.05 was selected as the significance level for all analyses.Group wise differences in demographics were tested using Chi-squared and analysis of variance (as applicable). Comparison between baseline and post-amphetamine scan variables  = 0.04, p = 0.96; effect of region, F  = 186.96, p < 0.001; genotype x region interaction, F  = 0.39, p = 0.98). The inclusion of smoking as a factor in the model did not alter the results . BPND derived using a simplified reference tissue method with the cerebellum as an input function did not alter the results As shown in 11C]FLB 457 BPND in five of the eight cortical regions of interest when data from all three genotypes were included (11C]FLB 457 BPND were observed between the genotypes (data shown in T and ΔBPP showed no significant effect for genotype consistent with that observed with ΔBPND. (data not shown). ΔBPND derived using the simplified reference tissue method did not alter the results .Consistent with previous studies –11, 19, included . No sign2/3 baseline receptor BPND nor amphetamine-induced ΔBPND in the cortical regions. The failure to demonstrate a relationship between COMT Val158Met genotype and cortical D2/3 receptor BPND, which is consistent with a previously published report, was expected [11C]FLB 457 are unclear. One factor could be the relatively high inter-individual variability associated with ΔBPND. This is likely due to the relatively lower baseline D2/3 BPND and decreased dopamine released in response to amphetamine in the cortex compared to that in the striatum [11C]FLB 457 in the cortex following an acute amphetamine challenge FLB 457-amphetamine data in 128 and 200 subjects respectively, as indicated by power calculations (β = 0.8). The heterogeneity of the subject population  and differences in scanning schedule (acquisition of baseline and post-amphetamine scans on the same vs. different day) may have also contributed to the variable Δ in BPND. Another factor that may have contributed to the inability to demonstrate an effect for COMT genotype on ΔBPND in our paradigm is the fact that the half-life for amphetamine-induced dopamine release is relatively prolonged in the cortex FLB 457 post-amphetamine scan  in our studies was designed to capture the peak dopamine release that follows the peak amphetamine plasma level (~ 3–4 hours), and not necessarily the clearance, which is measured following several half-lives. [11C]FLB 457 PET studies measuring time to restore baseline BPND rather than the magnitude Δ in BPND following an acute amphetamine challenge might be more relevant in detecting a difference in the clearance of dopamine as influenced by COMT Val158Met genotypes. It is also possible that alpha-methyl-para-tyrosine (AMPT) induced dopamine depletion PET studies that measure baseline dopamine levels may be more successful in detecting an effect for the COMT genotype. However, measuring baseline dopamine levels in the cortex with such an imaging paradigm has been unsuccessful due to technical challenges [1 receptor findings reported with COMT genotype because D1 receptors that are located in the extra synaptic space likely reflect slower adaptations, including changes in gene transcription in response to chronically altered tonic dopamine levels. It would also explain the lack of a relationship between COMT genotype and amphetamine-induced dopamine release, which is likely more reflective of the phasic DA release caused by behaviors.The results of this study demonstrate the COMT Val158Met genotype is predictive of neither Dexpected . Howeverexpected . This isexpected –22. The striatum . This ishallenge , 24. Here cortex . Studiesallenges , 27. ConND and questions related to how well the amphetamine-induced dopamine release paradigm captures the effect of the COMT genotypes on dopamine clearance. In conclusion, we were not able to demonstrate an effect for the COMT Val158Met genotype on either cortical D2/3 receptor BPND or amphetamine-induced dopamine transmission.To summarize, the strengths of this study include the use of a well-validated imaging paradigm to measure dopamine release in the cortex and inclusion of a reasonable sample size by PET study standards. The weaknesses of the study include increased variability associated with the dopamine release outcome measure ΔBP


Piper methysticum) are effective in alleviating anxiety in clinical trials. Despite the long-standing therapeutic interest in kava, the molecular target(s) of the pharmacologically active constituents, kavalactones have not been established. γ-Aminobutyric acid type A receptors (GABAARs) are assumed to be the in vivo molecular target of kavalactones based on data from binding assays, but evidence in support of a direct interaction between kavalactones and GABAARs is scarce and equivocal. In this study, we characterised the functional properties of the major anxiolytic kavalactone, kavain at human recombinant α1β2, β2γ2L, αxβ2γ2L , α1βxγ2L  and α4β2δ GABAARs expressed in Xenopus oocytes using the two-electrode voltage clamp technique. We found that kavain positively modulated all receptors regardless of the subunit composition, but the degree of enhancement was greater at α4β2δ than at α1β2γ2L GABAARs. The modulatory effect of kavain was unaffected by flumazenil, indicating that kavain did not enhance GABAARs via the classical benzodiazepine binding site. The β3N265M point mutation which has been previously shown to profoundly decrease anaesthetic sensitivity, also diminished kavain-mediated potentiation. To our knowledge, this study is the first report of the functional characteristics of a single kavalactone at distinct GABAAR subtypes, and presents the first experimental evidence in support of a direct interaction between a kavalactone and GABAARs.Extracts of the pepper plant kava ( Piper methysticum) to prepare a non-alcoholic psychoactive beverage, which is also called kava. Kava drinking is an integral component of the Pacific Islander culture, with kava playing roles as a sacred drug in religious rituals, a social lubricant at formal gatherings, and a medicine to induce relaxation and sleep [The use of intoxicating substances to enhance mood, alter consciousness and to achieve spiritual enlightenment is known in virtually every culture. Alcohol is the most commonly used intoxicant globally, with the exception in a few regions . For milnd sleep . The connd sleep , 4. In snd sleep . The recnd sleep .There is a long-standing interest in the use of kava in the treatment of anxiety. In clinical trials, kava extracts are superior to placebo in reducing anxiety, and are generally well tolerated with negligible to mild side effects –9. DespiARs), voltage-gated Na+ and Ca2+ channels, opioid μ and δ receptors, dopamine type-2 receptor, histamine type-1 and 2 receptors, cannabinoid type-1 receptor, and monoamine oxidase type B have been suggested to be the molecular targets for kavalactones [ARs are the main target for kavalactones.A group of structurally-related, lipophilic compounds known as kavalactones (or kavapyrones) is responsible for the clinical effects of kava . Kavain,lactones , 18–20. ARs are a class of physiologically and therapeutically important ligand-gated ion channels (LGICs). These pentameric receptors occur in the brain with diverse composition that arise from a large number of subunits . This structural heterogeneity confers highly complex pharmacology to GABAARs. An impressive range of clinically used therapeutics such as benzodiazepines, barbiturates and anaesthetics are known to bind to distinct allosteric sites found on GABAARs to modulate receptor function. To date, evidence suggestive of an interaction between kavalactones and GABAARs mainly comes from radioligand binding studies. In rodent brain membranes, kava extracts with enriched kavalactone content and pure kavalactones have been shown to enhance binding of orthosteric radioligands to native GABAARs [GABA GABAARs , 21–23. ARs has not been conclusively established due to several reasons. First, the contribution of non-kavalactone compounds in enhancing ligand binding cannot be ruled out with the use of kava extracts in some studies. Second, kavalactones were tested on a heterogeneous population of GABAARs isolated from rodent brain, thus the identity of specific GABAAR subtypes modulated by kavalactones is not known. Third, due to the weak affinity of kavalactones and the lipid-dependent activity reported by Davies et al. (1992) [ARs indirectly by altering the physicochemical properties of lipid membrane cannot be ruled out.Despite the findings of these studies, a direct interaction between kavalactones and GABA. (1992) , the posARs, we performed functional characterisation of the kavalactone kavain at 9 human GABAAR subtypes expressed in Xenopus oocytes. Kavain was selected for these studies due to its abundance in kava extracts commonly consumed [ARs occurs in a subtype non-selective and flumazenil-insensitive manner. We also present evidence, for the first time, that kavain action at GABAARs is attenuated by the anaesthetic-impairing β3N265M point mutation.To elucidate the molecular basis of the interaction between kavalactones and GABAconsumed , as wellconsumed –27. We d2), DMSO and all buffer ingredients mentioned in this study were purchased from Sigma-Aldrich ; diazepam was purchased from Apin Chemicals LTD ; allopregnanolone and DS2 benzamide) were purchased from Tocris Bioscience . Etomidate hydrochloride (HCl) was synthesised following protocols previously described in Janssen et al. [et al. [GABA, DL-kavain, flumazenil, propofol, zinc chloride . Linearised plasmid DNAs were used as templates for the synthesis of messenger RNAs (mRNAs) using the mMessage mMachine T7 transcript kit from Ambion . Mutations were introduced to various GABAAR subunits of interest using the QuikChange II Site-Directed Mutagenesis Kit. For the α1M236W point mutation, forward primer 5′-CCTGCCGTGCATATGGACAGTTATTCTC-3′ and reverse primer 5′-GAGAATAACTGTCCATATGCACGGCAGG-3′ were used. For the β2M286W point mutation, forward primer 5′-AGGCCATTGACATGTACCTGTGGGGGTGCTTTGTCTTCGT-3′ and reverse primer 5′- ACGAAGACAAAGCACCCCCACAGGTACATGTCAATGGCCT-3′ were used. For the β3N265M point mutation, forward primer 5′- ACAATGACAACCATCATGACCCACCTTCGGGAG-3′ and reverse primer 5′-CTCCCGAAGGTGGGTCATGATGGTTGTCATTGT-3′ were used.Human complementary DNAs (cDNAs) of GABAXenopus laevis frogs were approved by the Animal Ethics Committee of the University of Sydney (Reference number: 2013/5915). Xenopus laevis frogs were housed in tanks containing compatible triplets on a 12-hour light/dark cycle in a reticulated aquatic system of 15 cm water depth. The aquatic solution was maintained at 18–22°C and persistently filtered with 50 μm carbon filters and routinely monitored for pH, nitrate levels and water hardness. The frogs were fed Wardley’s cichlid floating pellets  twice weekly and Wardley’s reptile sticks with fortified calcium at least weekly. Tanks were cleaned twice weekly on the day after feeding to reduce nitrate levels. Mature female Xenopus laevis frogs were anaesthetised with 0.17% tricaine (buffered with 0.06% sodium bicarbonate) for 15 minutes, after which the loss of righting reflex was confirmed before transferring on to ice where surgeries were performed. A small (1–2 cm) abdominal incision was made through both the skin and muscle layer with surgical knives. Ovary lobes were removed with a pair of forceps, and kept in oocyte releasing 2 (OR2) buffer . The skin and muscle layer were sutured separately, and frogs were allowed to recover for six months before they were reselected for surgeries. A total of five recoverable surgeries were allowed on each frog, before a terminal surgery was performed, in which a lethal dose of tricaine (0.5%) was used. Removed ovary lobes were divided into small sections and treated with 2 mg/mL Collagenase A  in OR2 buffer for at least an hour to defolliculate the oocytes. The released oocytes were then rinsed in ND96 storage solution . Healthy-looking stage V–VI oocytes were isolated and kept in ND96 storage solution at 18°C until ready for injection.All the procedures involved in the use of ARs in Xenopus oocytes were performed as previously described [et al. (2016) [The expression and functional characterisation of recombinant GABAescribed . The sub. (2016) . The inj2, 1.8 mM CaCl2, 5 mM HEPES hemisodium; pH 7.4) at an approximate rate of 5 mL/min.Two-electrode voltage clamp recordings were performed on oocytes 2–5 days post-injection at room temperature (20–25°C) using a GeneClamp 500B amplifier  or a Warner OC-725C Oocyte Clamp . All experiments were conducted at a holding potential of –60 mV, and data were acquired with a PowerLab 2/25, and LabChart (version 5.0.2) software . The recording microelectrodes (0.2–1.1 MΩ) were generated from capillary glass  using a single-stage glass microelectrode puller , and were filled with 3 M KCl solution. The oocyte was continuously perfused with ND96 recording solution  was used for data analysis. All data are presented as mean ± standard error of the means (SEM). Raw data from GABA concentration-response experiments were fitted to a monophasic Hill equation, and the fitted maximal values were used for normalisation of each dataset. In this equation, I represents the current responses elicited by the co-application of GABA and modulator, IGABA represents the amplitude of the control GABA-elicited responses. Alternatively, the modulatory effect was expressed as the fold of potentiation, as shown in To illustrate the modulatory effect of a chemical of interest, the data were normalised and expressed as shown in vs. mutant), the means of the values obtained from individual dataset were analysed using either unpaired Student’s t test (comparing means for two groups) or one-way ANOVA with Tukey’s post hoc test (comparing every mean with every other mean in three or more groups). When comparing the effect before and after the addition of a drug, paired Student’s t test was used. Statistical significance was attained at p < 0.05.When comparing parameters across different groups , and found that kavain enhanced GABA-elicited responses in a concentration-dependent manner . The receptor subtypes investigated were α1β2, β2γ2L, αxβ2γ2L , α1βxγ2L  and α4β2δ  at α1β2γ2L GABAt manner . The modr n = 6; . We alsoARs respectively, and are characterised by distinct subcellular localisations and GABA sensitivities  without affecting the GABA potency significantly .We investigated the effect of combining benzodiazepine site ligands with kavain at α1β2γ2L GABAs n = 5; . In concARs. A low and a high concentration of the general anaesthetics were used  to produce the modulatory and agonist effects respectively.The effect of combining kavain and general anaesthetics (etomidate and propofol) on receptor modulation was also investigated at α1β2γ2L GABA3 responses efficaciously . At 30 μM, etomidate directly elicited current responses approximately 24 ± 4.2% of the maximal GABA amplitude (n = 6). The agonist effect was not significantly altered in the presence of 300 μM kavain .At 3 μM, etomidate enhanced the GABA EC3 responses efficaciously . Following exposure to 100 μM propofol, GABA-elicited current responses were highly variable, resulting in inconsistent measurement. Thus, the agonist effect of propofol was investigated in the absence of GABA. We found that when combined, the currents elicited by 300 μM kavain and 100 μM propofol were smaller than the application of propofol alone  than wild-type α1β2γ2L GABAARs  and 10 μM propofol  on GABA EC3 (1 μM) responses. In contrast, the agonist effects of 30 μM etomidate and 100 μM propofol appeared to be greater, but were not significantly different from wild-type receptors . Overall, these functional characteristics are consistent with previous studies [The co-expression of α1M236W with β2 and γ2L subunits resulted in receptors with higher GABA potency , which was significantly greater than that observed at wild-type receptors  and α1M236Wβ2γ2L  GABAARs . Note: kavain-elicited current amplitudes were not deducted from the measurement of kavain-induced GABA potentiation.Next, we investigated the effect of kavain alone and in combination with GABA EC50 = 25 μM; n = 5; p < 0.0001 for logEC50 comparisons vs. α1β2γ2L; unpaired t test; 3 (1 μM) was also significantly diminished . However, the direct activation caused by 100 μM propofol was unaffected . Similar propofol profile at α1β2M286Wγ2S receptors has been reported [3 responses; p < 0.0001; unpaired t test; 3-elicited current responses, however, was not significantly different from α1β2γ2L GABAARs . However, kavain modulation of GABA EC3 responses was significantly reduced from 260 ± 18% at α1β3γ2L to 110 ± 14% at α1β3N265Mγ2L receptors  and β  subunit isoforms in αβγ2L receptors, or replacing the γ2L subunit with δ (α4β2δ) did not result in significant differences in kavain potentiation.In this study, we characterised the functional effects of kavain at 9 different human GABAntiation . The conARs are functionally different  within minutes after i.p. injection [i.p.), clearly demonstrating pharmacokinetic synergism [et al. (1992) predicted higher brain concentrations (300 μM) with the administration of larger doses (300 mg/kg) of kavain [The low affinity of kavain detected in our study is in agreement with most studies conducted in the past using kava extracts or pure kavalactones , 22, 46.njection . A higheynergism . Davies f kavain , 48. Unff kavain .ARs  that line the transmembrane β+α‒ anaesthetic binding sites. Substituting the methionine residues with tryptophan decreases etomidate and propofol sensitivity, an effect which has been correlated with increased side-chain volume of tryptophan sterically hindering anaesthetic binding [The possibility of kavalactones interacting non-specifically with lipid membrane of neurons to exert their psychoactive effects has been raised , 22. HowARs . The exte agents . As such binding . Unlike  binding .50 > 200 μM) and steep concentration-response relationships, besides lacking subtype selectivity at GABAARs [ARs, the existence of multiple binding sites for kavain is highly probable. Future studies are warranted to provide a more comprehensive picture of the kavain binding site(s).We infer, based on the preliminary mutational evidence, that kavain binds to the transmembrane anaesthetic sites. Two observations support this notion: (1) The β3N265M point mutation impairs the sensitivity of anaesthetics agents that bind to homologous transmembrane cavities, but not benzodiazepines and neurosteroids which target binding sites of dissimilar locations. (2) Kavain displays remarkably similar pharmacological profile to the volatile anaesthetics which exhibit low affinity  interaction [ARs.As part of our efforts in characterising the interaction of kavain and other GABAumazenil . This fieraction . This fieraction , and diaeraction . As flumAR function by kavain occurs in a subtype non-selective and flumazenil-insensitive manner. Furthermore, we provide the first experimental evidence that supports a direct interaction of a major kavalactone with GABAARs in vitro. It is expected that the identification of specific GABAAR-enhancing kavalactones in the future will give impetus to the development and refinement of these kava-derived chemicals as novel anxiolytics.Despite the long history of kava consumption and the wealth of clinical evidence in favour of the efficacy of kavalactones in treating anxiety, there is a severe gap in our understanding of the molecular target(s) and the mechanism(s) of action of these psychoactive compounds. In this study, we have demonstrated that the enhancement of GABA


A protective ventilatory strategy should prevent VILI, but in patients with larger nonaerated areas hyperinflation may occur during tidal ventilation even during a protective ventilatory strategy . The gli2 100%, RR 10/minute); keeping Vt constant, PEEP was gradually increased from ZEEP to 22 cmH2O, unless there was occurrence of hypotension or SpO2 <90% or Pplat >45 cmH2O or G no more visible, and then similarly reduced from 22 cmH2O to ZEEP. The gliding was assessed at six points of intercostal spaces bilaterally and the movement of a hyperechoic point of pleura or a b-line was observed during tidal ventilation. For each step, the excursion of G during the inspiratory phase was measured and compared with the Cstat values. Statistical analysis was performed with the Pearson correlation coefficient (PCC).Ten patients with hypoxic respiratory failure (P/F <300) were ventilated in VcV , ranging from 0.537 (P = 0.017) to 0.964 (P < 0.0001).All patients completed the study without adverse events. In all patients we observed a reduction of G and Cstat at the increase of PEEP and specularly an increase of G and Cstat during the reduction of PEEP Figure . In fiveThe variations of G at different levels of PEEP are consensual with those of Cstat. The study of G during tidal ventilation could help to identify hyperinflation in nondependent lung regions and to optimize lung-protective ventilatory strategies.


No changes were found in the 3,4-dihydroxyphenylacetic acid to dopamine ratio, which increases when unpackaged dopamine is metabolized via monoamine oxidase.These findings suggest that inflammatory cytokines reduce the availability of dopamine precursors without affecting end-product synthesis or vesicular packaging and/or release and provide the foundation for future studies investigating therapeutic strategies that facilitate availability of dopamine precursors to improve depressive symptoms in patient populations with increased inflammation. Novel approaches to understanding the neural circuitry of behavior  have revealed that dopamine neurons play a pivotal role in multiple depressive symptoms . In addiInflammatory cytokines and other biomarkers of inflammation are reliably elevated in a significant proportion of depressed patients, and peripheral administration of inflammatory cytokines, including interferon (IFN)-alpha, is well known to induce depressive-like behavior in both humans and nonhuman primates . Indeed,Further relevant to dopamine and motivation and motor activity, neuroimaging studies in humans have found that administration of IFN-alpha or other stimuli that induce inflammatory cytokines  decrease neural activation of the ventral striatum to hedonic reward and alter activity of the substantia nigra, leading to depressive symptoms including anhedonia and psychomotor slowing . FurtherMacaca mulatta), 2 male (12–14kg) and 2 female (7–9kg), aged 12 to 14 years were individually housed in adjacent cages in same-sex colony rooms. Animals were fed Purina monkey chow twice daily supplemented with fresh fruits and vegetables and maintained on a 7 am-7 pm light-dark cycle. Prior to treatment initiation, animals were habituated to study procedures to minimize stress reactivity. IFN-alpha  20 MIU/m2 was subcutaneously administered in equivalent volumes (0.5–1.5mL) between 7 and 10 am 5 days per week for 4 weeks, similar to the treatment schedule of patients receiving IFN-alpha monotherapy for malignant melanoma, which has been previously reported to induce huddling, a depressive-like behavior, and decrease effort-based sucrose consumption in rhesus monkeys . After collection of data under control conditions, animals were given a recovery period of 3 to 4 weeks prior to initiation of IFN-alpha administration. Animals were assigned to stimulation with AMPH (100 μM) in both the presence and absence of L-DOPA (10 μM) using a counterbalanced design during the last 3 to 4 weeks of IFN-alpha administration, with sampling sessions occurring 10 to 12 days apart . Because of experimental complications during data collection under control conditions in one animal, n=3 for comparison of AMPH stimulation between IFN-alpha and control in the absence of L-DOPA  previously used in reverse in vivo microdialysis studies in rodents and monkeys in 2 animals to determine the lowest dose that increased extracellular dopamine >2-fold .Subjects were surgically prepared with guide cannulae implanted bilaterally above the caudate nucleus as previously described and in detail in the aterials . The cauaterials . Furtheraterials . Awake saterials . The dopt test. Statistical analyses were conducted using SPSS  and Sigma Stat  software packages.Two-way repeated-measures analysis of variance was used to assess dopamine responses before, during, and after AMPH in each treatment condition . Sphericity was computed using Mauchly’s W, and if sphericity was violated (Epsilon <0.75), the more conservative Greenhouse-Geisser correction was employed to adjust the degrees of freedom, thus preventing inflation of the F-ratio and reducing the Type I error rate. All tests of significance were 2-tailed with an alpha level of 0.05, and all post hoc analyses were conducted using Tukey’s test or paired P=.001) and a significant treatment ×time interaction  (P=.72) or treatment ×time interaction     . Adminis, P=.61) .P=.69) or treatment ×time interaction   .Reversal of the IFN-alpha–induced decrease in dopamine release by L-DOPA in the absence of an increase in DOPAC/dopamine provides evidence that cytokines affect dopamine precursors without affecting end-product synthesis or vesicular packaging or release mechanisms. These findings are consistent with observations of decreased CSF concentrations of dopamine metabolites (DOPAC and/or homovanillic acid) in both humans and monkeys exposed to chronic IFN-alpha .Administration of IFN-alpha induces a number of other inflammatory cytokines in the periphery and central nervous system (CNS) that are elevated in depression, including interleukin-6 and tumor necrosis factor . IFN-alpWe and others have previously reported evidence of reduced BH4 activity in IFN-alpha–treated patients . For exal-methylfolate, or S-adenosyl-methionine all have a role in the synthesis and/or regeneration of BH4. Even antioxidants, such as citric acid, have been shown to protect BH4 from oxidation and may be relevant to treatment of BH4 and dopamine deficiency (Together, these findings suggest that pharmacologic strategies that boost key components of dopamine synthesis may be efficacious, whereas dopamine reuptake inhibitors may be less effective, in treating depressive symptoms in patients with increased inflammation. Indeed, dopamine reuptake inhibitors have demonstrated limited efficacy in the treatment of depressive symptoms such as fatigue in cancer patients , as wellficiency . Finallyficiency .There are several limitations of this brief report that should be addressed, the first being the small samples size. However, 3 to 5 animals per group is common in nonhuman primate studies employing in vivo microdialysis techniques, and as with previous studies, a within-subject design was used to reduce effects of inter-subject variability and increase statistical power . An addiIn conclusion, the findings of this brief report indicate that cytokine-induced reductions in striatal dopamine release can be restored by administration of L-DOPA. These findings directly extend our previous findings of decreased dopamine release and decreased CSF dopamine metabolites in rhesus monkeys administered chronic IFN-alpha and complement neuroimaging studies in humans indicating that administration of cytokines or cytokine-inducers alter ventral striatal activation and dopamine metabolism to produce depressive symptoms of anhedonia and psychomotor slowing. Multiple pharmacological treatment strategies exist that may facilitate the availability of dopamine precursors, such as compounds that restore BH4 activity, and future studies are needed determine the potential for these therapies to improve depressive symptoms in patients with increased inflammation.http://www.ijnp.oxfordjournals.org/For supplementary material accompanying this paper, visit None.


Background. Pyogenic granuloma is a reactive tumor-like lesion commonly affecting the oral cavity. These lesions usually appear as localized solitary nodule with a sessile or pedunculated base and colour varying from red, purplish, or pink, depending on the vascularity of the lesion. Pyogenic granuloma shows predilection for gingiva and is usually slow growing, but at times it shows rapid growth. The natural course of this lesion can be categorized into three distinct phases, namely, (i) cellular phase, (ii) capillary phase/vascular phase, and (iii) involutionary phase. Histopathologically, pyogenic granuloma is classified into lobular capillary hemangioma (LCH) and non-lobular capillary hemangioma (non-LCH). Case Presentation. In this series, four cases (varied age groups and both genders) of pyogenic granuloma showing varying histopathological presentation in relation to its clinical course have been described. The lesion in its early phase reveals diffuse endothelial cells, with few budding into capillaries. Among the capillary phase, the LCH type shows numerous blood vessels organized into lobular aggregates whereas the non-LCH type does not show any such organization and resembles granulation tissue. The involutionary phase shows healing of the lesion and is characterized by extensive fibrosis in the connective tissue. Conclusion. In conclusion, knowledge of the various histopathological presentation of this lesion is necessary for proper identification. Pyogenic granuloma is a commonly occurring reactive lesion of the oral cavity. It is non-neoplastic in nature and is hence referred to as a tumor-like lesion . The terPyogenic granuloma involves the gingiva most frequently and presents as a nodular growth which may be slow growing or rapid in nature , 4. The A total of four cases have been selected for describing the various histopathological presentations. The clinical descriptions of these cases have been described in Clinically, these cases show general features like nodular presentation, showing similar size, occurring more commonly in females and located at gingival and extragingival sites. The histopathological presentation shows variations according to the various phases and has been described below.Case 1 shows histopathological features suggestive of the early stage/cellular phase. The gross specimen measured approximately 1 cm and showed a lobular surface . MicroscCase 2 shows features conforming to the established stage of pyogenic granuloma. Gross examination revealed the growth to be less than 1 cm in size . Low powCase 3 also describes an established stage of pyogenic granuloma. Gross specimen was smaller than 1 cm and revealed haemorrhagic areas . ScanninCase 4 describes the healing/involutionary phase of pyogenic granuloma. Gross specimen measured roughly 1 cm in size . MicroscPyogenic granuloma was first identified by Poncet and Dor in 1897 who described it as a vascularised mass and named it “Human Botryomycosis” , 9. Due Low-grade chronic irritation, trauma, and hormonal imbalances are said to be the main etiology for pyogenic granuloma which results in the overzealous proliferation of vascular type of connective tissue , 3, 10. Clinically, oral pyogenic granuloma appears as a nodular mass ranging from few millimetres to centimetres in size and are usually slow growing and asymptomatic , 11. TheThe microscopic picture of pyogenic granuloma in general shows exuberant granulation tissue which is covered by atrophic/hyperplastic epithelium that may be ulcerated at times and reveals fibrinous exudates. Presence of numerous endothelium-lined vascular spaces and proliferation of fibroblasts and budding endothelial cells are the characteristic features of pyogenic granuloma. Presence of mixed inflammatory cell infiltration is also observed . Cawson Sternberg et al. suggested three distinct phases to describe the course of pyogenic granuloma. The “early phase” reveals a compact cellular stroma with little lumen formation. The next phase described as the capillary phase reveals lobules which are highly vascular with abundant intraluminal red blood cells. The final phase referred to as “involutionary phase” shows intra- and perilobular fibrosis. This phase is suggestive of healing phase of pyogenic granuloma . ClinicaDepending on the different stages of pyogenic granuloma, certain lesions come in as differential diagnosis. Younger lesions may be mistaken for conventional granulation tissue histopathologically or hemangioma, Kaposi's sarcoma, and bacillary angiomatosis clinically , 20. HemPyogenic granuloma is a commonly occurring reactive lesion of the oral cavity and is non-neoplastic in nature. Presence of histopathological variation is related to its chronological phase. Knowledge of the same is essential for understanding the lesions. Also clinical correlation should be done for accurate diagnosis.