Nonalcoholic fatty liver disease (NAFLD) is increasingly regarded as a hepatic manifestation of metabolic syndrome. Though with high prevalence, the mechanism is poorly understood. This study aimed to investigate the effects of p21 on free fatty acid (FFA)-induced steatosis in L02 cells. We therefore analyzed the L02 cells with MG132 and siRNA treatment for different expression of p21 related to lipid accumulation and lipotoxicity. Cellular total lipid was stained by Oil Red O, while triglyceride content, cytotoxicity assays, lipid peroxidation markers and anti-oxidation levels were measured by enzymatic kits. Treatment with 1 mM FFA for 48 hr induced magnificent intracellular lipid accumulation and increased oxidative stress in p21 overload L02 cells compared to that in p21 knockdown L02 cells. By increasing oxidative stress and peroxidation, p21 accelerates FFA-induced lipotoxic effect in L02 cells and might provide information about potentially new targets for drug development and treatments of NAFLD. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver damage in individuals encompassing fatty liver (simple steatosis), steatohepatits (NASH) and cirrhosis in the absence of alcohol abuse These ‘two hits’ consist of the excessive hepatic fat accumulation owing to insulin resistance and oxidative stress Waf1/Cip1/Sdi1 is a well-characterized cyclin-dependent kinase inhibitor (CKI) that belongs to the Cip/Kip family of cdk inhibitors p21Cip1 mRNA levels were significantly higher in livers of OP (obesity prone) models compared with OR (obesity-resistant) models Recent studies have connected p21 with NAFLD. In ob/ob mice, NAFLD is associated with increased expression of p21, which together with STAT-3 overexpression impaired regeneration of fatty livers These findings suggest a possible link between p21 activity, ROS generation, LPO induction, and FFA-induced liver injury. The aim of this study was to investigate the role of p21 in the establishment of hepatocyte steatosis initiated by ROS, LPO and analyze the possible way.Cellular FFA loading was utilized because it appears to reproduce several of the key features of hepatic steatosis in human beings The human non-tumor cell line, L02, was obtained from China Cell Culture Center (Shanghai). The cell was maintained in an atmosphere of 5% CO2 at 37°C in DMEM  supplemented with 10% fetal bovine serum .Fat overloading induction of cells was done mainly according to previously established methods MG132 is a C-terminal peptide aldehyde and a proteasome substrate analog 4 cells per well into 6-well plates and allowed to adhere by culturing at 37°C, 5% CO2 for 4 hr. p21 siRNA and negative control siRNA were purchased as validated siRNAs (GenePharma). Transient siRNA transfections were carried out using Lipofectamine 2000 (Invitrogen) following the manufacturer's recommendation. The media was changed 12 hr post-transfection, and 24 hr post-transfection, the media was changed with the addition of a final concentration of 1 mM FFA for 48 hr.L02 cells were seeded at 7×10Cells were stained with Oil Red O to assess lipid content according to the Sigma protocol. Briefly, cells were washed twice with PBS and fixed with 10% neutral formalin for 15 min, then rinsed with PBS twice again. The fixed cells were stained with freshly prepared Oil Red O solution for 10 min at 37°C. Thereafter, cells were dyed with hematoxylin (JianCheng Bio-technology Institute), followed by washing with PBS before microscopic examination (Nikon microscope).Cellular triglyceride (TG) content was measured using an enzymatic kit (Applygen Technologies) following the manufacturer's instructions. Firstly, scraped cells from 6-well tissue culture plats, washed with PBS twice. Afterward, added 100 ul cell lysis buffer and incubated at 70°C for 10 min. Thereafter, centrifuged at 2000 rpm for 5 min, collected the supernatant and measure the TG content by the BCA method (Applygen Technologies).Cell toxicity was routinely measured by assaying aspartate aminotransferase (AST), alanine aminotransferase , glutathione (GSH) and oxidized glutathione (GSSG) using kits respectively , according to the manufacture's recommended protocol.Production of malondialdehyde (MDA), ROS, LPO and superoxide dismutase (SOD) were assayed using kits respectively , according to the manufacture's recommended protocol.5′-TGTCCGTCAGAACCCATGC-3′, reverse 5′-AAAGTCGAAGTTCCATCGCTC-3′; GAPDH, forward 5′-TCAACGACCACTTTGTCAAGCTCA-3′, reverse 5′- GCTGGTGGTCCAGGGGTCTTACT-3′.Total RNA from cells was isolated using Trizole reagent (Takara). Random-primed cDNA were generated by reverse transcription of total RNA samples with One Step PrimeScript RT-PCR Kit (Takara). A realtime-PCR analysis was performed with the ABI Prism 7500 sequence Detection System (Applied Biosystems) using SYBR Premix DimerEraser (Takara). GAPDH (for human genes) was used for internal control, respectively. Primers used were as follows: p21, forward After washing cells twice with PBS, whole cell lysates were extracted in M-PER mammalian protein extraction reagent (Thermo Scientific) by centrifugation . Cell lysates (20 µg) of total protein were loaded per well and separated on a 10% SDS polyacrylamide gel. Proteins were then transferred to PVDF membrane. Primary antibodies were: anti-p21 antibodies (Epitomics), anti-GAPDH . The secondary antibody was a goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology). The membrane was exposed to ECL Hyperfilm (Amersham Biosciences), and the film was developed. Each blot was stripped with a stripping solution  for 1 hr and re-probed with anti-p21 antibodies to the bands were quantified densitometrically.p values less than 0.05.Data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS (version 18). Groups were compared among themselves using the Student's t test. Differences were considered statistically significant at It is generally believed that fatty liver results from an imbalance between the hepatic uptake of FFAs, TG synthesis, and excretion As a cell cycle protein, p21 is regulated by proteasomal pathway. MG132 is a peptide aldehyde that inhibits 20S proteasome activity by covalently binding to the active site of the beta subunits and effectively blocks the proteolytic activity of the 26S proteasome complex To explore the role of p21 in FFA-treated L02 cells, we compared the cellular lipid accumulation in L02 cells treated with FFA in the presence or absence of MG132 for 48 hours. The results showed that p21 overload increased FFA induced lipid accumulation in L02 cells, which was confirmed by Oil Red O Staining . OblivioWe next measured the intracellular TG levels by an enzymatic kit. The TG content was increased by about 1.63 fold from 84.4 µg/mg protein in FFA-treated cells to 137.5 µg/mg protein in FFA plus MG132-treated cells . The resTo dissect the mechanisms underlying p21-mediated lipotoxic action in L02 cells, oxidative damage was assayed by measuring ROS, MDA, LPO, SOD, GSH and GSSG level. ROS, MDA, LPO and GSSG measurements showed that the level were increased by FFA treatment and were raised much more when added with MG132 . AccordiWe then used a pairs of siRNA duplexes to down-regulate p21 expression in L02 cell lines. Western blot analysis confirmed marked drop of p21 protein level by significant inhibitory efficiency of siRNA compared to the negative control . To inveAs depicted in The extent of oxidative stress in the p21 knockdown model of cell steatosis was compared to that negative control. As seen in Understanding the pathogenesis of NAFLD has recently assumed great importance with the recognition that it has a high prevalence and has the potential to progress to fibrosis and cirrhosis.NAFLD patients exhibit an elevated lipolysis and high circulating FFA levels In the process of FFA oxidation, the mitochondria leaks ROS mainly in the form of hydrogen peroxide Rather than being an ‘innocent bystander’, recent studies suggest that oxidative stress-induced lipid peroxidation may be a guilty party playing important roles in the progression of NAFLD. Furthermore, it is confirmed that p21 expression correlates with oxidative stress in cell death So it is reasonable to speculate a positive association between p21 and steatosis. The aim of the current study was to investigate the influence of p21 on NAFLD in a FFA-induced cell model, which may help elucidate the mechanism involved in the progression of the disease.In our attempts to characterize the influence of p21 on NAFLD progression, we found this to be ROS-dependent and potentiate FFA-induced steatosis. The p21 level was higher after FFA treatment compared to DMEM buffered L02 cells. MG132 leads to p21 overload since the ubiquitin-dependent proteolysis of p21 is blocked. Compared to FFA treated LO2 cells, those with FFA plus MG132 intervene presented more severe cellular steatosis. And there was close association between cellular steatosis and oxidative stress induced lipid peroxidation. The oxidative stress level, identified by ROS, MDA, LPO and GSSG, together with the cytotoxic marker (AST), increased in parallel with intracellular lipid vacuoles (confirmed by Oil Red O staining) and TG content. Meanwhile, the anti-oxidant markers  decreased accordingly.These findings suggest that FFA treatment in L02 cells causes lipid accumulation and oxidative stress, possibly mediated by p21, and it is a likely explanation for increased levels of p21 in NAFLD, reported previously.Thus, we assumed that p21 may catalyze the first hit of oxidative stress. According to this hypothesis, a critically down-regulate expression of p21, alleviates oxidative stress and lipid peroxidation. Mirroring this, this study demonstrated a knockdown of p21 expression in L02 cells, with less lipid vacuoles, decreased TG content, improved cytotoxic injury (AST), elevated anti-oxidant level  and alleviated oxidative stress  after 48 hr of FFA treatment compared to the negative control.Our data are consistent with the hypothesis that increase of p21 in L02 cells causes oxidative stress and lipid peroxidation, and accelerates FFA-induced lipotoxic effect. And the p21 silencing L02 cells were less sensitive to FFA to develop steatosis.The strong association of hepatocyte p21 expression with NASH, fibrosis stage and diabetes mellitus, has been studied a lot. Most of them attribute p21 as a hepatocyte senescence marker with an adverse liver-related outcome by its cell cycle arrest and apoptosis effect In conclusion, this study demonstrated a link between p21 expression, oxidative stress and lipid peroxidation in NAFLD and further, p21 played lipotoxic effect on FFA-induced steatosis in L02 cells. Analysis of p21 effect provided both global and specific information regarding the molecular events that cause this complex disease. The strong relation between cellular p21 expression and impaired fatty acid oxidation may be a potential target for therapeutic intervention. However, the precise mechanism by which p21 promotes hepatic steatosis remains to be elucidated, and further investigations are underway.


AbstractPoaceae bioinformatics. GrassBase and GrassWorld can continue to grow productively if the proliferation of alternative classifications and datasets can be brought together into a consensus system. If the datasets are reconciled instead of diverging further apart a long term cumulative process can bring knowledge together for great future utility. This paper presents the Poaceae as the first and largest model system for e-taxonomy and the study of classification development in plants. The origin, development, and content of both datasets is described and key contributors are noted. The challenges of alternative classifications, data divergence, collaborative contribution mechanisms, and software are outlined.GrassBase and GrassWorld are the largest structured descriptive datasets in plants, publishing descriptions of 11,290 species in the DELTA format. Twenty nine years of data compilation and maintenance have created a dataset which now underpins much of the  Grasses and floristic knowledge. Grasses (Poaceae) are undisputedly the most economically important family of flowering plants (www.fao.org), including wheat (Triticumaestivum L.), rice (Oryzasativa L.), maize (Zeamays L.), sugar cane (Saccharumofficinarum L.), bamboos, forage grasses, and lawn grasses. As a group so fundamental to the human civilisation the grasses have always been well studied and the cumulative body of knowledge on the circa 12,000 species  a radical modernisation of web presentation will be necessary, including links to observation data, plant ontogenies, and provision of machine readable data output.Poaceae taxonomic community are needed to take grass e-taxonomy forwards. Failure to plan and collaborate could lead to an increasing proliferation of contradictory classifications. Unique datasets of great value could be rendered obsolete if software development and database maintenance does not keep pace with technology platforms. Investment in community database integration and infrastructure could unlock untapped research and data mining potential of many historic datasets. The rich data and the long history of database compilation in the grasses present an unprecedented opportunity to study the development of classifications and to develop e-taxonomic models. The authors would like to invite potential collaborators to discuss dataset improvements and future plans.This paper argues that a collaborative approach and careful thought across the PageBreak


Melanopsin expressing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) entirely control the post-illumination pupil response (PIPR) from 6 s post-stimulus to the plateau during redilation after light offset. However, the photoreceptor contributions to the early redilation phase of the PIPR (< 6 s post-stimulus) have not been reported. Here, we evaluated the photoreceptor contributions to the early phase PIPR (0.6 s to 5.0 s) by measuring the spectral sensitivity of the criterion PIPR amplitude in response to 1 s light pulses at five narrowband stimulus wavelengths . The retinal irradiance producing a criterion PIPR was normalised to the peak and fitted by either a single photopigment nomogram or the combined melanopsin and rhodopsin spectral nomograms with the +L+M cone photopic luminous efficiency (Vλ) function. We show that the PIPR spectral sensitivity at times ≥ 1.7 s after light offset is best described by the melanopsin nomogram. At times < 1.7 s, the peak PIPR sensitivity shifts to longer wavelengths (range: 482 to 498 nm) and is best described by the combined photoreceptor nomogram, with major contributions from melanopsin and rhodopsin. This first report of melanopsin and rhodopsin contributions to the early phase PIPR is in line with the electrophysiological findings of ipRGC and rod signalling after the cessation of light stimuli and provides a cut-off time for isolating photoreceptor specific function in healthy and diseased eyes. In macaques, the pupil light reflex (PLR) measured during continuous light exposure and post light offset after pharmacological blockade of outer retinal rod and cone photoreceptors shows a sustained constriction that closely matches the spectral sensitivity of inner retinal melanopsin expressing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) peak ~482 nm) . In huma nm . In The PIPR has been measured in many studies using a high irradiance, short wavelength stimulus light near the melanopsin peak spectral response to directly assess melanopsin function as a biomarker for retinal disease . ResearThe data presented in this study are the results from a new analysis of previously recorded pupil traces ,18. The 2 = 0.005, F1,18 = 0.09, p = 0.77) in agreement with literature  [cone(λ)] [rod(λ)] with their relative contributions . The model was fitted to the data by adjusting the free parameters to minimise the sum of squares of the differences between S(λ) and the criterion PIPR. The k1 and k2 parameters represent the Quick pooling model of visual sensitivity  , the 10°cone(λ)]  and CIE sitivity ,44 for cStatistical data analysis was performed in GraphPad Prism . To determine the spectral nomogram that best described the PIPR, the deviation of the PIPR data from each nomogram was calculated. The agreement of either opn4, rhodopsin, Vλ or the combined opn4 + rhodopsin + Vλ nomograms with the criterion PIPR data was evaluated with Bland-Altman analysis, and the bias and 95% limits of agreement between the nomogram and criterion PIPR were reported . The BlaTo evaluate the differences in amplitude between 2, 3, 4, 5 and 6 s PIPR, a one-way repeated measures ANOVA  was performed. The CV of the PIPR amplitude was calculated as SD/Mean to determine the PIPR time metric with the lowest variability; intra-individual CV was based on at least two repeated measurements and inter-individual CV was based on the pupil data from 20 participants.When the criterion PIPR amplitude is measured for each primary light, the pupil light reflex traces overlap    compared 482 nm) , the bia 482 nm) . The qua 482 nm)  whereas A transition between the opn4 nomogram and the combined model as the better description of the criterion PIPR became evident at 1.7 s post-stimulus, with participant 32/F requiring a 0.49 rhodopsin contribution in the tertiary model, although there was only a small difference in the deviation  and bias-2.s-1 pulse in a cohort of 20 participants. The PIPR amplitude differed significantly between the measured times , with the mean PIPR amplitudes decreasing with increasing post-stimulus time  to a 1 s stimulus pulse measured ≥ 1.7 s post-stimulus is entirely described by the melanopsin (opn4) photopigment spectral nomogram peak at 482 nm) Figs –8. For p2 nm Figs-2.s-1) light is ~9 s [-2.s-1 above rod threshold) and melanopsin completely controls the PIPR thenceforward.The ipRGCs depolarise during light stimulation and repolarise slowly after light offset  to produ is ~9 s . Hence,  is ~9 s . In comp is ~9 s  and make is ~9 s  and the The post-stimulus time, when rhodopsin contribution to the PIPR in the opn4 + rhodopsin + Vλ model becomes zero and melanopsin entirely controls the model, differed by ~0.1 s between our two participants , in line with the electrophysiological observations of rod signalling shortly after the cessation of a light stimulus, and melanopsin solely drives the PIPR at longer post-stimulus times.


I2 statistic was used to assess heterogeneity. Study-level data were pooled using a random-effects model or a fixed-effects model (when less than 5 studies were included). We found that more time spent on near work activities was associated with higher odds of myopia  and that the odds of myopia increased by 2%  for every one diopter-hour (hr) more of near work per week. Therefore, the development of a strategy to reduce the impact of near work on myopia would be important for preventing myopia in children.Myopia has a multifactorial etiology, although environmental factors are predominant in determining its current patterns. Currently, associations between near work activities and myopia have not been consistently observed. Therefore, we performed a systematic review to quantify the effect of near work activities on myopia in children. Relevant articles published between 1989 and 2014 were identified in MEDLINE, Embase, and the Cochrane Library, and the citation lists were reviewed. Twelve cohort studies and 15 cross-sectional studies were included . The  The prevalence of myopia has increased dramatically in recent years around the world and, in some highly educated groups such as law and medical students, it now exceeds 80% , 2. ConsMyopia is generally believed to have a multifactorial etiology , and earNear work is considered as the activities done at short working distance such as reading, studying , computer use/playing video games, or watching TV, etc.) –13. Due We searched several databases , from April 1, 1989 to May 1, 2014, to identify potentially relevant articles. Our search included combined Medical Subject Headings and keywords for children with myopia (study population) and near work activities. The search terms included (“myopia” OR “short-sighted” OR “near-sighted” OR” refractive errors”) AND (“near work” OR “studying” OR “reading” OR “reading distance” OR “working distance”.) There were no language restrictions. After deleting duplicate articles, two authors independently screened the studies for inclusion, retrieved potentially relevant studies, and determined study eligibility. Then we reviewed the bibliographies of all selected articles to identify additional studies. Disagreements were resolved by consensus.Near work was defined as the sum of activities with short working distance such as reading, studying, writing, doing homework, watching TV, or playing video games, etc. We included studies that reported any near work activities as covariates with myopia, myopia incidence or progression as the outcome measure. We excluded studies that enrolled subjects older than 18 years of age.For each study, the following characteristics were extracted: (i) last name of first author, (ii) year of publication, (iii) study design, (iv) race/ethnicity of the study population if available, (v) number of subjects in the analysis, (vi) age range of subjects included in the studies, (vii) definition of myopia or myopia progression, (viii) definition of near work. (ix) effect estimate(s), and (x) which confounding factors was adjusted for.To quantify the effect of near work activity  on myopia development, some authors computed a weighted variable by adding three times reading, two times computer use, and two times video games use in hours per day as diopter hours . Each ar2 statistic was calculated to determine the proportion of inter-study variation due to heterogeneity, which suggested thresholds for low (25–49%), moderate (50–75%) and high (>75%) values. Study-level data were pooled using a random-effects model [To evaluate the effect of near work on myopia, the evaluated outcomes included prevalent myopia, myopic development or progression. We reported dichotomous outcomes as odds ratios (ORs) and continuous outcomes as the mean and their respective 95% confidence intervals (CIs). For studies that did not report estimate for general near work, we choose the activity with the shortest working distance to estimate the association. Similarly, we tried to include unadjusted data in our meta-analysis unless only adjusted outcomes being reported. The Its model , 19. In ts model –22 and Sts model , 23–25),The electronic database searches identified 14,069 citations. After evaluating these citations and their bibliographies, 27 studies met the inclusion criteria for this review  12–14, –43. The p = 0.0027) [2 = 37.36, p< 0.001) [p = 0.02) during the school year. In that study, the multivariate OR for the association between myopia and TV viewing was significantly higher than 1.0 [Fifteen cross-sectional studies investigated the relationship between near work activities and prevalence of myopia . Ten of  0.0027) . Among 1 0.0027) ;the resu 0.0027) . In 2353 0.0027) . In 1378than 1.0 . In 377 than 1.0 .p = 0.62), personal reading , watching TV  and playing video games/computer use  [p = 0.08) and 12-year-old (p = 0.8) subjects [p = 0.059) and no significant associations with other near work activities  [However, 5 other cross-sectional studies did not produce consistent results regarding whether near work activities were risk factors for myopia , 28, 43. = 0.11) . After a = 0.11) . Based op = 0.02) in the younger cohort (6 y/o at baseline) [p = 0.90) [p = 0.098) [Six cohort studies investigated the relationship between near work activities and the incidence of myopia . Only twaseline) . In contaseline) . In 514  = 0.90) . Among c= 0.098) . Wu et a= 0.098) .p≦0.01 [p = 0.07) [Six longitudinal studies demonstrated the relationship between near work activities and the progression of myopia . Two lonp≦0.01 . Scheima = 0.07) .I2 = 89.7%). The funnel plot showed that the distribution of studies was asymmetrical compared to the summary estimate  for every one diopter-hour more of near work per week  because it is an important cause of reduced vision in populations throughout the world . The preAccording to previous studies, juvenile-onset myopia typically develops at six to eight years of age and progresses at a rate of approximately 0.50 D per year through 15 to 16 years of age in Western children and approximately 1 D per year progression in Asian children –51. The The theory of hyperopic defocus from a deficient accommodative response in juvenile myopia is considered to be the association between near work in human myopia and the minus lens results from animal studies , 59. TheIn conclusion, this systematic review shows that near work activities were associated with myopia and that increased diopter-hrs of near work might increase myopia prevalence.S1 PRISMA Checklist(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.


Stress is related to heavy alcohol use and relapse in alcoholics. Using the reinstatement model, we have shown that corticotropin-releasing factor (CRF) underlies stress-induced relapse to alcohol seeking in laboratory rodents. Little is known about how other neurotransmitters interact with CRF in these effects. Dynorphin and its receptor  are involved in stress responses and in alcohol seeking. KOR and CRF receptors (CRF R) may interact in the production of stress-related behaviors but it is not known whether this interaction is involved in reinstatement of alcohol seeking.Male Long Evans rats were trained to self-administer alcohol (12% w/v). After extinction of responding, we determined the effects of the KOR agonist, U50,488  on reinstatement of alcohol seeking, and their sensitivity to the selective KOR antagonist nor-binaltorphimine dihydrochloride (nor-BNI) (10 mg/kg) administered at different times before U50,488. We then examined the effects of nor-BNI on reinstatement induced by the stressor yohimbine (1.25 mg/kg) and on reinstatement induced by exposure to alcohol-associated cues. Finally, we determined whether CRF R1 blockade with antalarmin  attenuates alcohol seeking induced by U50,488.U50,488 reinstated alcohol seeking. Prior treatment with nor-BNI 2, but not 24 h before administration of U50,488 or yohimbine blocked reinstatement induced by these drugs. Cue-induced reinstatement was blocked by nor-BNI administered 2 h prior to testing. Finally, U50,488-induced reinstatement was blocked by antalarmin.These data further support a role for KOR in reinstatement of alcohol seeking under nonstress and stressful conditions and that KOR and CRF R interact in these effects. We will then examine the effects of nor-BNI on reinstatement induced by the pharmacological stressor yohimbine, a drug that we have shown to induce reinstatement in a CRF R1-dependent manner. Nor-BNI will be administered at two different pretreatment times prior to U50,488 or yohimbine, as its selectivity for KOR versus other opioid receptors has been shown to vary in a time-dependent manner. Then we will examine whether reinstatement of alcohol induced by cues previously associated with alcohol seeking is blocked by nor-BNI. Finally, we will determine whether blockade of CRF R1 with antalarmin attenuates alcohol seeking induced by U50,488.The experimental procedures followed the “Guide for the care and use of laboratory animals” . Food and water were freely available in the home cage at all times and the temperature was maintained at 21 ± 1°C.Long Evans rats  weighing 200–250 g at the start of the experiment were used. The rats were individually housed under a 12:12 h light-dark cycle  activated an infusion pump , while responding on the other lever (an inactive lever) was recorded, but did not activate the pump. Activation of the infusion pump resulted in the delivery of 0.19 mL of alcohol into a drinking receptacle located between the two levers and initiated a 5-sec timeout period. During the timeout, the house light was turned off, a key light over the active lever was turned on, and white noise was emitted by a speaker. The beginning of the sessions was signaled by the illumination of a houselight located at the top of the self-administration chamber; at the end of the session, the houselight was turned off. When alcohol was left in the drinking receptacle after a self-administration session, it was measured and the volume was taken into account for intake calculation.Rats were trained to self-administer alcohol as described previously  and yohimbine was obtained from Sigma-Aldrich . The doses of yohimbine and antalarmin and their pretreatment times were based on our published studies  were followed by Newman-Keuls post hoc tests.Statistical analyses were performed separately on the numbers of responses made on the previously active and inactive levers during the reinstatement tests. Data were analyzed with analysis of variances (ANOVAs) and significant interactions  as the factor. Once the extinction criterion was reached, rats received vehicle (water) or one of the doses of U50,488, 30 min before 1-h reinstatement test sessions conducted in the operant chambers, in counterbalanced order with at least 2 days between each test. On the days between the drug tests, rats were injected i.p. with water and received drug-free extinction sessions.n = 7–8 per group) were trained to self-administer alcohol and received extinction sessions. In this and in the following experiments, rats were assigned to matched groups based on alcohol intake and extinction responding. A mixed design was used with the between factor of nor-BNI pretreatment condition  and within factor of U50,488 pretreatment condition . One group of animals received injections of nor-BNI vehicle or nor-BNI  2 h before the U50,488 vehicle or U50,488 injections. The other group received nor-BNI 24 h prior to U50,488 vehicle or U50,488 injections. Thirty minutes after the U50,488 vehicle or U50,488 injections, rats were placed in the operant chambers for the 1-h reinstatement test session. In order to minimize the use of animals in Experiments 2 and 3, we did not include a 24 h nor-BNI vehicle condition; we have found that baseline extinction responding is extremely stable from day to day once the extinction criterion is reached.Twenty-three rats (n = 12 per group), trained to self-administer alcohol and their responding was extinguished as above. A mixed design was used with the between factor of nor-BNI pretreatment condition  and within factor of Yohimbine condition . During testing, each rat was injected with the nor-BNI vehicle 2 h before, or nor-BNI , 2 or 24 h prior to injections of the yohimbine vehicle (water) or yohimbine. Forty-five minutes after the vehicle or yohimbine injections, rats were placed in the operant chambers for the 1-h reinstatement test session.The effects of nor-BNI on yohimbine-induced reinstatement was assessed in 36 rats (n = 11 per group), trained to self-administer alcohol and their responding extinguished as above, were used to determine the effects of nor-BNI on reinstatement induced by the light/tone cue previously associated with alcohol self-administration. A mixed design was used with the between factor of nor-BNI pretreatment condition  and within factor of Cue condition . During testing, each rat was injected with the nor-BNI vehicle or nor-BNI  2 h prior to 1-h test sessions. Rats were placed in the operant chambers and received one non-contingent presentation of the tone-light cue. Subsequent presses on the active lever resulted in cue presentation on a FR1 schedule during the remainder of the session. Extinction responding without cue presentation the previous day was used as the baseline. A 24 h nor-BNI condition was not included as such pretreatment did not affect U50,488- and yohimbine-induced reinstatement in Experiments 2 and 3.Twenty-two rats (n = 12 per group) were trained and their responding was extinguished as above and they were used to assess the effect of antalarmin on U50,488-induced reinstatement. A mixed design was used with the between factor of U50,488 dose  and within factor of Antalarmin dose . On the test days, each rat was injected with the antalarmin vehicle  or one of the doses of antalarmin, and 30 min later with vehicle or U50,488. Thirty minutes after the vehicle or U50,488 injections, rats were placed in the operant conditioning chambers for a 1-h reinstatement test session. There were at least 2 days between drug tests, and on these days animals were injected with water, i.p. and received extinction sessions.Twenty-four rats  = 10.33, P < 0.05]. The effects of U50,488 were significantly different from vehicle at the 5 mg/kg dose [P < 0.05], but not at the 2.5 mg/kg dose (P > 0.05). There were no significant effects of U50,488 on responding on inactive lever responding  = 3.9, P < 0.05]. In animals treated with nor-BNI vehicle, U50,488 significantly increased active lever pressing compared to those administered its vehicle (P < 0.05). The U50,488-induced increase was blocked by nor-BNI when it was injected 2 h (P < 0.05), but not 24 h prior to U50,488 (P > 0.05). Inactive lever pressing was not significantly affected by U50,488 or nor-BNI  = 5.6, P < 0.05]. In animals administered the vehicle for nor-BNI, yohimbine significantly reinstated alcohol seeking (P < 0.05). This reinstatement was significantly reduced when nor-BNI was administered 2 h prior to yohimbine (P < 0.05), but not when nor-BNI was administered 24 h before. Analysis of inactive lever data showed a significant effect of Yohimbine condition, due to the fact that inactive responding was overall slightly higher in animals administered yohimbine. This effect achieved statistical significance in the 24 h nor-BNI group (P < 0.05). . Table .F = 9.03, P < 0.05]. When animals were exposed to cues previously associated with alcohol delivery they showed a significant reinstatement of active lever pressing, compared to the no cue baseline condition (P < 0.05). This reinstatement was blocked in animals given nor-BNI 2 h prior to the test, compared to those administered vehicle (P < 0.05). There were no significant effects on inactive lever responding  = 3.46, P < 0.05]. Compared to animals treated with the vehicle, those administered U50,488 showed a significant reinstatement of alcohol seeking , but not by 10 mg/kg. There were no significant effects of either of the drugs on inactive lever responding  and receptor binding studies suggest that this antagonist causes a long-lasting antagonism of KOR receptors, that lasts days or weeks, and that its selectivity for KOR plateaus at 2 h and becomes maximal at 24 h after administration  has been studied in mice (Land et al. There is also a CRF-KOR interaction in the aversive responses elicited by stress, which involves, in contrast, CRF R2 receptors. Place aversion induced by either CRF or a CRF R2 agonist was blocked by a KOR antagonist, but KOR agonist-induced place aversion was unaffected by CRF R2 blockade (Land et al. Our findings of nor-BNI blockade of yohimbine-induced reinstatement are consistent with this proposed mechanism, as yohimbine produces reinstatement through a CRF R1-dependent mechanism. Our results on antalarmin-induced blockade of U50,488-induced reinstatement, however, suggest a different mechanism, with an opposite relationship between the two peptides. These latter data suggest instead that KOR stimulation evokes the release of CRF, which in turn stimulates CRF R to induce reinstatement. Antalarmin blocks the effects of this released CRF on the CRF R, thereby inhibiting the reinstatement response. A brain region in which this interaction might occur is the amygdala, a critical part of the circuitry involved in responses to stress (Johansen et al. These data provide further support for the important role of KOR in reinstatement of alcohol seeking under nonstress and stressful conditions. They also indicate an interaction between KOR and CRF in reinstatement of alcohol seeking. Further studies are necessary to elaborate the role of KOR and CRF R in stress-induced alcohol seeking. A key experiment we intend to conduct is to examine the effect of nor-BNI on reinstatement induced by the stressor, i.c.v. CRF.


Video games are accepted and used for a wide variety of applications. In the medical world, research on the positive effects of playing games on basic laparoscopic skills is rapidly increasing. Although these benefits have been proven several times, no institution actually uses video games for surgical training. This Short Communication describes some of the theoretical backgrounds, development and underlying educational foundations of a specifically designed video game and custom-made hardware that takes advantage of the positive effects of games on basic laparoscopic skills. The use of video games for training basic laparoscopic skills, such as inverted movements, eye–hand coordination, depth perception, and ambidexterity, is not an entirely new concept. In the past ten years, the combination of games and surgery has gained much interest, and the scientific literature on the subject is rapidly increasing. It is considered proven that video game experience correlates with laparoscopic skills , 2, and Schlickum et al.  showed tAlthough the positive effects of video games on the aforementioned skills can be considered legitimate, no actual game has been developed to benefit from these positive effects. One article written by Bokhari et al.  in 2010,Cutting Edge, a collaboration between the University Medical Center Groningen, game developer Grendel Games and the LIMIS Institute for Healthcare Innovations and Training, is creating a video game specifically aimed at laparoscopic training. By developing both advanced Wii Remote add-ons and custom video game software, we hope to create a method that is fun, cost-effective and able to help surgical novices train basic laparoscopic skills better than traditional games. Underground will differ from existing video games in the sense that the movements made during the game will have a lot more in common with laparoscopy than traditional games, and it is our theory that it can be validated in the same manner as virtual reality surgical simulators.In Underground (working title), players use two Wii Remote controllers in custom-made laparoscopic tool shells to play a game that is based on movements made during laparoscopic surgery. In contrast to simulators, the game does not contain actual medical content, but comprises a story-driven mode, based on a fictional world where the player has to help small robots to escape from a mine. To aid the robots in their escape, the player controls two large robotic arms and demolishes and rebuilds the environment of the mine Fig. .Fig. 1ScThe concept of a mine was chosen because laparoscopic surgeons also work in a primarily dark area and have to break things  before they can start to rebuild . In the process, the development of a traditional simulator was knowingly avoided.The gameplay of Underground consists of a mixture of action and puzzles in a sandbox-like environment. The main goal, escaping from the underground world, can be reached by solving puzzles that require basic laparoscopic skills, cognitive skills and problem-solving skills.During the game, the players learn to handle analogous versions of laparoscopic equipment under familiar circumstances such as the lack of depth perception and inverse movements. The game has four distinct themes, each with five different levels and a final boss. To defeat the boss at the end of each world, the player has to combine all the skills that were learned during the previous levels.To ensure that the learning goals  are met in the game, the game-based activities were aligned with the learning goal activities, using the constrained design approach to instructional computer games, as described by Shelton and Scoresby . One leaThe player is rewarded for handling the equipment carefully and minimizing collateral damage. Causing collateral damage results in the crumbling of walls or falling stalactites, impeding progress to the exit of the level for some robots. The less collateral damage you cause, the more robots you save.Although Underground is not an actual simulator, it will be validated through a traditional validation process, as described by the European Association of Endoscopic Surgeons in their consensus guidelines for the validation of virtual reality surgical simulators . In thisA prototype of the game, which contained a single level aimed at training all four learning goals, has proven to possess a solid construct and concurrent validity . This waFace validity of a near-final version of the game was obtained at the Chirurgendagen 2013, a three-day annual congress of the Netherlands Society of Surgery, by letting experts play the game and letting them fill in a questionnaire on the hardware and software, based on similar research in the field. The results are currently being analyzed and will be submitted for publication shortly.Finally, content validity, which deals with the correctness of medical content in a surgical simulator, cannot be tested in Underground (or any serious game for that matter) because the game completely lacks medical content, such as anatomy or procedural knowledge, on purpose. The authors will therefore refrain from testing this validity.Once the test validation on the final version of the game is finished, we can start looking at the actual effects of the game on basic laparoscopic skills, the so-called experimental validation. As noted before, video games can positively influence basic laparoscopic skills in both the long and short term –5. WhethGames could be used to train basic laparoscopic skills in surgical trainees, but to our knowledge no actual institution does so. Therefore, we are developing a motion-sensitive video game that uses custom-made Wii Remote add-ons that is specifically aimed at training those skills, hoping to be of better use than normal games and creating a fun and affordable training method. While the video game hardware and software is currently still under development, we have already started a traditional validation process, proving that our game has acceptable face, construct and concurrent validity in comparison to existing simulators. However, more research on both the test and experimental validity of the game should be carried out before we can actually use it in surgical training. The Wii U video game and its hardware are aimed for release at the end of 2013.


SmJAZ3 and SmJAZ9 were cloned from S. miltiorrhiza hairy roots and characterized. Expression profiles under methyl jasmonate (MJ) treatment revealed that SmJAZ3 and SmJAZ9 were both MJ-responsive. Subcellular localization assay showed that SmJAZ3 was located in nucleus while SmJAZ9 was preferentially in nucleus. Expression of SmJAZ3 and SmJAZ9 in S. miltiorrhiza hairy roots differently affected the production of tanshinone. Over-expression of SmJAZ3 or SmJAZ9 in hairy roots produced lower level of tanshinone compared with the control, tanshinone production was as low as 0.077 mg/g DW in line SmJAZ3-3 and 0.266 mg/g DW in line SmJAZ9-22. Whereas, down-regulation of SmJAZs enhanced tanshione production, the content of tanshinone increased to 2.48 fold in anti-SmJAZ3-3 line, and 1.35-fold in anti-SmJAZ9-23 line. Our work indicated that SmJAZ3 and SmJAZ9 are involved in regulation of tanshinone biosynthesis and act as repressive transcriptional regulators in the JA signaling pathway, which paves the way to further dissect molecular mechanism in details in the future.Jasmonic acid (JA) is an important plant hormone involved in regulation of many aspects of plant growth and development including secondary metabolism and JASMONATE ZIM-DOMAIN (JAZ) proteins are key components in JA signal processes. In this study, two new JAZ genes named  During the entire life of the plants, they are frequently exposed to various abiotic or biotic stresses such as high light, drought, salinity, and attack from herbivores or pathogens1234566910512141515Arabidopsis, they share a conserved TIFY×G motif within the ZIM (or TIFY) domain in the N-terminus and a SLX2FX2KRX2RX5PY consensus motif within the Jas domain in the C-terminus. ZIM domains mediate the homo- and heteromeric interactions among the JAZ proteins as well as their interaction with other transcription factors2021JAZ family, interact with MYC2 transcription factor and repress its function in JA signaling, and act as repressors17Salvia miltiorrhiza Bunge, a well-known traditional Chinese herb, plays a crucial role in clinical treatment of cardiovascular and cerebrovascular diseases23S. miltiorrhiza, one kind is the liposoluble tanshinone containing cryptotanshinone, tanshinone I, tanshinone IIA, dihydrotanshinone etc., which share a variety of biological activities including anti-oxidant, heart-protective, anti-ischemic, anti-bacteria and anti-tumor properties232526272426S. miltiorrhiza has been reported28SmHMGR, SmDXR, SmHMGS, SmGGPPS and SmCYP76AH1 have been successfully cloned from S. miltiorrhiza25273031S. miltiorrhiza. As a key regulator in plant JA signal pathway reported recently, JAZ repression proteins should have important regulatory role in tanshinone biosynthesis in S. miltiorrhiza. However, little is known about exact role of JAZ on tanshinone biosynthesis in S. miltiorrhiza. Herein, two genes of JAZ family were isolated from S. miltiorrhiza and their roles in regulating the tanshinone biosynthetic pathway are functionally identified through genetic engineering strategy.S. miltiorrhiza plants were grown in pots in the greenhouse under 25 °C with 16 h light/8 h dark period and relative air humidity of 60% or cultured on MS medium (pH5.8) containing 3% sugar and 0.8% agar as reported before2324S. miltiorrhiza plants were obtained from the Plant Garden of Shanghai Normal University and used for the analysis of expression pattern of SmJAZs in taproot, fibril, leaf, petiole, stem, seed and flower. The seeds of N. benthamiana, plasmid pCAMBIA2300, pGBKT7 and pGADT7 were provided by Professor Kexuan Tang from Shanghai Jiaotong University. Escherichia coli DH5α, Agrobacterium C58C1 and GV3101 were kept in our own laboratory. The plasmid of pMON530 was gifted by Professor Zhongnan Yang from Shanghai Normal University. The pMD-18 T vector and reverse-transcription PCR Kit were purchased from TaKaRa Biotechnology Co., Ltd. Primers synthesis and DNA sequencing was performed by Shanghai Sangon Biotechnological Company, China. Standards of cryptotanshinone, tanshinone I, tanshinone IIA, dihydrotanshinone were purchased from Aladdin, China. All other chemicals used were of analytical grade.S. miltiorrhiza were dried and pulverized in liquid nitrogen with mortar and pestle for extraction of the total RNA. Total RNA was used to synthesize the first strand cDNA with primer AP (5′-GGCCACGCGTCGACTAGTAC(T)16-3′) by using RT-PCR system . A local S. miltiorrhiza transcription database was built up from public transcripts from NCBI as well as our own transcriptomic data sequenced by hairy roots. Conserved domains of ZIM and Jas were utilized to search our transcriptome database to identify putative JAZs from S. miltiorrhiza with Blastx and Blastn. ORF Finder was used to identify the length of the ORF. Among the candidate JAZ sequences, SmJAZ3  was obtained with the specific forward primer SmJAZ3-F421 (5′-GCCGTTGGAACCACTGATTTTAG-3′) together with the primer AUAP. Primer pairs  were synthesized to amplify the whole ORF of SmJAZ9. The PCR amplification conditions were as follows: initial denaturation at 95 °C for 8 min, followed by 35 cycles denaturation at 95 °C for 30 s, annealing at 58 °C for 1 min and extension at 72 °C for 1 min 30 s.The hairy roots of SmJAZs. The nucleotide sequence and open reading frame (ORF) were analyzed by ORF Finder and the sequence comparison was conducted through database search using BLAST . Alignment of nucleotide and deduced amino acid sequences containing the two SmJAZs and other JAZs such as AtJAZs and OsJAZs were aligned with the ClustalW with default parameters. MEGA5.1 combined with CLUSTAL W alignments were used to construct the phylogenetic tree.Several bioinformatics softwares and websites were used for analysis of the two SmJAZs in different tissues  as well as its expression at 0 h, 0.5 h, 1.0 h, 2.0 h and 4.0 h after 100 μM MeJA treatment, plant materials were collected accordingly and total RNA was isolated according to the method described before23SmActinF: 5′- AGCACCGAGCAGCATGAAGATT-3′; SmActinR: 5′- AGCAAAGCAGCGAACGAAGAGT-3′) was performed as an internal control to estimate the expression level of the SmJAZs according to the relative quantitative analysis method (2−∆∆CT). Amplifications were performed under the following condition: 10 min denature at 95 °C, then 40 cycles of 15 s denature at 95 °C, 30 s annealing at 60 °C and 30 s extensions at 72 °C24To investigate the expression pattern of the BglII and KpnI site were added to the 5′- and 3′- of the ORF of SmJAZ3 and SmJAZ9 respectively via PCR. The PCR products were digested with BglII and KpnI and cloned into the vector pMON530. The constructed vectors were transferred into Agrobacterium strain ASE. Forty-day-old tobacco plants were used for the transient transformation experiment. After two days′ injection, localization of SmJAZs was observed using the confocal microscope (Carl Zeiss).SmJAZ3/SmJAZ9 gene or anti-SmJAZ3/anti-SmJAZ9 was amplified via PCR as indicated above. The vectors pCAMBIA1304 and pCAMBIA2300 were double-digested with Hind III and EcoR I. The smaller DNA fragment containing GUS gene expression cassette from pCAMBIA1304 was ligated with the large pCAMBIA2300 fragment to obtain the modified vector pCAMBIA2300+. The complete ORF of SmJAZs with restriction sites SpeI and BstEII were firstly cloned into pMD-18 T to generate pMD18T-SmJAZs. pCAMBIA2300+ and pMD18T-SmJAZs were then double-digested with SpeI and BstEII. The complete ORF of SmJAZs were then cloned into pCAMBIA2300+ to replace the GUS gene of the GUS gene expression cassette from pCAMBIA1304, under the control of CaMV35S promoter and the NOS terminator to generate pCAMBIA2300+-SmJAZ3 and pCAMBIA2300+-SmJAZ9. pCAMBIA2300+-anti-SmJAZ3 and pCAMBIA2300+- anti-SmJAZ9 were constructed using similar strategy with opposite direction of ORF in place of GUS gene. These above constructed plastids were transformed into A. rhizogenes strain C58C1 respectively. The aseptic leaves of 30 day-old S. miltiorrhiza were cultured in 1/2 MS medium in darkness for two days and then immersed in the Agrobacterium suspension for 10 min, then co-cultivated on MS solid medium23pCAMBIA2300 vector was used as a vector-only control. DNA isolation and PCR analysis procedure were carried out as we reported before2324The After 2 month culture, the hairy roots were dried at 50 °C in an oven and then ground into powder, 100 mg powder was placed in 16 mL solvent of methanol/dichloromethane  for tanshinone extraction, and then kept in dark for 24 hours after 1 hour′s ultrasonic process with frequency of 40000 Hz at room temperature . The substance was dried under vacuum and re-dissolved in 2 ml methanol. The solution was filtered and used for HPLC on Agilent 1260 apparatus equipped with a Waters reversed-phase C18 symmetry column. Acetonitrile-water  worked as the mobile phase at a flow rate of 1 mL/min with the detection wavelength at 270 nm523pGBKT7-SmJAZs and pGADT7-AtMYC2) were co-transformed into Saccharomyces cerevisiae AH109 cells following standard protocols5SmJAZ3 and SmJAZ9 cDNAs were cloned into pGBKT7 vector and the cDNA of AtMYC2 was inserted into pGADT7. Plasmids pGBKT7-AtJAZ9 and pGADT7-AtMYC2 were co-transformed into AH109 as the positive control. The empty vector pGADT7 and pGBKT7 constructs were co-transformed as the negative control. The indicated construct pairs were transformed into yeast AH109 and screened on yeast synthetic drop-out-Trp-Leu at 30 °C. Three days after transformation, yeast colonies were grown in selective liquid media for about 7 h, and the cell density was adjusted to OD600 = 1. A volume of 10 μL sample of the cell suspensions was plated out on yeast synthetic drop-out (SD/-Trp-Leu-His-Ade) to test protein interaction. Plates were incubated at 30 °C for 2–4 days.To assess possible protein interactions, the corresponding plasmids  using SPSS 11.5 software .S. miltiorrhiza JAZ genes which are induced by MJ and functionally identify their roles in tanshinone biosynthesis, conserved domains of ZIM and Jas were used to blast S. miltiorrhiza transcriptome database. Two JAZ fragments were found, one lacked 3′ terminal  and the other contained complete 921 bp ORF (later named JAZ9). Based on sequences of JAZ domains, a primer F421 was designed to amplify 3′ terminal cDNA of the JAZ3 together with the primer AUAP . To investigate the induction effect of MJ on SmJAZ3 and SmJAZ9 expression, 2-month-old S. miltiorrhiza hairy roots were treated with 100 μM MJ and analyzed at five time points . The expression of SmJAZ3 increased from 0.5 h and reached to the highest level at 1 h after MJ elicitation, which is nearly 6-fold higher than the control  in vector pMON530. Then the constructs were introduced into Agrobacterium strain ASE, respectively. Subcellular localization results showed that SmJAZ3 was only localized to the nucleus while SmJAZ9 was mainly in the nucleus but with some signal in the membrane in one-month-old tobacco  35 S promoter was transformed into Agrobacterium C58C1 and then transferred into S. miltiorrhiza to generate transgenic hairy root lines, respectively , 14/28 (50.0%), 16/36 (44.4%) and 16/36 (44.4%)  . Our resSmJAZ3 or SmJAZ9 can affect tanshinone biosynthetic genes, expression profiles of several key tanshinone biosynthetic genes such as SmGGPPS, SmDXS2 and SmKSL were examined, respectively. In the SmJAZ3 overexpressing S. miltiorrhiza hairy root lines, SmJAZ3 expression was up-regulated by 5–40 times in comparison to the control lines, while the transcripts of SmGGPPS, SmDXS2 and SmKSL were all reduced with different level in the SmJAZ3 overexpressing S. miltiorrhiza hairy root lines , tanshinone production in line SmJAZ3-3 was as low as 0.077 mg/g DW, a 94% decrease. Transgenic hairy roots lines harboring SmJAZ9 exhibited a slightly suppressive effect on the tanshinone biosynthesis, and the content of tanshinone decreased to 0.266 mg/g DW in SmJAZ9-22 line. Whereas, the content of tanshinone increased to 4.78 mg/g DW in anti-SmJAZ3-3 lines which was about 2.48-fold higher than the control, and it was about 3.22 mg/g in anti-SmJAZ9-23 lines with 1.35-fold higher than the control. The above results inferred that the inhibitory effect of SmJAZ3 on tanshinone biosynthesis is much stronger than SmJAZ9.Tanshinone is synthesized from universal precursor IPP that derived through two different pathways in separate cellular compartments, the MVA pathway occurring in the cytosol and MEP pathway occurring in the plastids, while the MEP plays a more important role in tanshinone biosynthesisS. miltiorrhiza so far), we fused the SmJAZ3, SmJAZ9 to the pGBKT7 vector (BD) and AtMYC2 to the pGADT7 vector (AD), followed by co-introduction of the above vectors into yeast cells AH109 for Y2H. Plasmids pGBKT7-AtJAZ9 and pGADT7-AtMYC2 were co-transformed into AH109 as the positive control and the empty vector pGADT7 was co-transformed with pGBKT7 constructs as a negative control  elicitation revealed that expression of SmJAZ3 and SmJAZ9 was quickly MJ-responsive, which suggested that SmJAZ3 and SmJAZ9 may be involved in MJ signal pathway and its related activated responses, in good agreement with those JAZs previously reported in model plant A. thalianaCOI1 and JAZ to generate protein complex and then 26 S proteomes were recruited to degrade JAZ proteins to liberate positive regulators such as MYC2 whose transcriptional activation was repressed by JAZs under normal condition57COI1-like protein interact with JAZs and make JAZ proteins degraded by 26 S proteomes in S. miltiorrhiza, and then some positive transcription regulators such as MYC2 were released from repression by JAZs to transcriptionally activate the expression of some tanshinone biosynthetic genes and correspondingly modulate tanshinone biosynthesis. SmJAZ9 could interact with AtMYC2, implied that the above hypothesis may exist in S. miltiorrhiza, which needed to be investigated further in the future. Over-expression of SmJAZ3 and SmJAZ9 in S. miltiorrhiza hairy roots resulted in reduced accumulation of tanshinone while down-regulation of SmJAZ3 and SmJAZ9 effectively improved the production of tanshiones. These indicated that SmJAZ3 and SmJAZ9 play important roles in tanshinone biosynthesis and act as repressive components in the JA signal pathway for tanshinone production, which paves a way to dissect molecular mechanism in details in the future.Being one kind of diterpenes with good pharmaceutical activities, tanshinone was essentially derived from two common precursors, isopentenl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)293436S. miltiorrhiza, which may be the target of JAZ proteins and play an important role in accumulation of tanshinone as positive regulator. Whether the SmJAZs have an interaction with the potential SmMYC2 needed to be further investigated. Thus work towards isolation and identification of MYC2-like transcription factor from S. miltiorrhiza may further decipher this action mechanism.JAZ proteins belong to a larger group of ZIM-domain proteins, named after the putative transcription factor Zinc-finger inflorescence meristem (ZIM). ZIM family members were assigned to this group according to a conserved 28 amino acid ZIM motif. In addition to ZIM, JAZ family members also have the highly conserved Jas motif of 26 amino located near the C-terminus, it is now well established that Jas motif participates in protein-protein interaction with both transcription factors like the member of bHLH family (MYC2) and COI1. In the present study, it was found that SmJAZ9 protein can interact with the AtMYC2 by yeast two hybrids (Y2H), which was consistent with former reports that the JAZ family has the character to interact with MYC238A. thaliana and some other model plants, several reports have revealed the direct involvement of JA and JAZ proteins in growth and development processes such as secondary growth4344SmJAZ3 in S. miltiorrhiza hairy roots down-regulated several tanshinone biosynthesis key genes such as SmDXS2, SmKSL and SmGGPPS in comparison to the control lines. At the same time, the production of tanshinone was decreased to 0.077 mg/g DW in JAZ3 over-expressing line JAZ3-3 in comparison to the control line (1.37 mg/g DW). While down regulated expression of JAZ3 in anti-JAZ3-3 line increased the content of tanshinone to 4.78 mg/g DW, which was 2.48-fold higher than the control. In transgenic SmJAZ9 line, average production of tanshinone was 1.32 mg/g DW, whereas, the content of tanshinone increased to 3.22 mg/g DW in anti-JAZ9-23 lines, a 1.35-fold increase as the control. These results reflect that the SmJAZ3 has much stronger suppressive effect on tanshinone biosynthesis than the SmJAZ9. Furthermore it was found that SmJAZ3 and SmJAZ9 acted as repressors in tanshinone biosynthesis. The above data testified the role of JA in regulating tanshinone biosynthesis genes and thereby affecting the production of tanshinone in S. miltiorrhiza. However, further investigations are needed to figure out whether SmJAZs interact with SmMYC2 or other transcription factors, how and which SmJAZ they interact with. We also need to further elucidate how the JA signaling functions or whether it cross talks with other signaling pathways in S. miltiorrhiza. Overall, this study extends our understanding in the molecular regulation of tanshinone biosynthesis induced by MJ signal. Our work indicated JAZs such as SmJAZ3 and SmJAZ9 play important role in regulation of tanshinone biosynthesis, it may be involved in other secondary metabolism. The isolation and functional characterization of the two JAZs from S. miltiorrhiza paves a way to further dissect molecular mechanism and manipulate tanshinone production in plant.In How to cite this article: Shi, M. et al. Methyl jasmonate induction of tanshinone biosynthesis in Salvia miltiorrhiza hairy roots is mediated by JASMONATE ZIM-DOMAIN repressor proteins. Sci. Rep.6, 20919; doi: 10.1038/srep20919 (2016).


To determine factors associated with future falls and/or near falls in people with mild PD.The study included 141 participants with PD. Mean (SD) age and PD-duration were 68 (9.7) and 4 years (3.9), respectively. Their median (q1–q3) UPDRS III score was 13 (8-18). Those >80 years of age, requiring support in standing or unable to understand instructions were excluded. Self-administered questionnaires targeted freezing of gait, turning hesitations, walking difficulties in daily life, fatigue, fear of falling, independence in activities of daily living, dyskinesia, demographics, falls/near falls history, balance problems while dual tasking and pain. Clinical assessments addressed functional balance performance, retropulsion, comfortable gait speed, motor symptoms and cognition. All falls and near falls were subsequently registered in a diary during a six-month period. Risk factors for prospective falls and/or near falls were determined using logistic regression.Sixty-three participants (45%) experienced ≥1 fall and/or near fall. Three factors were independent predictors of falls and/or near falls: fear of falling  history of near falls  and retropulsion . The strongest contributing factor was fear of falling, followed by a history of near falls and retropulsion.Fear of falling seems to be an important issue to address already in mild PD as well as asking about prior near falls. Postural instability is one of the cardinal signs of Parkinson’s disease (PD). People with PD are particularly unstable backwards –3 which In the postural instability and gait difficulty (PIGD) subtype of PD, walking difficulties and balance demanding activities may be affected already early on ,7. Gait Falls are one of the most disabling features of PD and occur in 35–90% of patients, among whom 18–65% experience recurrent falls . It is aThe Regional Ethical Review Board in Lund, Sweden approved the study (Dnr 2011/768). All participants gave written informed consent.All people diagnosed with PD receiving care at a south Swedish university hospital during 2007–2012 were considered eligible for inclusion (n = 349). Exclusion criteria were age above 80 years old (n = 116), inability to stand without support (n = 22), unable to understand instructions (n = 14) or having severe comorbidity (n = 11). Of the remaining 186 potential participants, 40 (16 women) declined participation. Those who declined did not differ significantly  from the 146 participants with respect to age and PD duration.Anti-parkinsonian medications were recorded from medical records. All participants were assessed during an outpatient visit, which was scheduled at a time of day when the participant usually reported to feel at best.First, the participants completed self-administered questionnaires targeting freezing of gait, turning hesitations –26, walkAll participants then self-rated their present motor status as “good/on”, “on with dyskinesias”, or “bad/off”. This was followed by clinical assessments  administIn the last six months, have you fallen in such a way that your body hit the ground?), history of near falls , balance problems while dual-tasking  and pain (Do you presently suffer from pain?).Additional self-administered questions were then administered. These targeted demographic data  and the presence or absence of dyskinesia  Table 1Table 1. an unexpected event in which the participants come to rest on the ground, floor, or lower level” [a fall initiated but arrested by support from the wall, railing, other person etc.” [By using a diary, participants were instructed to register all consecutive falls and near falls for six months. At the outpatient visit, the definitions of a fall and a near fall were thoroughly described to all participants. Falls were described and defined as “r level” . Near faon etc.” .Did you fall in such a way that your body hit the ground?” The corresponding question about a near fall incident was phrased: “Were you close to falling, but managed to brace yourself at the last moment  clarified whether the incident was a fall or a near fall. The question in relation to a fall was phrased as follows: “All participants were telephoned monthly to ensure that registrations were completed according to instructions. During the last telephone call, they were requested to return the diary in a pre-stamped envelope.Data were checked regarding underlying assumptions and described accordingly using IBM SPSS version 21. Normally distributed interval/ratio level variables were described using means and SDs. In other cases, medians (q1–q3) were used. Categorical variables were described using n (%). The alpha level of significance was set at 0.05 (2-tailed). Anti-parkinsonian medications were expressed as daily levodopa equivalent (LDE) doses (mg/day) .Logistic regression analysis was performed in order to establish risk factors for prospective falls and/or near falls (dependent variable). Initially, simple logistic regression analysis was used for factors (independent variables) that were considered potentially important for falls and/or near falls. Variables that were significant at the alpha level of 0.2 were subsequently included as independent variables in a multiple logistic regression analysis in order to identify those independently associated with prospective falls and/or near falls. The <0.2 P-value threshold was chosen in order to avoid leaving a confounding variable out. Both forward and backward methods were used . The final model was controlled for age and gender.In order to facilitate comparisons with prior studies, we also explored factors associated with prospective falls only . The statistical procedure was otherwise identical to the main analysis (see above).Five participants did not complete the prospective 6-month follow-up of falls and/or near falls due to e.g. developing severe comorbidities. The final sample  had a mean  age and PD duration of 68  and 4  years, respectively. Their median (q1–q3) UPDRS III score was 13 (8–18). Further details are provided in At the time of assessments, 123 out of the 141 participants (87%) rated their motor status as”on”, whereas 12 (9%) rated it as “on with dyskinesias”, and 6 (4%) rated it as”off”.During the 6-month follow-up, 63 out of 141 (45%) participants experienced at least one fall and/or near fall. Forty-five out of 141 participants (32%) reported falls, of whom 26 (58%) reported more than one fall ; on average they reported 5 falls . In total, 44 participants (31%) reported near falls. Eighteen of those reported only near falls whereas 26 also reported falls. Twenty-six out of the 44 (59%) reported more than one near fall; they reported on average 11 near fall incidences . The total number of all reported incidences was 452 (n = 63); 158 (35%) of those were falls whereas 294 (65%) were near falls.Simple logistic regression analyses identified 16 independent variables associated with prospective falls/near falls at P<0.2 . Table 4Simple logistic regression analyses identified 15 independent variables associated with prospective falls at P<0.2; the identified variables were the same as in This study comprehensively investigated contributing factors for experiencing future falls and/or near falls in people mildly affected by PD. When using multivariate analyses, three contributing factors were identified. The strongest factor was FOF, followed by a history of near falls and having retropulsion. That is, FOF seems to be an important issue to address already in mild PD as well as asking about prior near falls. Our findings may thus have important clinical implications since these aspects may not be addressed in those having mild PD. Although several prospective studies have investigated contributing factors for experiencing future falls  has been suggested to be preferably to MMSE ,51. In aThis study identified three contributing factors for experiencing future falls and/or near falls in people mildly affected by their PD. The strongest factor was FOF, followed by a history of near falls and having retropulsion. That is, FOF seems to be an important issue to address already in mild PD as well as asking about prior near falls. A history of near falls appears to be a stronger predictor for future falls than a history of falls. This highlights the need for addressing near falls in more depth in larger longitudinal studies including a broader range of PD severities.


Alcohol misuse imposes substantial harm on Indigenous Australians whose health status is poorer than non-Indigenous Australians. Although Indigenous youth are over represented in Indigenous alcohol harms, few interventions addressing alcohol-related harm among Indigenous youth have been evaluated. Given this paucity of evidence, a survey was designed to evaluate the effects of a whole-of-community, anti-binge drinking intervention for young people in an Indigenous community in far north Queensland, Australia.A cross sectional, baseline-post intervention study assessed the impact of a two year anti-binge drinking intervention targeting young people (18–24 years). A survey was developed and implemented at baseline and again two-years post-intervention, administered by young local people employed as research assistants. Survey respondents were recruited through snowballing techniques. Survey items asked about respondents’ knowledge of binge drinking and standard drinks, involvement in alcohol-free social activities, frequency of short-term risky drinking (binge drinking), and mean alcohol expenditure during short-term risky drinking occasions.The intervention was called Beat da Binge. Two major events and multiple minor activities each year were implemented, focusing on drinking education, alcohol-free community-wide social events, and youth-specific sporting and social activities to facilitate self-empowerment.Beat da Binge was associated with a statistically significant 10 % reduction in the proportion of survey respondents who reported that they had engaged in an episode of short-term risky drinking, in the frequency of short-term risky drinking for all beverage types except wine (ranging from 4 % to 31 % reductions), in mean expenditure on alcohol during short-term risky drinking sessions ($6.25) and in the proportion of activities with family/friends that usually include alcohol (7 %). There were also statistically significant increases in awareness of binge drinking and standard drinks (28 % and 21 % respectively). In addition to alcohol-specific outcomes, there was a statistically significant 8 % increase in the proportions of respondents engaged in training as their main weekday activity, which was partly off-set by a 13 % reduction in those whose main weekday activity was family care or home-related tasks.Reductions in the proportion of survey respondents who reported binge drinking, along with increases in awareness and involvement in alcohol-free social activities suggest the community-based intervention was effective. The potential impact of sample selection and self-reporting limitations on results need further investigation. There is an urgent need for Indigenous, community-driven public health programs that are well evaluated to both improve Indigenous health and the strength of the current evidence base to inform future community interventions. Among Indigenous Australians, short-term risky drinking, currently defined by the National Health and Medical Research Council (NHMRC) of Australia as the consumption of more than four standard drinks in a single drinking occasion , appearsDespite the recognised extent of these harms, the specific alcohol consumption and harm characteristics of Indigenous young people living in a diverse range of communities have not been adequately described . In geneIn 2008, the Australian Government provided funding through a National Binge Drinking Strategy for programs designed to reduce rates of short-term risky drinking among Indigenous young people. Yarrabah, an Indigenous community approximately a one-hour drive from Cairns in Queensland, successfully applied for funding to develop and implement a community-based intervention targeting short-term risky binge drinking among their young people. Key stakeholders in Yarrabah were concerned about the apparent increase in risky alcohol consumption among their young people following the cessation of funding for the Australian Government’s Community Development Employment Program (CDEP). The lead organisation for this intervention in Yarrabah (Gindaja Treatment Centre) invited researchers from James Cook University (JCU) in Cairns, the National Drug and Alcohol Research Centre (NDARC) at the University of New South Wales (UNSW) in Sydney, and the Hunter Medical Research Institute (HMRI) at the University of Newcastle, to partner with them to evaluate their program.This study aims to evaluate the effects of a whole-of-community, anti-binge drinking intervention for young people in Yarrabah.The protocol for the research project was approved by JCU Human Research Ethics Committee .In 2011, Yarrabah had a population of 2,409: 97 % were Indigenous and half the population was aged 25 years or less, with a median age of 21 years . AccordiGiven the uniqueness of the intervention to Yarrabah and for practical limitations, largely funding constraints, a baseline/post evaluation design was agreed using self-reported outcomes.Beat da Binge, a two-year project targeting binge drinking, commenced in April 2010. Key features of the intervention are that it was community-driven, utilised participatory approaches, actively engaged young people in its design, implementation and evaluation, and sought to create a partnership with researchers . InterveMajor events included Foundation Day (the commemoration of the founding of the mission in 1893) and NAIDOC week . Activities coinciding with major events were the distribution of T-shirts, leaflets and other resources that specifically conveyed messages about safer drinking practices, high-risk times for risky drinking and harms, and provided ideas for alternatives to drinking. Project staff was also available to actively promote and discuss these messages. Similar activities were implemented as minor community events in two ways, first, by integrating them into existing events, such as domestic violence week, drug action week, child protection week, carols by candlelight, rugby league carnival day, Christmas movie night, the Bishop Malcolm Indigenous carnival, and the healthy community initiative of community survival day. Second, a range of events were offered with co-sponsorship from local organisations, including music, sporting and cultural events, a ‘dive-in’ movie night, Seahawks chickettes activity, self-defence training and restraint techniques (START), taekwondo, a touch-football camp, and boxing.Baseline and post-intervention surveys were developed to target 18–24 year olds in Yarrabah. This age group was chosen to coincide with the minimum legal drinking age (lower end of the range) and because approximately half the population of Yarrabah is aged less than 25 years (upper end of the range). The Beat da Binge project officers, who were local residents, trained as research assistants, consulted with people in this age range in Yarrabah to identify potential survey items and potential ways of phrasing those items. In partnership with researchers, those potential items were then aligned with survey items that have some evidence for their reliability, validity or comparability with existing surveys. Finally, they were organised into broad domains, including demographics  , which wItems for behaviours directly related to alcohol consumption included the amount of money normally spent on alcohol in a drinking session and the proportion of activities with family and friends that would normally include drinking alcohol.The details of the procedure are provided elsewhere . In summχ2) tests, Student’s t-test test and Odds Ratios (OR). Statistical significance was set as p<0.05 (95 % level of significance). All analyses were conducted using STATA [Data collected from both the baseline and post-intervention surveys were summarised and differences reported. Differences in the proportion of respondents from baseline to post-intervention are reported for: involvement in at least one session of short-term risky drinking in the previous 12 months; involvement in at least one session of short-term risky drinking in the previous 12 months by beverage type; and, for those who had engaged in at least one session of short-term risky drinking in the previous 12 months, mean expenditure on alcohol during short-term risky drinking sessions, awareness of what binge drinking is, familiarity with standard drinks and the proportion of activities with family or friends that include alcohol. Finally, as a measure of exposure to the intervention, the proportion of post-intervention respondents who indicated they had heard of Beat da Binge is reported. Secondary outcomes include changes in: mean expenditure on alcohol during binge drinking sessions; binge drinking awareness; familiarity with standard drinks; and the extent to which activities with family and friends included alcohol. Inferential statistical analysis were conducted using Chi-square  and post- (n=154) intervention surveys reported similar demographic characteristics. Respondents for the post-intervention survey were statistically significantly less likely to be engaged in family care or home-related tasks as their main weekday activity  and more likely to be engaged in training . For alcohol use, there was a statistically significant 10 % reduction in the proportion of respondents who reported engaging in short-term risky drinking after the implementation of the Beat da Binge program, relative to the baseline proportion .Table n=152) to post (n=92) implementation of the Beat da Binge program in the proportion of respondents who engaged in short-term risky drinking, across all beverage types except wine: beer  and the proportion of respondents who reported that their activities with family/friends usually include alcohol , in mean expenditure on alcohol during short-term risky drinking sessions ($6.25) and in the proportion of activities with family/friends that usually include alcohol (7 %). There were also statistically significant increases in awareness of binge drinking and standard drinks (28 % and 21 % respectively). In addition to alcohol-specific outcomes, there was a statistically significant 8 % increase in the proportions of respondents engaged in training as their main weekday activity, which was partly off-set by a 13 % reduction in those whose main weekday activity was family care or home-related tasks.The baseline/post evaluation design was the most pragmatic option but its primary limitation is that it cannot rule out the possibility that a factor other than the Beat da Binge program was causally related to the observed outcomes, even though there was no other intervention implemented in Yarrabah during the project period. The challenges of using a systematic or random sampling in an outer regional Indigenous community forced the study to use convenience sampling approach and similar challenges have been faced in a remote Indigenous study on substance use . NeverthWhile national data are available on the extent and nature of the Indigenous youth binge drinking problem, there is a dearth of community specific data that would allow programs to be targeted to local risk factors and harms, and to evaluate community-level interventions . DespiteCommunity stakeholders do not dispute the findings of this study and indicated, during presentation of study results, that the reported changes are possibly true and that they have observed positive changes amongst youths. Beyond the potential benefits of reduced proportions of short-term risky drinkers among young people in Yarrabah, this study suggests that projects led and designed by Indigenous communities, and evaluated in partnership with researchers, can be potentially effective. This integrated approach may provide a more effective blue-print for reducing alcohol-related harm than the traditional government- or researcher-designed policies and programs that typically allow insufficient community input. Local leaders in Yarrabah consider that this community-driven approach, with active engagement of young people in its design, implementation and evaluation, is a key reason for the positive outcomes observed. Such locally-developed interventions and evaluations not only provide appropriate education, in this case regarding binge drinking, standard drinks and alcohol-related harms, but may also contribute to improving the social determinants of health by providing training and employment to young people as casual researchers and increasing their locus of control. There is international evidence that the empowering nature of such initiatives can, in itself, lead to improved health outcomes, and that empowerment is a viable public health strategy .This study reports on the collection and use of community specific data to assess the impact of a community designed and implemented intervention for Indigenous young people engaging in short-term risky drinking. The primary outcome of a statistically significant 10 % reduction in the proportion of Indigenous young people who reported that they had engaged in an episode of short-term risky drinking suggests that locally developed interventions can be potentially effective, and that encouraging partnerships between Indigenous communities and researchers to evaluate community-led intervention is feasible. Furthermore, statistically significant secondary outcomes included increased awareness of binge drinking and standard drinks, and greater engagement in social activities that did not involve alcohol consumption. The potential impact of sample selection and self-reporting limitations on results need further investigation. This study provides further evidence of the feasibility and potential effectiveness of Indigenous-led, community-based intervention and evaluation.


Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.The Formosan subterranean termite,  Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm areas of high humidity, approximately 35° north and south of the equator, which includes the southeastern United States [The Formosan subterranean termite (FST), d States . Workersd States , 3. Gut d States , 5, 6.Previously, we provided proof of concept for a novel strategy for termite control using paratransgenesis , 9, 10. Since this product is highly specific to killing protozoa and its applicability is thus restricted to the control of subterranean termites, which rely on protozoa for survival, we decided to explore possibilities to develop a more broadly applicable paratransgenic system. Previous experiments showed that injection of low doses of Melittin into the hind gut of FST not only kills the protozoa but also leads to necrosis of gut tissue . MelittiKluyveromyces lactis (New England Biolabs Inc. (NEB), MA, USA) was genetically engineered to express and secrete the lytic peptide Melittin . The Melittin gene was PCR amplified (primers: 5´GTAAAACGACGGCCAGT3´and 5´CAGGAAACAGCTATGAC3´) and the amplified fragment was cloned into the pKLAC2 expression vector  according to the manufacturer’s manual . The constructs were isolated and linearized with SacII and the expression cassettes were introduced into the chromosome of K. lactis cells at the LAC4 locus according to the manufacturer’s protocols (https://www.neb.com/products/e1000-k-lactis-protein-expression-kit). Yeast strains were grown on yeast carbon base (YCB) agar medium containing 5mM acetamide at 30°C for 48 hrs. Single colonies were resuspended in 2 ml YPGal medium and incubated for 48 hrs at 30°C while shaking at 250 rpm. Yeast cells were harvested by centrifugation at 7000 X g for 30 sec and the culture supernatants were transferred to fresh tubes.The commercially available yeast RQQ-NH2, ). Codon ails see ). The pKK. lactis cells was verified via PCR. Single colonies were suspended in 25μl of 1M sorbitol containing 2 mg/ml lyticase and incubated at 30°C. After 1 h, the lyticase-treated cells were lysed at 98°C for 10 min in a PTC-200 thermocyler . PCR was performed according to the K. lactis instruction manual with the primers supplied with the K. lactis kit. Integration of the expression cassette at the LAC4 locus in the K. lactis genome was verified by the amplification of a 2.4 kb product (https://www.neb.com/products/e1000-k-lactis-protein-expression-kit).Correct integration of the expression cassettes into the genome of ® Bis_Tris Precast Gels  by the presence of a protein band in the 2.8 kDA region that did not appear in the controls. A non-lethal K. lactis strain expressing a far red fluorescent protein, mPlum  was previously engineered by Sethi et al. [Expressed Melittin was tentatively confirmed in the supernatant of engineered yeast cultures via NuPAGEi et al.  and usedTetrahymena pyriformis . Fifty microliter of the culture supernatants was incubated with 50 μl of T. pyriformis culture with a concentration of 10 cells/μl. After 24 h, live protozoa were counted using a Sedgewick-Rafter cell  under a microscope  at 200 X magnification. The Melittin expressing yeast strain that was most effective against Tetrahymena in vitro was chosen for further evaluation in termite feeding assays.Toxic activity of the culture supernatants of yeast strains was measured against free-living aerobic protozoa of the species Yeast strains were freeze-dried overnight using a lyophilizer and mixed separately at the rate of 1 part of yeast (0.25 mg) to 10 parts (2.5g) of α-cellulose powder and moistened with 6 ml of autoclaved tap water. Freeze drying does not impact viability and gene expression of yeast and yeast as a bait ingredient does not impact consumption .Bait disks (1.5 x 0.5 cm) were cut out the mixture with a cork borer . TriplicFive worker guts per treatment and replicate were extirpated using sterile forceps at days 2, 4, 6, 10, 13, 17, 20, 27 and 34. Hind guts were opened in an anaerobic glove box in sparged Trager U media on a glass slide using fine probes [K. lactis were qualitatively confirmed by the presence of blue colonies after plating the gut contents on Kluyveromyces differential medium [Uptake and retainment of l medium . Gene exl medium .™, Walmart, Baton Rouge, LA) to make gut contents visible. At days 1, 2, 7 and 20, five termite workers per treatment were observed under a stereo microscope  for traces of colored gut content outside of the gut. Guts were also extirpated to test for traces of blue gut content in the body cavity. Results were recorded via a digital camera  attached to the stereoscope.Bait disks and feeding assays were prepared for one termite colony as described above to test effects of baits containing Melittin-expressing yeast versus a control of plain α-cellulose on gut integrity. Water for moistening the bait disks was laced with blue food color  and were thus combined. General Linear Model Analysis showed that colony origin, treatment and days of feeding had significant effects on gut and protozoa health (P ≤ 0.001) with no significant interactions (P ≥ 0.05) between these variables except for treatment*days . Previous studies have shown that lytic peptide concentrations as low as 1 μM are sufficient to kill termite gut protozoa when injected into the hind gut of workers [All three treatments differed significantly in their effects on gut and protozoa health . As shown by the non-overlapping 95% confidence intervals in  workers , 13); hoUntil day 36 (Colony 1) and day 44 (Colony 2), the controls do not differ from each other in their effect on gut health. However, towards the end of the experiment, the bait with yeast expressing mPlum caused decline in gut and protozoa health, but never caused complete defaunation in contrast to the loss of all protozoa caused by the treatment with Melittin-expressing yeast at day 54 and 56 for colony 2 and colony 1, respectively .Although previous studies  have notThe results revealed that Melittin expressing yeast defaunates guts of termite workers, similar to what has been previously shown with other lytic peptides applied in their pure form , 10 or ein vivo or the guts rupturing during the gut pulling procedure because Melittin weakened the gut walls. In either case, differences between treatments and controls in dye distribution and presence of dye residue in the abdomen due to gut rupture suggest mechanical or metabolic changes associated with the ingestion of Melittin producing yeast. This supports the finding of the previous experiment based on observation of gut integrity and protozoa health .


There are errors in the author affiliations. The publisher apologizes for the error. The affiliations should appear as shown here:1, Kerri A. Nottage2, Elliott W. Cole1, Jane S. Hankins2, James G. Gurney1,3Vikki G. Nolan1 Division of Epidemiology, Biostatistics and Environmental Health, School of Public Health, University of Memphis, Memphis, TN, USA, 2 Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA, 3 Department of Epidemiology and Cancer Control, St. Jude Children’s Research Hospital, Memphis, TN, USA.