Drosophila melanogaster as the model organism to explore the molecular mechanisms underlying the development of penetration resistance to AVMs. We clearly show that the chitin layer is thickened and the efflux transporter P-glycoprotein (P-gp) is overexpressed in the AVM-resistant larvae epidermis. We reveal that the activation of the transcription factor Relish by the over-activated epidermal growth factor receptor (EGFR)/AKT/ERK pathway induces the overexpression of the chitin synthases DmeCHS1/2 and P-gp in the resistant larvae. Interestingly, we discover for the first time, to the best of our knowledge, that AVM directly interacts with EGFR and leads to the activation of the EGFR/AKT/ERK pathway, which activates the transcription factor Relish and induces the overexpression of DmeCHS1/2 and P-gp. These findings provide new insights into the molecular mechanisms underlying the development of penetration resistance to drugs.With the widespread use of avermectins (AVMs) for managing parasitic and agricultural pests, the resistance of worms and insects to AVMs has emerged as a serious threat to human health and agriculture worldwide. The reduced penetration of AVMs is one of the main reasons for the development of the resistance to the chemicals. However, the detailed molecular mechanisms remain elusive. Here, we use the larvae of  With thrmitilis –7, and aγ-aminobutyric acid-gated chloride channel and result in the release of chloride in insects and worms [S-transferase can result in metabolism resistance to AVMs in insects [Numerous studies have revealed two important mechanisms for the development of resistance to AVMs, i.e. target insensitivity and increased metabolism of AVMs ,9. AVMs nd worms ,11. Thisnd worms ,13. Genend worms ,14. Meannd worms –17. Elev insects .Besides these two mechanisms, reduced penetration of insecticides in insects is another mechanism underlying the development of the resistance to insecticides –23. BecaDrosophila melanogaster is a widely used model organism for insects. In addition, the Drosophila larvae are similar to nematode worms in numerous biological processes [Drosophila larvae as the model organism in this study for both worms and insects to explore the mechanisms underlying the development of penetration resistance to AVM. In addition, although the Drosophila adults were used in our previous study [D. melanogaster as the model organism in this study. Here, we identify a clear molecular mechanism underlying the penetration resistance to AVM in Drosophila.rocesses –27. Becarocesses ,29, we uus study , we foun2.2.1.Drosophila larvae and adults with AVM. We found that the larvae died significantly faster than the adults when they were exposed to the same doses of AVM (a). Meanwhile, we found that the resistance ratio of the larvae was drastically higher than that of the adults . These findings indicate that the larvae are more sensitive to AVM than adults. As a result, Drosophila larvae were chosen as the model organism in this study. Indeed, when exposed to the same concentrations of AVM, the AVM-susceptible larvae (S larvae) died significantly faster than the AVM-resistant larvae , which suggests that R larvae are resistant to AVM toxicity.We first sought to compare the sensitivities of AVM-susceptible strain s of AVM a. Meanwhd). Previous studies have shown that AVMs mainly exist in the original form in the body of vertebrate animals [Drosophila have not been reported as yet. Thus, in this study, only the original form of AVM was detected by HPLC, partly owing to the short treatment time. Immunofluorescence analysis also showed that the amount of AVM accumulated in the fat body of R larvae was lower than that in S larvae, after the larvae were treated with the same concentration of AVM for the same time . These results indicate that AVM can penetrate more easily into S larvae, which leads to the faster death of S larvae. Thus, the reduced penetration of AVM into R larvae results in their resistance to AVM.High-performance liquid chromatography (HPLC) analysis results showed that the permeability of AVM in the larval body of the resistant strain was only half of that in the susceptible strain d. Previo animals ,32, and ame time e,f. Thes2.2.a). In addition, electron microscopy showed that the chitin layer in R larvae was approximately twice as thick as that in S larvae (b). The alimentary canal is another important barrier that restricts the diffusion of AVM. The peritrophic matrix (PM) is composed of chitin and glycoproteins and lines the insect intestinal lumen, and the PM can protect the midgut epithelium from mechanical damage, pathogens and toxins [Epidermis is the first and most important barrier that restricts the diffusion of chemicals including AVMs into the animal body. To investigate whether the penetration resistance to AVM is related to the epidermis, the structure of S and R larvae body wall was examined with the optical microscope. The semi-thin slice results showed that R larvae body wall was about twice as thick as S larvae body wall a. In addS larvae b. The ald toxins . The eleD. melanogaster larvae, there are two genes for chitin synthases, i.e. DmeCHS1 and DmeCHS2, which belong to the insect CHS-A and CHS-B gene families, respectively [DmeCHS1 and DmeCHS2 are mainly expressed in the epidermis and other tissues, respectively. The RT-PCR and q-PCR analysis results showed that mRNA levels of the two chitin synthases, DmeCHS1 and DmeCHS2, in R larvae were higher than those in S larvae (c), especially for DmeCHS1. These results indicate that the overexpression of chitin synthases probably contributes to the thickened chitin layer in R larvae.In ectively ,34. DmeCS larvae c, especib). In conclusion, the higher expression of P-gp and chitin synthases in resistant larvae probably leads to the reduction of AVM permeability in R larvae.In the epidermal cells of animals, P-glycoprotein (P-gp) is a key factor that regulates the penetration of AVM, because it can transport AVM out of cells . P-gp is32.3.Drosophila adults [Drosophila larvae, immunofluorescence analysis and western blotting analysis were used.Chitin synthases are upregulated during development and epidermal wound-healing , and thea adults . In ordea,c). In addition, P-gp and p-EGFR were mainly expressed in the epidermis of larvae and upregulated in the epidermis of R larvae (b). P-gp expression in the susceptible strain was inhibited after the treatment with the EGFR phosphorylation inhibitor lapatinib, AKT phosphorylation inhibitor wortmanin and ERK phosphorylation inhibitor U0126 (d). Furthermore, these inhibitors reduced the mRNA levels of DmeCHS1 and DmeCHS2 in S larvae (e). These results indicate that the EGFR/AKT/ERK pathway regulates the expression of P-gp and chitin synthases DmeCHS1 and DmeCHS2.We found that the EGFR/AKT/ERK pathway was activated in epidermal cells of the resistant strain a,c. In aR larvae b. P-gp eor U0126 d. FurtheS larvae e. These DmeCHS1 and DmeCHS2 in the resistant strain, the larvae of the two strains were treated with AVM and lapatinib. Western blotting analysis and q-PCR assay results showed that AVM enhanced P-gp expression and DmeCHS1/2 mRNA levels in both the two strains of larvae . Moreover, lapatinib inhibited the basal level of P-gp expression and DmeCHS1/2 mRNA levels in both S and R larvae, and further inhibited the AVM-induced overexpression of P-gp and DmeCHS1/2. Notably, the expression of P-gp and DmeCHS1/2 in R larvae was suppressed by lapatinib to levels comparable with those in S larvae . In addition, wortmanin and U0126 had similar effects in regulating P-gp expression compared with lapatinib. Both wortmanin and U0126 reduced P-gp and DmeCHS1/2 mRNA levels in S and R larvae and suppressed the overexpression of P-gp and DmeCHS1/2 in R larvae . Altogether, these findings suggest that the overexpression of P-gp and chitin synthases DmeCHS1/2 in R larvae is induced by the activation of the EGFR/AKT/ERK pathway.To demonstrate whether the activation of the EGFR/AKT/ERK pathway indeed induces the overexpression of the proteins P-gp, f larvae f,g. MoreS larvae f,g. In a2.4.DmeCHS1/2, we detected the activity of transcription factor Relish (NF-κB) in the larvae. The full-length Relish (110 kDa) is not functional. Upon activation and cleavage, the N-terminal fragment of Relish migrates into the nucleus and serves as a transcriptional factor, whereas the C-terminal fragment (49 kDa) remains in the cytoplasm, and can be used as an indicator for Relish activation [a). To determine whether activation of Relish leads to the overexpression of P-gp and DmeCHS1/2, the larvae of the two strains were exposed to different concentrations of Relish activation inhibitor pyrrolidinedithiocarbamic acid (PDTC). PDTC treatment decreased P-gp expression in both of the S and R larvae and decreased DmeCHS1/2 mRNA levels in R larvae . Meanwhile, PDTC inhibited P-gp expression in S and R larvae treated with AVM (c).To determine how the activation of the EGFR/AKT/ERK pathway induces the overexpression of P-gp and tivation . CompareR larvae b,d. Meanwith AVM c.Figurea). In addition, lapatinib, wortmanin and U0126 suppressed the expression and activation of Relish in both the S and R larvae, whereas AVM induced the activation of Relish (b–d). Moreover, in S2 cells, AVM induced the activation of Relish dose-dependently and lapatinib, wortmanin and U0126 suppressed the activation of Relish induced by AVM (e). Taken together, our results suggest that AVM and the EGFR/AKT/ERK pathway induces the activation of Relish, which regulates the overexpression of P-gp and DmeCHS1/2.We next investigated the relationship between the EGFR/AKT/ERK pathway and Relish. In the susceptible strain, lapatinib, wortmanin and U0126 inhibited the activation of Relish dose-dependently a. In addf Relish b–d. Mored by AVM e. Taken 2.5.a,b). In addition, lapatinib inhibited the activation of the EGFR/AKT/ERK pathway induced by AVM (c). These results suggest that AVM activates the EGFR/AKT/ERK pathway immediately upon contacting the cells, which raises the possibility that AVM directly interacts with the membrane protein EGFR when it contacts cells. To test this possibility, we analysed the co-localization of AVM and EGFR. EGFR and p-EGFR in S2 cells co-localized with AVM after cells were exposed to AVM for 30 min. However, pretreatment with lapatinib prevented p-EGFR from co-localizing with AVM (d). Moreover, in primary epidermal cells from wing imaginal discs and epidermal cells of S larvae, AVM induced the activation of EGFR and co-localized with p-EGFR, and pretreatment with lapatinib blocked these effects . Furthermore, we found that EGFR and p-EGFR co-immunoprecipitated with AVM in S2 cells and their interactions were disrupted by lapatinib pretreatment (g). In summary, these observations suggest that AVM interacts with EGFR directly, which induces the activation of the EGFR/AKT/ERK pathway.To explore the mechanism of the activation of the EGFR/AKT/ERK pathway in R larvae and determine whether AVM directly or indirectly activates EGFR, we examined the activation of the EGFR/AKT/ERK pathway in S2 cells exposed to AVM. The treatment of AVM dose-dependently and time-dependently activated the EGFR/AKT/ERK pathway within 1 h a,b. In ad by AVM c. These with AVM d. Moreov effects e,f. Furtreatment g. In sumDrosophila EGFR. We found that the AVM most likely bound to the domain II of EGFR , which is different from EGF [To further confirm the interaction mode of AVM and EGFR, we docked the AVM B1a onto the three-dimensional structures of the ectodomain of from EGF .3.D. melanogaster larvae. In addition, AVM interacts with EGFR directly and activates the EGFR/AKT/ERK signalling pathway in Drosophila epidermal cells.Our study reveals that the EGFR/AKT/ERK/Relish signalling pathway plays pivotal roles in regulating the penetration resistance to AVM in DmeCHS1/2 in AVM-resistant Drosophila larvae compared with that in the susceptible larvae. However, an earlier study showed that AVMs can inhibit chitin synthesis in Mucor miehei and Artemia salina [Drosophila larvae and Mucor miehei may not be the same.This study shows firstly that the decrease of epidermal penetration of AVM is related to the upregulation of a salina . The difDrosophila adults and larvae. The upregulation of the AVM efflux-transporter P-gp in BBB was found to confer the resistance to AVM in Drosophila adults [Drosophila larvae have an immature one [b), whereas P-gp is mainly expressed in the head of adults [There are notable differences in P-gp expression between a adults . Unlike ture one –43. P-gpf adults . Our resDrosophila adults [Identification of the signalling pathway to regulate the overexpression of P-gp and chitin synthases is crucial for illustrating the mechanism of penetration resistance. Our findings are consistent with earlier reports showing that P-gp expression is regulated by the EGFR signalling pathway ,45. The a adults . Howevera adults ,48. ThusκB) was activated by EGFR and induced the overexpression of chitin synthases and P-gp. Relish (NF-κB) can be activated in a wound-healing model of Drosophila larvae [κB can induce the cleavage of Relish and the nuclear translocation of the N-terminal fragment of Relish [2+ signalling activated Relish and the overexpression of P-gp upon AVM treatment [2+ signalling pathway has crosstalk with the EGFR signalling pathway, which merits further investigation.In this study, we discovered that the transcription factor Relish . This immunizing antigen was then injected into rabbits, and the polyclonal antibody was acquired [AVM (containing 93% avermectin B1a and 7% avermectin B1b) was obtained from the ZND Bio-technology Co., Ltd. , and the anti-AVM antibody was a gift from Professor Shen in CAU . The procedures for preparing the anti-AVM antibody were as follows. First, the structure of AVM was modified by succinylation. Then, the 4″-acquired .Lapatinib (EGFR phosphorylation inhibitor) was purchased from Selleck . Wortmanin (phosphoinositide-3-kinase (PI3K) inhibitor) and U0126 (mitogen-activated protein kinase kinase (MEK) inhibitor) were purchased from Kinasechem (UK). The Relish inhibitor pyrrolidinedithiocarbamic acid (PDTC) was purchased from Sigma-Aldrich . The mouse monoclonal anti-P-gp antibody (C219) was purchased from Calbiochem . Anti-p-AKT antibody, anti-p-ERK antibody, anti-AKT antibody and anti-ERK antibody were purchased from Cell Signaling Technology . Anti-EGFR antibody was purchased from Santa Cruz Biotechnology . Anti-p-Tyr antibody was purchased from BD Biosciences . Anti-Relish antibody was purchased from the Developmental Studies Hybridoma Bank .4.2.w1118 strain D. melanogaster was used as the AVM-susceptible strain (S). To obtain the AVM-resistant strain (R), we exposed susceptible D. melanogaster to AVM in standard fly medium prepared according to the method used previously [The eviously . AVM con4.3.Different concentrations of AVM were dissolved in acetone. The third-instar larvae of the same size were chosen. The adults of the same sizes were also used. A drop of AVM solution (0.25 µl) was applied with a hand microapplicator  to the dorsum of the larvae and the abdomen of adults lightly anaesthetized on ice. Control larvae and adults were treated in the same way with acetone. Larvae and adults were fed with fresh medium and kept at 25°C and 70% humidity. For each assay, 50 third-instar larvae and adults were treated, and the whole assay was repeated three times.4.4.50 of Drosophila adults and larvae to AVM was determined by using the topical drug treatment. Each treatment group contains thirty larvae or adults of the same size. Each Drosophila larva or adult was spotted with 0.25 µl different concentrations of AVM and cultured in the standard medium for 24 h. The AVM was dissolved in acetone. The LD50 was calculated according to the mortality of Drosophila at different concentrations of AVM. The resistance ratio was the ratio of the LD50 of the R strain and the LD50 of the S strain.The LD4.5.Drosophila wing epithelial cells were obtained by culturing the imaginal discs of larvae according to the previous report [S2 cell line was a gift from Professor Li . S2 cells were cultured in Hyclone insect medium  at 26°C. The primary s report .4.6.Drosophila larvae were treated with various concentrations of AVM, lapatinib, wortmanin, U0126 and PDTC. The chemicals were dissolved in DMSO to make stock solution and then diluted in double distilled H2O (ddH2O) to make working solutions, which were mixed with standard fly medium . Drosophila larvae, susceptible or resistant to AVM, were transferred from standard medium into chemical-containing medium. After 48 h treatment, the larvae were collected and washed in ddH2O, dried by using absorbent paper and weighed. The larvae samples were then prepared for western blotting analysis or HPLC analysis.4.7.Cells or larvae were lysed in buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1% sodium deoxycholate, 1 mM PMSF and 1% protease inhibitors. Lysates were centrifuged at 12 000 r.p.m., 4°C for 15 min. Supernatants were boiled with the loading buffer for 5 min. The protein samples were electrophoresed in SDS–polyacrylamide gels and then transferred onto Millipore PVDF membranes . The membranes were blocked in PBST buffer containing 5% fat-free milk (w/v) for 1 h at room temperature (RT), incubated with the corresponding antibody at 4°C overnight, then incubated with the secondary antibody conjugated to horseradish peroxidase for 2 h at RT. Membranes were stained with standard ECL reagents purchased from ComWin Biotech  and then photographed by DNR MicroChemi4.2 system .4.8.The larvae tissue frozen sections and S2 cells were fixed in 4% paraformaldehyde for 10 min at RT, blocked in PBST containing 3% BSA for 1 h at 37°C, incubated with the first antibody at 37°C for 1 h, then incubated with the appropriate fluorescent probe-labelled secondary antibody for 1 h at 37°C. The nuclei were stained with Hoechst33258 for 10 min at RT. All images were acquired using a Carl Zeiss LSM710 laser scanning confocal microscope .4.9.DmeCHS1 and DmeCHS2 in Drosophila was determined by quantitative PCR using a TaKaRa SYBR Premix Ex Taq™ (Tli RNaseH Plus) PCR kit . Each group of treated larvae weighing 100 mg was homogenized with 1 ml Trizol reagent  in a glass homogenizer on ice. Then, the mixture was placed at RT for 10 min. 200 µl CHCl3 was added, and the tubes were vortexed for 1 min and centrifuged at 12 000 r.p.m. for 15 min at 4°C. The supernatant was transferred into a new centrifuge tube, mixed with 500 µl isopropanol, vortexed for 1 min and centrifuged at 12 000 r.p.m. for 10 min at 4°C. The precipitant was washed with 70% ethanol twice, dried and dissolved in 0.1% DEPC H2O. The total RNA concentration was measured using a Biophotometer plus . Total RNA (1 µg) was reverse-transcribed into cDNA by using a M-MuLV reverse transcriptase assay kit . Quantitative PCR assay was carried out with a MX3000P real-time PCR thermocycler . The PCR primers used are listed in the electronic supplementary material, table S1.The relative mRNA level of 4.10.4, dehydrated stepwise in ethanol solutions and embedded in resin. After 48 h polymerization at 60°C, the tissues were sliced at 200 or 100 nm thickness. The 200 nm slices were stained by 1% toluidine blue (dissolved in 1% Borax solution) for 1 min and observed using an optical microscope . The 100 nm slices were plated on the copper network, stained with uranyl acetate for 10 min in the dark, washed with PBS three times, stained with lead citrate for 30 min and washed with PBS. The images were acquired using a JEOL-1010 transmission electron microscope .The larvae were dissected in PBS on ice to obtain the larva body wall. The tissues were fixed in the fixative solution containing 4% paraformaldehyde and 2.5% glutaraldehyde for 24 h. Then, the tissues were fixed in 1% OsO4.11.2O, each larva was spotted by 0.25 µl of 10 mM AVM solution and cultured in common corn medium for 2 h. The larvae bodies were washed with acetonitrile three times to obtain the sample for AVM on the surface of the larvae body. Then, the larvae were homogenized with acetonitrile on ice and centrifuged at 12 000 r.p.m. for 15 min at 4°C. The supernatant was used as the sample for AVM that has penetrated into the larvae body.After 1 h starvation in ddHN-methyl imidazole (NMIM)–acetonitrile (ACN) method. First, 10 µl of a mixture of 1-methylimidazole and acetonitrile  was added to the sample vials. Then, 20 µl of a mixture of TFAA and acetonitrile  was added to the sample vials. The sample vials were vortexed for 30 s and derivatized for 15 min in the dark. After the derivatization, 30 µl methanol was added to the sample vials, and then vortexed for 1 min. The AVM residues were detected with the Agilent 1100 series HPLC system . The amount of AVM residue was calculated according to the standard curve. AVM permeability was calculated as the ratio of the amount of internalized AVM and the sum of the amount of internalized AVM and AVM on the surface of the larvae body.All the samples were filtered with 0.22 µm filters, blow-dried with nitrogen and then dissolved in 60 µl acetonitrile. Then the samples and the internalized samples were treated with the trifluoroacetic anhydride (TFAA)–4.12.Drosophila EGFR was carried on the AutoDock. The molecule files were modified for docking by using ADT software [Drosophila EGFR ectodomain is 3I2T. The AVM B1a was chosen as flexible ligand, and EGFR was chosen as rigid receptor. Ten models with the lower binding energy were shown in ADT. The docking model with the lowest binding energy was chosen as a result.The Docking analysis of AVM B1a and software . The PDB4.13.t-tests were used to compare paired data and a one-way analysis of variance followed by Dunnett's test was used for multiple comparisons. Values of p < 0.05 were considered significant; values of p < 0.01 were considered extremely significant.All experiments were repeated at least three times. Data were expressed as the means ± standard error. For statistical analysis, Student's


N = 154) were randomly assigned to play a violent-sexist game, a violent-only game, or a non-violent game. After gameplay, measures of identification with the game character, traditional masculine beliefs, and empathy for female violence victims were assessed. We found that participants’ gender and their identification with the violent male video game character moderated the effects of the exposure to sexist-violent video games on masculine beliefs. Our results supported the prediction that playing violent-sexist video games increases masculine beliefs, which occurred for male  participants who were highly identified with the game character. Masculine beliefs, in turn, negatively predicted empathic feelings for female violence victims. Overall, our study shows who is most affected by the exposure to sexist-violent video games, and why the effects occur. (200 words)Empathy—putting oneself in another’s shoes—has been described as the “social glue” that holds society together. This study investigates how exposure to sexist video games can decrease empathy for female violence victims. We hypothesized that playing violent-sexist video games would increase endorsement of masculine beliefs, especially among participants who highly identify with dominant and aggressive male game characters. We also hypothesized that the endorsement of masculine beliefs would reduce empathy toward female violence victims. Participants ( Not only is empathy hard to outsource and automate, but it makes the world a better place.”—Daniel H. Pink, authorEmpathy is an emotional response that corresponds to the feelings of another person, such as feeling distress when seeing another person in distress. Empathy does indeed make the world “a better place” to live and it is one of the best predictors of prosocial behavior ,2,3. NumGrand Theft Auto (GTA) games, female characters are treated as sex objects rather than as individuals worthy of respect. GTA main male characters are always depicted as hyper-masculine, dominant, and aggressive men. In contrast, the female characters are portrayed as sexual objects—usually prostitutes or pole-dancers—who are peripheral to the game narrative and whose sole purpose is to entertain the main male characters  but not for females [-0.0406 (95% CI = -0.1318 to 0.0097)] who played with a violent-sexist game. No effect was found for males [-0.0486 (95% CI = -0.1733 to 0.0363)] or females [-0.0294 (95% CI = -0.0799 to 0.0041)] in the violent-only game condition. Similarly, no indirect effect of type of video game on empathy was found for males [-0.0874 (95% CI = -0.0134 to 0.3363)] or females [-0.0181 (95% CI = -0.0783 to 0.286)] in the nonviolent game condition. Further, conditional indirect effects of type of video game on empathy for female violence victims were significant only for males in the violent-sexist game condition at values of identification with the game character greater than 4.2583 [-0.1920  is masculine beliefs. Results showed that masculine beliefs were negatively related to empathic feelings for female violence victims. To our knowledge, the present research is the first to elucidate the underlying mechanism that links violent video games playing to desensitization of violence against women.Speaking to the specificity of our effects, it is noteworthy that type of video game played directly affected identification with the game character and masculine beliefs, whereas it did not directly affect empathy for female violence victims. However, we found an indirect conditional effect of violent-sexist games  on empathy, which consistently emerged through the mediation of masculine beliefs and the moderation of identification with the game character. Accordingly, the effects were statistically significant only for highly identified male participants who played the GTA games, which are both violent and sexist. We found no significant effects for violent-only or nonviolent video games on masculine beliefs or empathic feelings. As in previous research , genderThis investigation of virtual representations of males and females in video games is extremely relevant, because video games have distinct features compared to other forms of media  and diffOur study, like all studies, has limitations. Few main limitations stand out. First, we used a self-report measure of empathy. Future research should consider other measures of empathy that are less subject to demand characteristics, such as physiological measures .Second, we examined only two moderators  and only one mediator . Future research should examine other possible moderators  and mediators .Third, our study was based on a short exposure to violent-sexist video games . Although it is impressive that we were able to obtain significant effects after such a brief exposure, we do not know what the consequences would be for longer exposures. If the effects occur after only 25 minutes of play in a laboratory experiment, they are probably magnified after longer periods of play outside the lab. Indeed, individuals usually play video games for much longer periods of time . PrFourth, we did not measure sexist thoughts after a certain amount of time, so we cannot ascertain if and how long the observed effects last. However, previous experimental research has shown that the effects of violent video games can last at least 24 hours after gameplay if players ruminate about the content of the game ,43.GTA is a well-known game, and the simple act of playing it could have primed masculine and sexist thoughts regardless of the actual gameplay. Indeed, media audiences often make their assessment of characters and narratives using existing schemes rather than actual on-screen action/content [GTA. Furthermore, future studies should test whether the empathy reduction linked with violent-sexist video games is specific to female violence victims or whether it extends to male violence victims. Future research should also examine video games with female characters that are not depicted in a sexualized manner.Fifth, /content . Thus, fSixth, we did not actually test whether feelings of empathy mediate aggression against women. As a first step, this study focused on empathy for female violence victims. Future studies should explore whether violent-sexist video games also increase aggression against women.One of the best predictors of aggression against girls and women is lack of empathy . The preS1 Appendixnot at all to 7 = very much; Cronbach α = .79).Participants indicated how much pain they thought the girl was feeling, and how much they thought she was suffering (1 = (DOCX)Click here for additional data file.


The aim of this study was to evaluate the efficacy of slanted recession of the lateral rectus muscle for intermittent exotropia with convergence insufficiency. This prospective study included 31 patients who underwent slanted lateral rectus recession for intermittent exotropia with convergence insufficiency between June 2010 and June 2012. Following parameters were recorded and analyzed: patient sex, age, preoperative and postoperative near and distance ocular alignment, and changes in stereopsis. The mean age of the patients was 9.2 years. The preoperative mean deviation angle was 32.4 PD at distance and 43.4 PD at near. After 6 months, slanted lateral rectus recession reduced the deviation angles to 2 PD at distance and 3.4 PD at near. In addition, the mean difference between distance and near deviation angles was significantly reduced from 11 PD to 1.4 PD at 6 months postoperatively. Slanted lateral rectus recession for intermittent exotropia with convergence insufficiency in children successfully reduced the distance and near exodeviations and the near-distance difference without increasing the risk of long-term postoperative esotropia or diplopia. The convergence-insufficiency type of intermittent exotropia (IXT) is characterized by a greater exodeviation at near than at distance by at least 10 prism diopters (PD) . This tyThe surgical procedures recommended for this type of IXT include bilateral lateral rectus (LR) recession with or without a slanting procedure, bilateral medial rectus (MR) resection with or without a slanting procedure, LR recession with MR resection, or MR resection with an adjustable suture , 5, 7–12Among the surgical procedures, slanted LR recession for the convergence-insufficiency type of IXT, which was introduced by Snir et al. , demonstThis prospective institutional study consisted of patients with an IXT greater at near than at distance by 10 PD or more. Thirty-one patients who underwent bilateral slanted LR recession for IXT with convergence insufficiency between June 2010 and June 2012 were included. All patients had a follow-up duration greater than 6 months. The research was approved by the institutional review board of our institution before this study began and adhered to the Declaration of Helsinki for the protection of human subjects. Informed consent was obtained from the parents of all patients prior to surgery.Patients with associated vertical, torsional, and A-V pattern of strabismus, previous ocular or strabismus surgery, ocular or neurologic pathologic conditions, amblyopia, or a postoperative follow-up duration less than 6 months were excluded from the study. Preoperatively, no patients underwent occlusion therapy or myopic overcorrection or received a prescription for prism eyeglasses.All patients underwent full ophthalmologic and orthoptic evaluations, including best-corrected visual acuity and cycloplegic refraction (with cyclopentolate 1% instilled twice). The distance (6 m) and near (1/3 m) deviations were measured with the alternating prism cover test by using accommodative targets in all the nine diagnostic positions of gaze, while the patients were wearing their best optical correction. Abnormalities induction and version were checked. Torsion was measured with a double Maddox rod test and fundus photograph. A binocular test for fusion and stereopsis was performed for all patients. Fusion was evaluated by the Worth four-dot test at distance and near, and stereopsis was evaluated by the Titmus test at near.All patients were operated on by the same surgeon (BYC). Under general anesthesia, slanted LR recession was performed through a temporal limbal peritomy. In the slanted LR recession, the new insertion was created in an oblique fashion in comparison with the original insertion . The uppAll patients were examined at 1 day, 1 week, 1 month, 3 months, and 6 months postoperatively and at their last visit. The examination included the angles of deviation at distance and near, the extent of the difference between those two deviation angles, and checking whether there was postoperative diplopia, torsion, or A-V pattern of strabismus. The criteria for successful surgery were a postoperative deviation angle at near and distance of less than or equal to 8 PD and a difference between the near and distance deviation angles of less than or equal to 8 PD. The impact of slanted LR recession on the reduction of the near-distance difference was calculated as follows: the mean reduction of near-distance difference in exodeviation after slanted surgery was divided by the mean amount of slanted recession, which was defined as the mean difference between the new upper and lower poles of the slanted LR muscle compared with their original insertions [t-tests were used to compare the preoperative and postoperative angles of deviation at distance and near and the differences between them. Differences were considered significant if the P value was less than 0.05. The statistical analyses were performed using SPSS software .Paired In this prospective study, the study group was composed of 31 patients, including 14 males and 17 females, with a mean age of 9.2 years . The mean duration of follow-up was 9.7 ± 5.5 months  .The study group showed a significant postoperative reduction in the means of both the distance and near exodeviation. The preoperative mean exodeviation was 32.4 ± 4.8 PD  at distance and 43.4 ± 3.2 PD  at near. After 3 months, slanted LR recession reduced the mean exodeviation to 2.2 ± 3.8 PD  at distance and 3.1 ± 4.4 PD  at near, and, after 6 months, the procedure reduced the mean exodeviation to 2.0 ± 4.3 PD  at distance and 3.4 ± 5.0 PD  at near. At 6 months postoperatively, the number of patients with a distance deviation of less than or equal to 8 PD was 28 (90.3%), and the number of patients with a near deviation of less than or equal to 8 PD was 26 (83.9%).P < 0.001)  preoperatively to 1.4 ± 2.2 PD  at 6 months postoperatively (< 0.001) . Each miP < 0.05). At the last follow-up, none of the patients demonstrated abduction limitation, diplopia, consecutive esotropia, torsion, or A-V pattern misalignment.A successful outcome was obtained in 26 of the 31 (83.9%) patients. The 5 patients who did not satisfy the criteria for a successful outcome demonstrated undercorrection of the exodeviation but still showed a postoperative reduction in the difference between the distance and near exodeviations of less than or equal to 8 PD. The mean stereopsis significantly increased from 496.7 arcsec preoperatively to 160 arcsec at 6 months postoperatively  or postoperative diplopia , 14, 15.Among six patients who underwent bilateral MR resections ranging from 4 to 6 mm, von Noorden  found thTo increase the surgical success rate for IXT with convergence insufficiency, Kraft et al.  performeA comparison of our postoperative results with previous reports yields several significant points. First, 26 patients (83.9%) demonstrated residual near and distance exodeviations with less than or equal to 8 PD after slanted LR recession. This success rate of slanted LR recession is relatively superior to the success rates that have been reported using other surgical procedures for IXT with convergence insufficiency, which are between 18% and 76.2% , 12. SecThis study has important limitations, but most stem from its small sample size and the absence of a control group. In addition, children included in this study had relatively smaller near-distance difference of 11 PD than those of other studies (11.3 PD~14.5 PD) , 7–9, 16Interestingly, our results are different from those of van der Meulen-Schot et al. , who didIn conclusion, slanted LR recession for IXT with convergence insufficiency in children successfully reduced the distance and near exodeviations and the near-distance difference without increasing the risk of long-term postoperative esotropia or diplopia.


This paper presents a two-level scheduling scheme for video transmission over downlink orthogonal frequency-division multiple access (OFDMA) networks. It aims to maximize the aggregate quality of the video users subject to the playback delay and resource constraints, by exploiting the multiuser diversity and the video characteristics. The upper level schedules the transmission of video packets among multiple users based on an overall target bit-error-rate (BER), the importance level of packet and resource consumption efficiency factor. Instead, the lower level renders unequal error protection (UEP) in terms of target BER among the scheduled packets by solving a weighted sum distortion minimization problem, where each user weight reflects the total importance level of the packets that has been scheduled for that user. Frequency-selective power is then water-filled over all the assigned subcarriers in order to leverage the potential channel coding gain. Realistic simulation results demonstrate that the proposed scheme significantly outperforms the state-of-the-art scheduling scheme by up to 6.8 dB in terms of peak-signal-to-noise-ratio (PSNR). Further test evaluates the suitability of equal power allocation which is the common assumption in the literature. With the rapid growth of video traffic over wireless networks, mobile operators are expected to support a large number of video subscribers simultaneously. The quality-of-service (QoS) provisioning of these services confronts two major technical hurdles. First, video communication is bandwidth intensive and delay sensitive while the inherent limited wireless resources are shared by multiple users. Second, loss of different video packets induces different amounts of distortion in the received video. When transmitting video over error-prone wireless channels, the impact of channel errors on the video can be extremely severe. To optimize the received video quality, it is essential to allocate radio resources to video packets in an efficient and robust way by taking into account the channel conditions and the video characteristics.Resource allocation (RA) for orthogonal frequency-division multiple access (OFDMA) systems has spurred significant attention from both industry and academia communities due to high spectral efficiency and fine granularity resource control . Conventional RA schemes treat video bitstreams in a content-agnostic fashion . AccordiSeveral recent RA works have explicitly selected video quality as their QoS performance metric. These works can be categorized as content-aware RA methods. The core idea is to determine the importance level of each packet. In , a channDespite the significant gains accrued by the content-aware RA schemes in –11, theiIn this paper, we propose a two-level multiuser video transmission framework which optimizes the packet scheduling and resource allocation. The ultimate goal is to maximize the average video quality of all users under the playback delay and resource constraints. This is achieved by jointly exploiting the multiuser diversity and the importance of each packet. Real encoded video bitstreams with practical modulation and coding schemes are considered. With regard to the coding aspect, a coded orthogonal frequency-division multiplexing (OFDM) system is considered. In general, any OFDM system that combines channel coding and interleaving techniques can be classified as coded OFDM systems. This technique is very effective in combating the burst errors stemming from the OFDM frequency selectivity that compromises the channel coding performance .The main contributions of this paper are as follows. First, most existing RA works treat all the video packets with the same target BER. In contrast, the proposed RA allocates resources in such a way that more (less) important video packets are assigned with stricter (looser) target BERs. This can be viewed as a form of unequal error protection (UEP) which is established for its superior performance in transmission over noisy networks. Second, an overall target BER is considered to control how many average packets can be sent within one transmission interval. As observed in , the traThird, in a practical wireless video transmission system, this paper aims to cast some light on the suitability of EPA, which is the most prevalently used alleviation in the RA literature . In factThe rest of the paper is organized as follows. Section 2 presents the system specifications, models and assumptions. Section 3 describes the two-level scheduling design. Section 4 reports simulation details and results. Section 5 concludes the paper.K different video streams, each to a designated user as depicted in m of user k is stamped with the importance level Qk,m (described later), packet size Rk,m and waiting time Tk,m. These parameters are collected as video content information (VCI). Note that any packet that misses its decoding deadline Tmax will be discarded by the BS.We consider a downlink transmission scenario, where a base station (BS) transmits Ptot and a total bandwidth BT. There are N subcarriers to be shared by all users. Each subcarrier occupies a bandwidth of Bn = BT/N and experiences flat fading under the assumption that Bn is smaller than the coherence bandwidth in a frequency-selective wireless channel. At the receiver, additive white Gaussian noise (AWGN) of power spectral density N0 with mean zero and variance σ2 = N0Bn is present. For user k on subcarrier n, the channel gain and the corresponding channel-to-noise ratio (CNR) are denoted by Hk,n and γk,n = |Hk,n|2/σ2, respectively.All scheduled video sessions are served via a downlink OFDMA system with a total power L OFDMA symbols for payload transmission. Note that although “frame” is a more accurate way to refer to the physical layer service data unit (SDU), we use “subframe” to avoid confusion between MAC and video frames. Then, the achievable transmission bits at subframe t rk,n[t] is expressed asαk,n and sk,n denote realistic coding gap and transmission power allocated on subcarrier n of user k, respectively. If square M-QAM modulation with Gray bit mapping is adopted, αk,n is provided byrk,n[t] should be restricted to integer form due to discrete modulation levels Γk,n which is given byrk,n[t], the number of video bits that can be loaded onto subcarrier n is expressed byc is the coding rate.We assume that the BS has perfect knowledge of channel state information (CSI) and the channel conditions remain static over one time transmission interval (TTI). The TTI corresponds to the time length of a single media access control (MAC) subframe with k,∀n.(2). Note thρk,n as the indicator for subcarrier allocation, where ρk,n = 1 implies that subcarrier n is assigned to user k; otherwise, ρk,n equals to zero. As each subcarrier cannot be shared by multiple users, the binary variable ρk,n should satisfyDenote k be the set of subcarriers assigned to user k. Then, only subcarrier n with ρk,n = 1 is included in Ωk. To ease the notational burden, let overall CNR as a matrix γ with [γ]k,n= γk,n, rate allocation as a matrix r with [r]k,n= rk,n, subcarrier assignment as a matrix ρ with [ρ]k,n = ρk,n, power allocation as a matrix s with [s]k,n = sk,n, and coding gap as a matrix α with [α]k,n = αk,n.Let ΩBased on the video content information and the channel state information, the proposed two-level scheduler (shaded area) makes decision once every subframe. The upper level schedules the transmission of video packets among multiple users based on an overall target bit-error-rate (BER). Instead, the lower level assigns subcarriers to the scheduled users, as well as adjusts modulation and power levels of each subcarrier. The ultimate goal is to maximize the average overall quality of all users while adhering to resource and playback constraints. Once the optimization decision is made, each scheduled user flow undergoes bit-interleaved coded modulation (BICM) in isolation due to its strong forward error protection in frequency-selective fading environments .k are aggregated into a bitstream which will be convolutionally encoded into coded bits and then interleaved across several subcarriers that has been assigned to that corresponding user. The number of interleaved bits that can be loaded onto each subcarrier depends on the calculated modulation constellation size. Second, at the receiver, hard-decision demodulation is performed on the received signal of each subcarrier, followed by de-interleaving in order to assemble the coded bits. Third, the concatenate video bitstream is obtained by passing the coded bits to the Viterbi decoder. Finally, the bitstream is unmerged into video packets where only error-free packets are passed onto the video decoder and corrupted ones are considered lost.Let us now scrutinize the BICM operation in the context of video delivery. First, the selected packets from video Qk,m as the quality increment of decoded video k caused by the transmission of packet m. Mathematically, it can be expressed asm after the transmission of Πk(m−1)≜{πk,1,πk,2,…,πk,m−1}, respectively. τk,m is the indicator for packet scheduling, where τk,m = 1 implies that packet m of user k is scheduled at the current subframe. Otherwise, τk,m = 0. Πk(m−1) denotes the reordered set of packets in user queue k such that πk,1 is the first packet to be transmitted from that queue.Error concealment invokes when there is any lost packet in order to mask the visual artifacts. We choose the copy-from-previous (CP) error concealment method due to its simplicity and efficiency . In partQk,m in f−1  ∈ {1,2,α N} for ∀n4: Sort Ak.5: Using form = 1 to Mk6:   n* such that Jk,m = n*7:   Find end for8:   end for9:  k*,m* = arg maxk,m and set τk*,m* = 110: Find Mk* = Mk*−m*; N = N−{Ik*(1), Ik*(2), …, Ik*}11: Update until no more packet to be streamed or the condition of line 07 is not satisfied for ∀k∀m12: overall target BER may change not only the number of scheduled packets, but also the packet order to be transmitted. This is because Jk,m is associated with the transmission rate that varies accordingly with the overall target BER. Hence, k = μk be the multiplier vector. Then, the problem To solve N ≥ 8, which renders the duality gap negligible + is defined as max. Then, we derive the subcarrier assignment n = 1, …, N, g(μ) is computed from μ* that maximizes μ can be done with the subgradient method in which the subgradient for queue k is defined asg(μ) = ℒ. Then, μ is updated byi is the iteration index, δi is a positive step size which follows diminishing step size policy [g(μ) for queue k at current iteration i. The same iterative procedure is repeated until the subgradients of all queues converge to zero. Finally, μ = μ*allows us to attain near optimal solution ρ* ≈ ρ′ and α* ≈ α′ because the strong duality holds. The complete steps are given in Algorithm 2. Denote Z1 as the number of iterations needed to update To solve e policy , and ΔGkAlgorithm 2: Subcarrier Assignment.i = 1, μi>0, and choose 0 < ϵ ≪ 1 as the stopping criterion.1: Initialization: Set repeat2: ρk,n = 0∀k,∀n.3: Set forn = 1 to N4: fork = 1 to K5:  6:  Compute end for7:  8: Set end for9: i = i+110:k11: Update until 12: b represents an even number starting from b = 2 and stands for the number of bits. bmax is the maximum number of bits and its corresponding k,n= 0 indicates no transmission.So far we assume continuous version of modulation levels. In practice, the level can be only selected from the following set k,n. However, it may not yield good video quality as the Viterbi decoder views the whole subchannel (those subcarriers that have been assigned to user k) as a channel with fluctuating BERs associated with SNRk,n = sk,nγk,n. It is well known that channel coding performs poorly in such environments and frequency-selective power allocation (FPA) can enhance the performance [Based on the objective function in formance .k| comparisons to search for the most power-efficient subcarrier. Once this technique is performed for every scheduled user, all subcarriers’ power is normalized such that Ptot is satisfied. The overall complexity is given as By using the greedy bit loading algorithm in , we assiThe power allocation strategy deserves further attention. So far in the RA literature, EPA is the most commonly used assumption to alleviate the computational complexity. While such simplification enjoys the (near) optimal performance in efficiency-oriented resource allocation policies, this is not the case for fairness-oriented schemes . The reaQk,m estimation requires the expected distortion accumulated up to the previous frame f−1, which is given bym of frame f−1. If the packet is dropped by the BS, k,n and sk,n) by executing the proposed two-level scheduling scheme. From Qk,m of the new frame would react to become larger in order to reflect its urgency in terms of BER protection level.Recall that foreman, carphone, coastguard, silent, mobile and news in QCIF (176 × 144) format are used. The video sequences are encoded in H.264 . For each video, the PSNR as reported below is averaged over 100 frames and over five sets of decoded video corresponding to five random error patterns for each time slot. All results are included in dataset  sequences such as foreman and carphone but also in slow-motion (simple) videos such as news. The packets of complex video are generally more important that those of simple video. Thus, many complex packets are likely to be transmitted where the benefit of protecting these packets via stricter target BER is more visible. On the other hand, when the simple packets are scheduled, they are deemed extremely important since many inter-coded packets rely on them. The effect of error propagation can last for a long period if these packets are distorted. UEP-CA-like schemes recognize this fact by assigning stricter target BERs to them.overall target BER = 10−2. For easeof exposition, we only provide the outcome from the 10th to 90th frame.To gain insight of the performances from another perspective, From th frame for the UEP-CA. There is a steep rise and drop centered at that point. The rising STD prior to the pivotal point (75th~79th) indicates that certain users were either suffering resource starvation or lacking of BER protection at that time. This issue would be compensated by Algorithm 2 where stricter BERs would be assigned to 81st~85th frames of those users.Additionally, a very meaningful phenomenon is found at the STD curves around the 80We have proposed a two-level scheduling framework for transmitting H.264 videos over multiuser coded OFDM systems, by taking into account the channel conditions, the video information and multiple target BERs. The main contribution lies in how UEP of video packets is rendered via subcarrier assignment and power allocation strategies. A novel weighted sum distortion minimization problem was formulated and frequency-selective power allocation was employed. Bit-level simulation results demonstrate that the proposed schemes remarkably outperform the advanced schemes, with the PSNR gain as high as 6.8. Furthermore, via comparison with equal power allocation, we recommend that frequency-selective power allocation is necessary to fully exploit the potential channel coding gain.Applying the natural logarithm on both sides of BERk,n to getWe differentiate After some algebraic operations, αk,n/∂BERk,n) is always positive for the feasible BER range is 0 < BERk,n < 0.02, it is clear that αk,n is an increasing function of BERk,n.As the gradient (∂S1 Dataset(XLSX)Click here for additional data file.


Gmax) on a scanner influence the sensitivity to a range of axon diameters. Multishell high-angular-diffusion-imaging (HARDI) protocols for Gmax of 60, 140, 200, and 300 mT/m were optimized for the pulsed-gradient-spin-echo (PGSE) sequence. Data were acquired on a fixed monkey brain and Monte-Carlo simulations supported the results. Increasing Gmax reduces within-voxel variation of the axon diameter index and improves contrast beyond what is achievable with higher signal-to-noise ratio. Simulations reveal an upper bound on the axon diameter (∼10 μm) that pulsed-gradient-spin-echo measurements are sensitive to, due to a trade-off between short T2 and the long diffusion time needed to probe larger axon diameters. A lower bound (∼2.5 μm) slightly dependent on Gmax was evident, below which axon diameters are identifiable as small, but impossible to differentiate. These results emphasize the key-role of Gmax for enhancing contrast between axon diameter distributions and are, therefore, relevant in general for microstructure imaging methods and highlight the need for increased Gmax on future commercial systems.The ActiveAx technique fits the minimal model of white matter diffusion to diffusion MRI data acquired using optimized protocols that provide orientationally invariant indices of axon diameter and density. We investigated how limitations of the available maximal gradient strength ( A variety of geometrical models that relate the diffusion MRI signal directly to white matter microstructural features, such as axon diameter and fiber density, have been proposed Alexander a′. The authors suggest the mean axon diameter weighted by volume, which they call α, as an interpretation of the axon diameter index, a′. They, moreover, verified that both statistics discriminate naturally arising ADDs and correlate with one another for distributions that lie within a window of axon diameters to which the imaging protocol is sensitive. This window of sensitivity depends on the set of diffusion MRI data acquired and hence the imaging protocol, which is constrained by hardware limitations as well as the sequence used. In particular, the maximum available gradient strength determines the smallest diameters that can be discriminated; the largest diameters that we can estimate reliably depend on the largest diffusion times we can measure, which in turn depend on the relaxation time constants. The protocol may also affect the level of sensitivity to each axon diameter within the window causing deviations of the axon diameter index, a′, from the idealized index, α.A key compromise in ActiveAx compared to the orientationally specific AxCaliber technique Gmax and the pulsed-gradient-spin-echo (PGSE) sequence. Diffusion MRI data were acquired ex vivo on a fixed monkey brain using an experimental MR scanner. Axon diameter index maps for various Gmax were calculated and compared. We also repeated the Monte-Carlo simulation experiments in Ref. Here, we study experimentally the dependence of the axon diameter index on imaging protocols obtained from the active-imaging optimization using various −9 m2 s−1, which is a typical measured apparent diffusion coefficient parallel to the fiber direction in dense white matter at room temperature in our ex vivo sample, and the intracellular fraction was f1 = 0.7 as in Ref. M of distinct b-values found. Typically, the procedure in Ref. M = 3 or 4 unique nonzero b-values. For each unique nonzero b-value, we assigned a HARDI shell, we added a shell with b = 0 s/mm2 and initialized each shell with equal numbers of measurements totaling 360. A greedy search algorithm then found, at each step, the reassignment of one measurement to a new shell that gave the largest increase in the objective function. The process terminated when no reassignment improved the score.Optimized imaging protocols based on the PGSE diffusion-weighted (dw) sequence were generated using the active-imaging optimization and a priori axon diameters of 1, 4, and 20 μm. A priori tissue–water diffusivity was set to 0.45 × 10Gmax constrained to 60, 140, 200, and 300 mT/m was generated. This yielded four protocols, one for each Gmax, which we refer to as ActiveAx060, ActiveAx140, etc. The protocols are shown in Table For ex vivo imaging, a series of optimized imaging protocols with T2, which was set to 50 ms. T1 was 800 ms. We also generated for comparison two further optimized imaging protocols with Gmax fixed to 300 mT/m and artificially higher T2 constants of 400 and 4000 ms  in each protocol using an estimate of One normal young perfusion-fixed Vervet monkey brain (32 months of age) was included in the project and was borrowed for MR scanning from the Montreal Brain Bank Ex vivo diffusion MRI was performed on an experimental 4.7T Varian Imaging System with a bore size of 120 mm and a maximum gradient strength of 400 mT/m with a slew rate of 2000 T/m/s. We used a quadrature volume radio frequency coil and a conventional diffusion weighted PGSE sequence with single-line read out.To ensure a high quality of the diffusion MRI data set acquired, we followed the guidelines for the preparation and MR scanning stages (III and IV) in Ref. Gmax protocols  shown in Table http://dig.drcmr.dk). Common scan parameters were isotropic 0.5 mm voxels, no gap between slices, number of excitations = 1, repetition time = 5600 ms, and sagittal slices. Session I included 20 slices for all Gmax, whereas session II included 30 slices. For all imaging protocols, the middle slice was aligned with the mid-sagittal plane of the brain. Maximal SNR was ensured by minimizing the TE to 67.2, 49.5, 43.0, and 35.9 ms for Gmax of 60, 140, 200, and 300 mT/m, respectively. All data were visually quality inspected. Visual inspection of acquired diffusion weighted MRI datasets revealed that no within session postprocessing was needed to correct for image motion or artifacts before fitting the data to the tissue model.All diffusion MRI datasets for the four optimized a′, from fitting the MMWMD model. For each substrate and each protocol, we obtain noise-free data from the Monte-Carlo simulation and fit a′ 100 times with different independent Rician noise trials with SNR of 20 in the nondiffusion-weighted signal for each protocol. Finally, the experiment was repeated assuming SNR of 20 at TE = 62.7 ms  and adjusting SNR for the shorter TE of the other protocols assuming T2 = 50 ms.The Camino Synthesized data were also generated, in a similar way, for a range of single axon diameters: 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 8.0, and 10 μm but with fixed intracylinder volume fraction of 0.7.We concentrate here on voxels in which we expect a homogeneous fiber orientation. Thus, we used the same exclusion criteria as in Ref. In this study, we focused on three large and highly dense fiber tracts with different orientations: the corpus callosum (CC), left–right oriented, the corticospinal tract (CST), mostly superior–inferior oriented, and the cingulum (CIN), anterior–posterior oriented. Two sets of regions of interest (ROIs) were drawn, one for each session.http://www.fil.ion.ucl.ac.uk/spm/software/spm).The CC was subdivided into 10 regions as described in Ref. SNR was calculated as described in Refs.b-values (M = 3) acquired as HARDI protocols with the number of dw directions (N) distributed in the range 80–106 for all shells and about 70 repetitions of b = 0 s/mm2. The optimized configurations tend to favor more directions at the lowest b-value than at the higher b-values. In practice, however, the difference in angular resolution using 80 or 106 dw directions is minor.The optimized protocols using the PGSE sequence in Table td, and the wavenumber, q, in the optimized protocols show a weak but systematic trend across Gmax: the lowest b-value has a short td and a long 1/q, the medial b-value has a long td and long 1/q, and the largest b-value a long td and short 1/q. Table td and 1/q combinations that emerges when artificially increasing T2 to 400 and 4000 ms. Table q-values as Gmax increases. However, Δ also decreases, so that higher SNR can be expected for higher Gmax as TE decreases. Tables From Table Gmax as shown in Gmax is much stronger than SNR. In both cases, variation for a Gmax of 140 mT/m and higher, gradually decreases as Gmax increases . With the addition of noise the lower bound cannot be identified from variation but is still apparent from the mean values of a′ (blue crosses). Both the lower bound . These large diameters have a strong effect on α but are out of range of the diffusion times in the ActiveAx protocols and therefore do not contribute to a′. a′ in T2  becomes noticeably broader than that for the mid-range diameters . This suggests that, although larger axons do influence the signal enough to recover their diameter, the relatively short diffusion times cause fewer spins to experience the effect of a boundary, and the signal-contrast is weaker than for smaller axons diameters. This same effect causes the deviation of a′ from α that we observe for the data points in a′, are preferentially sensitive to mid-range axon diameters.Gmax of 60, 140, 200, and 300 mT/m, respectively, thus SNR increases as expected with higher Gmax as TE decreases.The SNR measured in CC for sessions I and II is  and  for Gmax shown in Gmax. Regional clusters of different axon diameter indices, especially in splenium, fornix, and anterior commissure, become clear as Gmax increases, especially for Gmax > 140 mT/m. The simulations in a′ at higher Gmax, and it is clearly seen from The voxel-wise axon diameter indices in the midsagittal plane of CC for all b = 0 s/mm2 image in session I, which prevents successful fitting of the model in a few CC voxels; the figure shows those voxels as zero diameter (black), like the background.A small amount of Gibbs ringing occurred in the Gmax settings  plays a key-role in providing useful contrast in diffusion based axon diameter index maps. Our results provide new insights into the performance of current microstructure imaging techniques. Although we have used the ActiveAx technique We show by combining simulations on microstructural substrates and lengthy ex vivo scanning sessions, normally not practically possible, how the available gradient strength  needed to obtain sufficient diffusion weighting  at this low Gmax. This increases TE and thus reduces SNR. However, SNR, which can be increased in practice by repeating measurements or increasing voxel size, is not the most important parameter reducing performance at Gmax = 60 mT/m compared to higher Gmax. A more fundamental limitation is that long pulses prevent short diffusion times thereby reducing sensitivity to smaller axon diameters, reducing anatomical contrast.The variation of the axon diameter index for the lowest Gmax, at least up to the maximum used here (300 mT/m). It seems likely that even higher Gmax will provide further details. The results suggest using a Gmax of at least 140 mT/m whenever possible, as at this gradient strength many anatomical details that are not discernible at lower Gmax will then appear. However, even the lowest Gmax of 60 mT/m  still produces useful contrast, providing unique and specific microstructural differences that are not extractable from diffusion tensor imaging (DTI).Our results suggest always using the highest possible Gmax of 300 mT/m, it can be hard to distinguish, using a′, a particular ADD with α = 3 μm from a particular distribution with α = 5 μm. However, the ADDs used in T2, Gmax, and SNR impact upon a′. A wide range of distributions can have any particular value of α. Other pairs of distributions with α = 3 and 5 μm are distinguishable; the idealized delta distributions (single axon diameters) in a′ for 3 and 5 μm clearly separate. In combination, those results suggest that the width of the ADD plays an important role in the contrast we get from a′: the tighter the distributions, the more discriminating a′. The relatively large variation of a′ in The simulations in Gmax stresses the fact that it is not a fully quantitative measurement. However, the contrast is important even if the absolute values are harder to interpret. The axon diameter index map may be considered an axon-diameter-weighted image rather than a quantitative map of mean axon diameter. Nevertheless, the contrast is a unique and useful tool for studying brain anatomy nondestructively as, for example, the axon diameter index captures the same broad trends in axon size as reported in histological studies The variation of the axon diameter index across Simulations provide unique insight into the window of sensitivity of ActiveAx and other axon diameter imaging techniques that use PGSE. In particular, (i) the existence of a lower bound of axon diameters below which small axon diameters are identified as small but cannot be distinguished from one another; and (ii) the suggestion of an upper bound above which sensitivity is reduced because of insufficient diffusion time.a′, although a wide ADD may also cause departures owing to variation in sensitivity across its support. However, the most significant departures occur when the distribution extends above the upper bound, α is larger than a′, because larger axons have a stronger influence on α than they have on a′ due to the relative short diffusion time.When the ADD is entirely below this upper bound, α usually provides a good interpretation of a′ for any ADD that is more accurate than α by including a modulation by a sensitivity function f over axon diameters in the original expression for α  da = ∫ p (a) da = 1. For α, f is constant. For a′, f(a) appears to approximate the variance of the posterior distribution at a. The results for ActiveAx060 in a′ in The simulation experiments with truncated distributions of axon diameters  highlighT2 of the sample, which can be significantly reduced (30–50 ms) for fixed brain tissue. If we ignore the effects of T2, as in T2 is low.Sensitivity to large diameters is limited by the If equal sensitivity across the range of diameters is important, one might adapt the optimization from Ref. a′ in the monkey brain data are likely to match α. Should we need to increase sensitivity to larger axons, or if T2 is even lower, a stimulated echo sequence is expected to be beneficial, as it enables long diffusion times with short TE. Indeed, some earlier methods In practice, axons with diameter over 10 μm are rare, so the values of Gmax rather than an artifact of the optimization algorithms; note that we included in the optimization of the imaging protocols an a priori axon diameter of 1 μm, which is below all lower bounds. The lower bound arises, because with small axons, the largest possible internal perpendicular displacements are not large enough to cause detectable signal attenuation for any configuration of the PGSE sequence with limited Gmax. As the gradient strength increases, PGSE configurations can be found that produce signal attenuation for smaller axons, because we can combine higher diffusion weighting with shorter diffusion times. However, the lower bound decreases only slowly as Gmax increases. Replacing the rectangular pulses with switching or oscillating gradient waveforms enables diffusion weighting with shorter effective diffusion times Gmax. Recently, Drobnjak et al. Simulations elucidated the existence of a lower bound of a few micrometers below which axon diameters are identified as small but not discriminated. The lower bound is a fundamental consequence of limited T2 is also typically reduced T2 in vivo. However, the lower bound for correctly detecting small axons is likely to be similar in vivo and ex vivo for the same Gmax. The importance of a high Gmax to ensure accuracy and minimal variation of estimation to microstructural details in vivo applies to all scanners  independent of field strength. We hope that the major improvements we demonstrate from increasing Gmax will help expedite the development of stronger gradient systems for commercial human scanners.Here, we assumed that the microstructural environment and its length scales and restrictions are preserved ex vivo Gmax increases, whereas higher SNR is less important. We conclude that a Gmax of 300 mT/m reveals anatomical details not seen at lower Gmax values (even with higher SNR), and one should, therefore, use the highest possible Gmax in these types of studies.The influence of the available gradient field strength on estimated axon diameter indices was investigated for the first time. Fixed monkey brain results show clear improvements in regional contrast in axon diameter index maps as


Malnutrition is a prevalent condition in hospitalized children. Our aims were to evaluate the nutritional state and to validate the STRONGkids risk assessment tool in a hospitalized paediatric population in Romania. This is a prospective single-centre study in a tertiary teaching hospital in Romania (May 2011-January 2012). We calculated the STRONGkids score and measured the children's height and weight. Standard deviation <-2 for weight-for-height and height-for-age was considered to indicate acute or chronic malnutrition respectively. Two hundred seventy-one children were included, with median age of 5.2 years and median hospital stay of 2.01 days. Prevalence of malnutrition and severe malnutrition was 37% and 15% respectively. Using the STRONGkids screening tool, 58% of the children were found at risk of malnutrition (24% were at high risk). The kappa coefficient of agreement between STRONGkids and WHO malnutrition classification was 0.61. When a low serum protein level was used in upgrading STRONGkids risk category, kappa increased significantly to 0.71 (p=0.001). A modified STRONGkids score, incorporating total serum protein levels, performs well in predicting malnutrition in hospitalized paediatric population in Romania. Malnutrition in children results in impaired growth, development, poor health, and overall decreased wellbeing . NutritiManagement and prevention of malnutrition in hospitalized children rely on early identification of those at risk in order to implement early nutritional interventions. For this purpose, several risk assessment tools have been developed ,13-15. TThis nutritional score identifies three risk categories  and correlates well with WHO malnutrition standards . Its appConsecutive children admitted to Targu Mures University Hospital, a tertiary paediatric teaching hospital located in Targu Mures, Romania, were enrolled in this study between 1 May 2011 and 30 January 2012. We calculated the STRONGkids score and measured the height and weight of children admitted to the hospital; z-score [also called standard deviation (SD) score] quantifies the deviation of an individual's value from the median value of a reference population, divided by the standard deviation of the reference population . We used this score to describe how far a measurement stands from the median (average) . Standarth percentile for children of the same age and sex) and those with active malignancy admitted during this time period were excluded.Inclusion criteria were age between 1 and 17 year(s) and duration of hospitalization of at least 24 hours. Obese children .2). Mid-upper arm-circumference (MUAC) in cm and tricipital skinfold (TSF) in cm were also recorded in the first 24 hours of hospital stay. Height and weight were measured according to standard procedures  unrelated to the study. Weight in kg, and height in cm were measured and recorded for all children at the time of admission to the hospital. Body mass index was calculated as weight divided by square of height , Mariakerke, Belgium] for a descriptive analysis of the population and the assessment of the malnutrition risk. Categorical variables were summarized as percentages and compared with Fisher's exact test for two groups and chi-square tests for three or more groups. Continuous variables were presented as medians with interquartile ranges and were compared using the Kruskal-Wallis non-parametric test or the Mann-Whitney two-sample test. Agreement between the STRONGkids and WHO malnutrition class was assessed with the kappa method of inter-rater agreement. A kappa level over 0.7 was considered clinically significantly. The significance level was set at p<0.05.Four hundred and fifteen children were hospitalized between 1 May 2011 and 30 January 2012 at our paediatric hospital; 326 children were found eligible for the study, of whom 271 agreed to participate. Baseline characteristics of the cohort are presented in The median (range) WFH z-score and HFA z-score were 0.1  and −0.34 . The anthropometric parameters of the nutritional subgroups are presented in According to the WHO classification of malnutrition, 100 children (37%) were malnourished, and of them, 41 (15%) had severe malnutrition. 48 (17.71%) had acute malnutrition (WFH z-score <-2 SD), of whom 17 (6.3%) were considered severe, and 37 (13.65%) had chronic malnutrition or stunting (HFA z-score <-2 SD), of whom 24 (8.9%) were considered severe. There were no significant differences in HFA z-score or WFH z-score between genders , 92 (33.9%) and 64 (23.7%) children had a low, moderate and high risk of malnutrition respectively. Those at high risk of malnutrition had lower median BMI, TSF, and MUAC.Acute malnutrition (WFH z-score <-2 SD) was present in 89% of children with scores above 4, and in 10% of those with a score of 3. Chronic malnutrition (HFA z-score <-2 SD) was present in 16.2% of children with a score of 2, in 21.6% of those with a score of 3, and in 57% of those with scores above 4. Only 5.4% of those with score 0 or 1 had chronic malnutrition.The kappa statistic for the correlation between STRONGkids and WHO malnutrition class was 0.61. When presence of a protein level of <6 mg/dL was used in upgrading the risk to the next category in order of severity of the STRONGkids tool assessment, the agreement between the STRONGkids and WHO malnutrition class improved significantly  .Smaller age and a longer duration of hospitalization were both associated with a higher malnutrition score (p=0.0001). As expected, children with underlying gastrointestinal disorders were most likely to have acute malnutrition (p=0.0051).Children with normal nutritional status, who had a low STRONGkids score, had a significantly lower median BMI than those in the normal WHO category (0.7 vs 0.5 respectively) (p=0.0001); however, MUAC, TSF, and serum proteins did not differ significantly.Our study aimed to describe the prevalence of malnutrition in a cohort of hospitalized children at an academic hospital in Romania and to evaluate the ability of the STRONGkids screening tool to identify malnutrition risk in this population. In our study, the prevalence of malnutrition 37%) was significantly higher than that reported by others in both developed countries -7 and so% was sigChildren with a higher risk of malnutrition often have worse outcomes  and longet al.  to the nutritional risk assessment, significantly improved the score's performance in our population. In contrast, the adjustment of cutoff points in the study of Moeeni r STAMP. Our study has several limitations. This is a single-centre experience and may not be representative of the whole Romanian paediatric population; thus, the results may not be extrapolated to other populations without further study. We chose a Caucasian, relatively uniform population from central Romania, without information whether the environment, geographical conditions, or eating habits might influence the results.The test administration and anthropometric measurements were all performed by a single operator, and test reliability might have been improved by addition of another operator. The gold standard for the nutritional evaluation was WHO definition of malnutrition , and theA modified STRONGkids tool, incorporating serum protein levels, could be useful to identify hospitalized children at risk of malnutrition in a tertiary paediatric hospital in Romania. Further research is needed to evaluate whether nutritional risk assessments and targeted nutritional interventions help improve important patient-centred outcomes in hospitalized children.


Mycobacterium ulcerans, the etiologic agent of Buruli ulcer, has been detected on aquatic plants in endemic tropical regions. Here, we tested the effect of several tropical plant extracts on the growth of M. ulcerans and the closely related Mycobacterium marinum. M. ulcerans and M. marinum were inoculated on Middlebrook 7H11 medium with and without extracts from tropical aquatic plants, including Ammannia gracilis, Crinum calamistratum, Echinodorus africanus, Vallisneria nana and Vallisneria torta. Delay of detection of the first colony and the number of colonies at day 7 (M. marinum) or day 16 (M. ulcerans) were used as endpoints. The first M. ulcerans colonies were detected at 8 ± 0 days on control Middlebrook 7H11 medium, 6.34 ± 0.75 days on A. gracilis-enriched medium (p<0.01), 6 ± 1 days on E. africanus- and V. torta-enriched media (p<0.01), 6 ± 0 days on V. nana-enriched medium (p<0.01) and 5.67 ± 0.47 days on C. calamistratum-enriched medium (p<0.01). Furthermore, the number of detected colonies was significantly increased in C. calamistratum- and E. africanus-enriched media at each time point compared to Middlebrook 7H11 (p<0.05). V. nana- and V. torta-enriched media significantly increased the number of detected colonies starting from day 6 and day 10, respectively (p<0.001). At the opposite, A. gracilis-enriched medium significantly decreased the number of detected colonies starting from day 8 PI (p<0.05). In conclusion, some aquatic plant extracts, could be added as adjuvants to the Middlebrook 7H11 medium for the culturing of M. marinum and M. ulcerans. Mycobacterium ulcerans; genomic analysis of M. ulcerans indicated that it emerged from Mycobacterium marinum [M. marinum is an aquatic mycobacterium hosted by ectothermic animals, mainly fish [M. marinum causes skin granulomas, including vivarium granuloma [Buruli Ulcer is the third most common mycobacteriosis in the world . It is r marinum ,3 after  marinum . M. marinly fish ,6. Afterranuloma ,8.M. ulcerans-specific sequences, including IS2404, IS2606 and ketoreductase-B domain of the mycolactone polyketide synthase genes [M. ulcerans is not a routine technique for the diagnosis of Buruli ulcer, as M. ulcerans is a slow-growing organism with a doubling time of more than 30 hours. The reservoir, sources and routes of transmission of M. ulcerans remain elusive, although epidemiological observations suggest stagnant aquatic environments as potential reservoirs [M. ulcerans [M. ulcerans DNA was significantly associated with specific communities of submerged terrestrial plants, in Ghana [M. ulcerans DNA was detected in stagnant water covering decaying organic materials [M. ulcerans isolate has been isolated from an environmental source, and none have been isolated from plants [Gerris collected in Benin, after inoculation of liquid Middlebrook medium, three passages in mouse footpad and subculture on Löwenstein-Jensen medium [The laboratory diagnosis of Buruli ulcer relies on the PCR-based detection of se genes . Isolatiservoirs ,11 and iulcerans . Also, tin Ghana . Recentlaterials . Howeverm plants . Later en medium .M. ulcerans and M. marinum was modulated by aquatic plants prevalent in some Buruli ulcer regions. We grew mycobacteria in Middlebrook 7H11-based medium complemented with plant extracts and monitored the growth of mycobacterial colonies using autofluorescence of bacilli.In this study, we further explored whether the growth of M. marinum clinical isolate [rpoB gene sequence analysis [M. ulcerans ATCC19423T strain Agy99  [M. ulcerans isolate was manipulated in a Biosafety Level 3 laboratory, Medical School, Marseilles, France. After production in broth, cultured mycobacteria were passed three times through a 30-gauge needle in order to disperse aggregates, and mycobacteria were suspended in phosphate buffered saline  in a tube containing glass beads. The tube was vigorously vortexed in order to further separate any bacterial aggregates. The suspension was calibrated at 105 mycobacteria/mL by observing and counting dispersed bacteria after Ziehl-Neelsen staining and by measuring the optical density of the suspension . Inoculum was expressed in mycobacteria / mL.An  isolate  was idenanalysis . M. ulcelineage)  was isolM. marinum and M. ulcerans strain, 100 μL of a 105 mycobacteria/mL bacterial suspension were seeded on a plate containing Middlebrook 7H11 medium, incubated at 32° C. Negative control plates were inoculated with sterile PBS. Two days, four days, eight days and sixteen days after inoculation, colonies were observed both by the naked-eye and using a MZ-FLIII fluorescence microscope  equipped with a GFP filter and an ICA digital camera (Leica) to detect mycobacterial autofluorescence. Counting of fluorescent colonies was performed using the Leica Application Suite software (Leica). Experiments were performed in three replicates.For each M. ulcerans  in presence of Campygen microaerophilic atmosphere generators (Oxoid). Negative control plates inoculated with sterile PBS were incubated in parallel in the ratio of one negative control plate for six inoculated plates. Cultures were observed by autofluorescence and naked-eye every day for seven days for M. marinum and every two days for 16 days for M. ulcerans in order to numerate the colonies. The counting of fluorescent colonies was performed using Leica Application Suite software (Leica). Colonies were identified using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) [M. marinum) or day 16 post-inoculation (M. ulcerans) were used as two endpoints to compare the growth of mycobacteria on Middlebrook 7H11 medium with and without tropical aquatic plant extracts.A total of 19 g of Middlebrook 7H11 agar powder (Becton Dickinson) was mixed with 10 mL of 50% glycerol  and suspended in 885 mL of water. The mixture was autoclaved at 121°C for 10 min, and 5 mL of a 0.22 μm-filtered 200 g/L tryptone solution (Becton Dickinson) and 100 mL OADC supplement were added to obtain 1 L of base medium. Tropical aquatic plants  were selected based on the widespread growth in Buruli ulcer-endemic regions and for requiring growth conditions compatible with those of ulcerans  (S1 File-TOF-MS) . All expp value of <0.05 was considered significant.The Two-Sample Independent t-Test was used to compare delays of positivity and kinetics of growth. A M. marinum, naked-eye and autofluorescence detected no colony at day 2, 10.6±1.5 versus 30±2 colonies at day 4 (P<0.05), and 67.3±2 versus 133±2.64 colonies at day 8 (P<0.05). Colonies were too numerous to be counted at day 16. As for M. ulcerans ATCC 19423T, the same comparison yielded no colonies at day 2 and day 4, 0 versus 35±3.6 colonies at day 8 (P<0.05) and 124±4.16 versus 247.67±5.68 colonies at day 16.Negative control plates remained free of colonies by naked-eye and autofluorescence reading. As for M. marinum and M. ulcerans were observed as spontaneously fluorescent, independently from the medium. The fluorescence system could detect 30 μm-diameter colonies which were undetectable by naked eye. Autofluorescence significantly reduced (p<0.001) the delay of mycobacteria colony detection in Middlebrook 7H11 medium and in all tested vegetal-enriched media  and 2.19 ± 0.37 days on C. calamistratum-enriched medium (p<0.03). All plant extracts significantly decreased the delay of M. ulcerans colony detection compared to Middlebrook 7H11 medium alone. The delay of detection of the first M. ulcerans colony was 8 ± 0 days on Middlebrook 7H11, 6.34 ± 0.75 days on A. gracilis-enriched medium (p<0.01), 6 ± 1 days on E. africanus- and V. torta-enriched media (p<0.01), 6 ± 0 days on V. nana-enriched medium (p<0.01), and 5.67 ± 0.47 days on C. calamistratum-enriched medium (p<0.01).Using autofluorescence, the delay of detection of the first M. marinum, A. gracilis-enriched medium and C. calamistratum-enriched medium significantly increased the number of detected colonies at each time point compared to Middlebrook 7H11 medium (p<0.05). E. africanus-enriched medium significantly increased the number of detected colonies starting from day 5 PI (p<0.05) (For (p<0.05) .C. calamistratum- and E. africanus-enriched media significantly increased the number of detected M. ulcerans colonies at each time point compared to Middlebrook 7H11 medium (p<0.05) . A. gracilis-enriched medium significantly decreased the number of detected colonies starting from day 8 PI (p<0.05).Similarly, (p<0.05) . V. nanaM. marinum and M. ulcerans microcolonies. Autofluorescence has been previously described for Mycobacterium tuberculosis and M. marinum [M. ulcerans. Here, we validated this new method of detection of M. ulcerans microcolonies, following our recent validation for Mycobacterium tuberculosis [M. ulcerans and M. marinum can be detected using fluorescence lighting thanks to the natural autofluorescence of these mycobacteria. The fastidiousness of M. ulcerans culture makes this procedure of particular interest from a diagnostic perspective.In this study, we took advantage of mycobacterial autofluorescence to detect  marinum ,20, but rculosis  due to trculosis ,23. Neverculosis . These dM. ulcerans and M. marinum. C. calamistratum and E. africanus were native to Cameroon before they became endemic in Central Africa, which is the region with the highest number of Buruli ulcer cases in the world [V. nana and V. torta exhibited a significant stimulating effect on the growth of M. ulcerans. V. nana is found in Queensland , and V. torta is found in Japan and southeast Asia, which are regions where Buruli ulcer cases have been described [M. ulcerans. These data agree with the previously reported observation that some aquatic plants stimulate growth and biofilm formation of M. ulcerans [Using this protocol, we observed that two tropical plant extracts significantly stimulated the growth of he world ,25. Likeescribed  or suspeulcerans .A. gracilis even significantly decreased the growth of M. ulcerans.The characterization of growth-stimulating factors contained in the plant extracts was beyond the scope of the present study. These factors are thermoresistant, as the stimulating extracts have been autoclaved prior to their incorporation into the culture medium. These factors are most likely specific, as we observed that the growth kinetics differed between plants, and M. marinum and one isolate of M. ulcerans have been tested, and the data reported here should be regarded as preliminary. However, the similar results obtained from both M. marinum and M. ulcerans suggest that these data may be extrapolated to M. ulcerans at large, as M. ulcerans originated from M. marinum [i.e., environmental ecology and transmission mode(s) of M. ulcerans. These data suggest that C. calamistratum and E. africanus extracts could be used as adjuvants to the Middlebrook medium for cultivating M. marinum and M. ulcerans from environmental and clinical sources.In this study, only one isolate of  marinum . These d marinum , i.e., eS1 File(DOCX)Click here for additional data file.