To examine levels of psychological distress and help seeking behaviour in resettled refugees attending English tuition classes in Australia, and their associations with participants’ demographic characteristics.Data was collected by bilingual interviewers between March and November 2013. A volunteer sample of attendees of Adult Migrant English Programs (AMEP) in Western Sydney were recruited. Participants were two hundred and twenty five Iraqi refugees resettled in Western Sydney, who had left Iraq no earlier than 1991, were fluent in Arabic and/or English, and were between the ages of 18 and 70. The chief outcome measures used were the Kessler Psychological Distress Scale (K-10) as well as The Harvard Trauma Questionnaire (HTQ).χ2 (3) = 8.26, p = .04). Of those participants with clinically significant PTSD symptomatology according to the HTQ, only 32.9% reported ever having ever sought help for a mental health problem.On the K-10, 39.8% of participants had severe psychological distress, 19.4% moderate distress, and 40.7% had low to mild distress. Ninety-five percent of participants reported having experienced one or more potentially traumatic event (PTE) as defined by the HTQ prior to leaving Iraq, with a mean of 14.28 events (SD = 8.69). Thirty-one percent of participants met the threshold (≥2.5) for clinically significant PTSD symptomatology, with a significantly higher occurrence among participants with lower education attainment (The high level of distress found in this sample, combined with low uptake of mental health care, highlights the need for programs targeted to promote help-seeking among Iraqi refugees who have resettled in Australia. Further, the higher level of PTSD symptomatology found amongst those with lower education attainment has mental health promotion and treatment implications. Specifically, in designing service and treatment programs, consideration should be given to the possible impact excessive levels of psychological distress may have on learning in refugees, to ensure that those who have been unable to develop proficiency in the English language receive effective care. Each year Australia offers protection to approximately 13,500 refugees and to date Australia has resettled 700,000 since the end of World War II [ opinion”. This pePsychological distress, in particular Posttraumatic Stress Disorder (PTSD) and symptoms of depression, is known to be common in refugee populations –5. In a After resettlement, the physical and psychological health of refugees presents a new area of concern. Refugees experience a complex set of mental health issues owing to their pre-migration experiences, as well as post-migration cultural dislocation . These mOne group of potentially severely traumatized refugees are those who originate from Iraq. Iraq has been Australia’s primary refugee country of origin since 2008, with 14,378 Iraqi refugees resettled in Australia since this time . The ongAs part of the resettlement process, host nations often provide a variety of services including language tuition for those with low proficiency in the host language. In Australia, Adult Migrant English Program (AMEP) classes are delivered by service providers around Australia in over 250 locations. The program involves attending 510 h of English language training, career counselling and task-based learning aimed at facilitating living and settlement in Australia . AlthougGiven that Iraqi continue to constitute a significant proportion of those seeking asylum, in light of the continuing conflict within Iraq, information about this population’s mental health status and help-seeking behaviour is important for health professionals and for public health administrators. Consequently, the goal of this study was to examine levels of general psychological distress, including PTSD symptomatology, and help seeking behaviour in a volunteer sample of Iraqi refugees attending English tuition classes in Australia. We were also interested to consider associations between mental health and help-seeking and individuals’ demographic characteristics, since associations of this kind might indicate specific targets for health promotion and/or early intervention programs .Participants were resettled Iraqi refugees attending the Adult Migrant English Program (AMEP) at three different colleges located in the cities of Auburn, Fairfield and Liverpool which are in the Western Sydney region of Australia. AMEP is a federally funded English language tuition program that is offered to all migrants and humanitarian entrants to Australia who do not have functional English. Participation entailed the completion of a pen-and-paper, respondent-based survey, conducted on the college campuses by means of in-person interviews. Bilingual investigators attended the colleges between March and November 2013 and informed potential participants of the project through the distribution of flyers. The flyers outlined the aims and nature of the research and indicated that all participants would receive a food gift voucher in the amount of AUD $25.00 upon completion of the survey in appreciation of their time. Written consent was obtained from all participants, following a more detailed description of the survey content, prior to commencement of each interview. Inclusion criteria were having been born in Iraq, having left Iraq no earlier than 1991, being fluent in Arabic and/or English, and being between the ages of 18 and 70 years. Approval for the study was obtained from the University of Western Sydney Human Research Ethics Committee.The survey included, in addition to demographic information and measures of mental health as described below, a “mental health literacy” component in which a vignette was presented of a fictional Iraqi refugee, who had been exposed to trauma prior to leaving Iraq and who was suffering symptoms of PTSD, followed by a series of questions concerning the problem described . One of To assess the general symptoms of anxiety and depression, the Kessler Psychological Distress Scale (K10)  was usedThe Harvard Trauma Questionnaire (HTQ)  is a widStatistical analysis was carried out using IBM SPSS Statistics version 20.0 . ContinuInterviews were conducted with a total of 225 participants over a 10-month data collection period. Their demographic characteristics are shown in Table χ2 (6) = 16.01, p = .01. As shown in Table z = 2.4). There was no association between distress levels and age χ2 (4) = 1.54, p = .82, sex χ2 (4) = 2.08, p = .72, religion χ2 (4) = 4.83, p = .31, or marital status χ2 (6) = 6.12, p = .41.According to the cut-off scores for the K10 used in the Australian National Survey of Mental Health and Well-Being (NSMHWB)  39.8 % oM = 14.28, SD = 8.69). The most commonly reported events were being forced to flee country (81.9 %), being confined to home because of chaos and violence outside (77.9 %), and being oppressed because of ethnicity, religion or sect (69.9 %). The total number of PTEs experienced did not differ significantly by sex, t (222) = 0.75, p = .45), education, F  = 1.88, p = .13, religion, H (2) = 3.46, p = .18, or marital status, F  = 0.51, p = .51. Total PTEs did, however, vary as a function of age, in that older participants (those older than 50 years) experienced more PTEs, F  = 13.98, p < .001.Ninety-five per cent of participants reported having experienced one or more PTE’s prior to leaving Iraq (χ2 (3) = 8.26, p = .04. As noted in Table z = 2.0). There was no association between PTSD symptoms and age, χ2 (2) = .698, p = .71, sex χ2 (2) = 0.52, p = .47, religion χ2 (2) = 0.34, p = .85, or marital status χ2 (3) = 7.88, p = 0.96.Thirty-one per cent of participants met the threshold ≥2.5) for clinically significant PTSD symptomatology according to the HTQ. In comparison, reported rates of PTSD in NSMHW ranged from 6.4 % for 12 months prevalence to 12.2% for lifetime presence, which are between three to five fold less than our sample. There was a significant association between PTSD symptoms and education .5 for clχ2 (2) = 2.80, p = .25, sex χ2 (2) = 0.45, p = .80, religion χ2 (2) = 1.84, p = .40, education χ2 (3) = 1.27, p = .74, marital status χ2 (3) = 6.75, p = .08, or distress level χ2 (2) = 2.61, p = .27. However, there was a significant association between help-seeking behaviour and PTSD symptomology χ2 (1) = 11.26, p < .01. Significantly more participants with probable PTSD than expected sought help for a mental health problem (z = 2.5). Nevertheless, only 32.9 % of participants experiencing PTSD symptomology according to the HTQ reported having ever sought help for a mental health problem. Of those with probable PTSD, the most common type of help sought was from a family member (23.1%), followed by GP (21.5%), psychiatrist (13.8%), psychologist (12.3%) and religious leader (10.8%). It is interesting to note that only 9.2% of participants reported seeking help from specialist trauma and torture mental health services.Nineteen percent of participants reported having ever sought help for a mental health problem, such as the one described in the vignette. Of these, the top four sources for seeking help were family member (27.2 %), psychiatrist (16.1%), GP (14.4 %) and psychologist (13.8%). It is interesting to note that 6.5% of participants sought help from their religious leader or clergyman. There was no association between help-seeking behaviour and age This study sought to report on trauma exposure and associated psychological symptomatology and help seeking in Iraqi refugees who were attending English tuition classes. This refugee population represents one of the largest groups of refugees currently being resettled in Australia and hence their psychological profile is important from a service provision perspective. Our results indicated that more than 90% of participants reported exposure to at least one human rights violation, 39% reported severe psychological distress and 31% had clinically significant symptoms of PTSD. Less than one in four participants (19.5%) reported ever having sought help for a trauma related mental health problem and, of those participants who had sought such help, only a small minority had consulted a mental health professional (13.8%). Such significantly high levels of psychological distress and trauma exposure amongst the participants bears direct relevance in their ability to effectively engage with and participate in the English tuition classes.Although the levels of distress and exposure to trauma observed among participants in the current study are comparable to those observed in other resettled refugee populations , 29, theA secondary aim of the current study was to examine associations between distress and PTSD symptomatology and demographic variables and thereby identify subgroups of the refugee population likely to be most vulnerable. Results of this analysis indicated participants with little or no formal education had particularly high levels of distress and traumatic symptomology. This potentially bears consideration in the development of refugee specific service provision and mental health treatment. Firstly, aside from specialist torture and trauma services, many of those who are involved in the resettlement process in Australia  may have a brief understanding of the refugee process but limited understating of the resultant psychological conditions. As such, these frontline workers are poorly equipped to deal with the psychological distress and emergent mental health needs. We propose that additional training and resources, such as a culturally adapted Mental Health First Aid program, be provided to those who work with refugees thereby promoting early identification and first aid in a timely and culturally appropriate manner. A second consideration bears direct relevance to the type of psychological treatment offered to traumatized refugees. Although trauma-focused cognitive behavioural therapy may usually be considered the most effective treatment for PTSD in most traumatized populations , its effLimitations of the current study should be noted. These include the use of self-report measures of mental health, the inclusion of a measure of general psychological distress rather than separate measures of anxiety and affective disorders, and the collection of only basic information concerning help-seeking behaviour. Strengths of the study, in our view, were the relatively large sample size, which permitted analysis of associations between demographic characteristics and measures of mental health and the administration of the survey instrument by means of in-person interviews. Although participants were volunteers attending the AMEP classes, by recruiting from colleges in western Sydney which has the highest concentration of resettling Iraqi refugees in Australia, an attempt at redress representativeness was made.In sum, Iraqi refugees attending English classes are likely to have very high levels of distress and PTSD symptomatology and very low uptake of mental health care even among severely distressed individuals. Those with minimal education may be a particularly vulnerable subgroup. There is a need for research to identify aspects of mental health literacy likely to be conducive to low or inappropriate help-seeking in resettled refugee populations and to use this information to inform culturally sensitive health promotion and early intervention programs.


Dysregulation of brain-derived neurotrophic factor (BDNF), behavioral inhibition temperament (BI), and small hippocampal volume have been linked to anxiety disorders. Individuals with BI show facilitated acquisition of the classically conditioned eyeblink response (CCER) as compared to non-BI individuals, and a similar pattern is seen in an animal model of BI, the Wistar-Kyoto (WKY) rat. The present study examined the role of hippocampal BDNF in the facilitated delay CCER of WKY rats. Consistent with earlier work, acquisition was facilitated in WKY rats compared to the Sprague Dawley (SD) rats. Facilitated acquisition was associated with increased BDNF, TrkB, and Arc mRNA in the dentate gyrus of SD rats, but learning-induced increases in BDNF and Arc mRNA were significantly smaller in WKY rats. To determine whether reduced hippocampal BDNF in WKY rats was a contributing factor for their facilitated CCER, BDNF or saline infusions were given bilaterally into the dentate gyrus region 1 h prior to training. BDNF infusion did not alter the acquisition of SD rats, but significantly dampened the acquisition of CCER in the WKY rats, such that acquisition was similar to SD rats. Together, these results suggest that inherent differences in the BDNF system play a critical role in the facilitated associative learning exhibited by WKY rats, and potentially individuals with BI. Facilitated associative learning may represent a vulnerability factor in the development of anxiety disorders. Anxiety is the most commonly treated and prescribed for psychiatric condition in today’s society. Determining who is susceptible to developing anxiety disorders and how these vulnerabilities impact treatment efficacy is currently an active area of research. Individual differences play a crucial role in whether a person develops an anxiety disorder or not. Epidemiologic studies indicate that exposure to early childhood trauma and chronic stress increases one’s risk to developing anxiety disorders, whereas a behaviorally inhibited temperament, a small hippocampal volume, and more recently, dysfunction of hippocampal brain-derived neurotrophic factor (BDNF) are associated with inherent vulnerabilities. While various risk or vulnerability factors have been identified, the mechanisms by which they confer vulnerability are still unknown , 2.Brain-derived neurotrophic factor is a neurotrophin that influences cell growth, cell differentiation, and synaptic modification , 4 and iThe link between an abnormal BDNF system and anxiety vulnerability may be through the hippocampus. A small hippocampal volume and impaired hippocampal-dependent learning are likely pre-existing conditions in those that develop PTSD, suggesting that a dysfunctional hippocampus is a vulnerability factor for PTSD , 20. PTSSimilar to humans, BDNF is important for normal function of the hippocampus in animals. A low amount of BDNF is associated with a smaller hippocampal volume . BDNF isBehavioral inhibition is a temperament characterized by withdrawal from and avoidance of novel social and non-social interactions  and is aReflective of altered hippocampal function in behavioral inhibition is the facilitation of non-hippocampal-dependent associative learning in individuals with inhibited temperament. The delay paradigm of classical conditioning of the eyeblink response (CCER) does not require the hippocampus , in contThe WKY rat demonstrates inhibited temperament as evidenced by reduced exploration in the open-field test , 48 and In summary, an impaired BDNF system is a vulnerability factor for anxiety disorders and affects normal hippocampal function. Inhibited temperament is also a vulnerability factor for anxiety disorders and is associated with facilitated acquisition of delay CCER in humans and animals. The present study was conducted to determine whether an impaired hippocampal BDNF system underlies facilitated CCER that is associated with inhibited temperament and anxiety vulnerability.Subjects were male SD and WKY rats obtained from Charles River, Kingston, NY, USA. They were approximately 3 months in age at the time of testing and maintained on a 12-h light/dark cycle with onset of light at 0700 h. All animals were tested during the light phase. Rats were housed individually in standard cages (16.5 in × 8.5 in × 8 in) with ad lib access to food and water and were acclimated upon arrival for at least 5 days prior to experimentation. All experiments were carried out in accordance with the Institutional Animal Care and Use Committee of the East Orange, New Jersey Health Care System, Veterans Affairs Medical Center.Sprague Dawley and WKY rats were anesthetized with Nembutal (50 mg/kg i.p.), and supplemented as necessary. Guide cannulas  were implanted bilaterally  directed at the dentate gyrus region of the hippocampus. Each guide cannula was fixed to skull screws (stainless steel) using dental acrylic cement. A stylet was inserted into the guide cannula to keep the cannula patent.Electrodes were implanted into the periorbital muscles for eyeblink conditioning. Four Teflon-coated, stainless steel wires  had the insulation stripped from one end that was inserted into the muscle. The other end of the wire was inserted into a plastic connector  that was glued to three to four skull screws using dental acrylic. Two wires were used to record electromyography (EMG) and the other two wires delivered electrical stimulation.Following the surgical procedure, sutures were used as needed and rats were post-operatively treated with flunixin meglumine  for 2 days. Rats were allowed at least 4 days to recover from surgery.Eyeblink conditioning was conducted in a sound-attenuated chamber (27 cm × 29 cm × 43 cm) with a viewing window . The EMG signals were recorded from electrodes that were connected to a differential AC amplifier through a cable attached to the plastic connector on the rat’s head. EMG signals were filtered (300–500 Hz) and amplified . Electrical stimulation of the periorbital muscles was delivered by a stimulus isolation unit . A computer equipped with an A/D board and LabView software  controlled stimuli presentation and recording of EMG signals digitized at a sampling rate of 1000 Hz. One day prior to conditioning, freely moving rats were habituated to the apparatus for 30 min. During habituation, EMG signal quality was determined. Rats were conditioned for 1 or 2 days following habituation.Rats were conditioned using a delay conditioning paradigm. Rats received 100 conditional stimulus (CS)-unconditional stimulus (US) paired trials per day. An auditory stimulus  served as the CS. Electrical stimulation of the periorbital muscles  served as the US. CS and US co-terminated. The inter-trial interval (ITI) ranged from 15 to 35 s with an average of 25 s.Electromyography was analyzed to determine the occurrence of eyeblinks using a custom designed script in S-Plus . For each trial, the 250 ms prior to the presentation of the CS was used as a baseline for each trial. An eyeblink, conditioned response (CR), was designated when the EMG activity exceeded a threshold amplitude following the CS onset and prior to the US onset. Threshold amplitude was equal to the mean amplitude of the baseline plus four standard deviations of the baseline activity. Any response recorded during the first 30 ms of the CS onset (250–280 ms) was not counted as a CR, as this time frame typically indicates an orienting response and represents less than 10% of eyeblinks. To evaluate the rate of acquisition, trials were grouped into five blocks of 20 trials per day. Analysis of variance (ANOVA) with repeated measures was used to analyze CR.For animals receiving infusions prior to eyeblink conditioning, an infusion cannula  attached to a Hamilton syringe via polyethylene tubing  was inserted into the guide cannula. Sterile saline (0.5 μl) or rhBDNF  was administered (0.1 μl/min) into the dentate gyrus region of the hippocampus. After drug administration, the infusion cannula was allowed to remain in place for 5 min, and then removed and replaced with a stylet. Infusions were given approximately 45 min (40–50 min range) prior to the start of the eyeblink conditioning session. Saline or BDNF was infused prior sessions 1 and 2 of conditioning.Animals for RT-PCR analysis were sacrificed and the hippocampus was extracted approximately 1 h after Day 1 of eyeblink conditioning. Because BDNF levels fluctuate throughout the day, tissue collection was confined to 3 h after the onset of the light cycle, approximately between 10:00 a.m. and 1:00 p.m. After decapitation and rapid removal of the brain, CA1, CA3, and dentate gyrus regions of both hippocampi were dissected rapidly on ice, placed in microcentrifuge tubes, and stored in dry ice. Net wet tissue weight of the tissue was recorded. Samples were stored at −80°C pending analysis.® containing 3 μl of cDNA, 10 μl Taqman Universal PCR master mix, 1 μl of Taqman probe , 1 μl of Bovine Serum Albumin , and 5 μl dH20.mRNA for BDNF, TrkB (high affinity BDNF receptor), and the immediate early gene Arc (activity-regulated cytoskeleton-associated protein) was measured using RT-PCR. Total RNA was isolated from the dentate gyrus by submerging in Trizol reagent and adding Zirconium disruption beads . Supernatant was further processed and DNase treated as per manufacturer’s instructions . The RNA concentration was quantified using the NanoDrop Spectrophotometer . Total RNA was reverse transcribed by first denaturing 1 μg sample and 1 μl of 300 ng/μL RT primer at 65°C for 5 min and then chilling on ice. Next, 6 μl of 5× Superscript Buffer, 1.5 μl 0.1M DTT, 1.5 μl 10 mM dNTPs, 1 μl Superase In, and 1 μl Superscript III  were added to the samples and incubated at 25°C for 10 min, followed by 45°C for 2 h. The RT reaction was terminated by heating at 70°C for 15 min and the cDNA stored at −20°C. RT-PCR was performed using Roche LightcyclerThe cycle threshold (CT) value was determined for each probe. Data for each target gene were assayed in duplicate and averaged, target values were normalized to the mean of the housekeeping gene 18S ribosomal RNA, which showed the lowest amount of variability across strain and treatment. Fold differences between samples for each gene product were calculated as follows:p < 0.05) and trending (p < 0.1) results are reported.Statistical analyses were conducted using Statistical Package for the Social Sciences . All results were considered significant at α = 0.05. Behavioral data for mRNA analysis were evaluated with a mixed design ANOVA for CR probability with blocks as a within-subject factor and strain as a between-subject factor. Average CR probability was calculated for blocks consisting of 20 trials, resulting in five blocks per session. Behavioral data for BDNF administration had a similar experimental design, but with the addition of treatment as a between subjects factor. mRNA data were analyzed using an ANOVA with strain and conditioning as between subjects factors. Separate analyses were conducted for BDNF, TrkB, and Arc mRNA in each hippocampal subregion. Only significant (n = 7) and WKY (n = 8) rats were trained in one session of delay classical conditioning of the eyeblink response followed by sacrifice for assessment of hippocampal BDNF, TrkB, and Arc mRNA. Due to problems with EMG recording, 1 SD and 2 WKY rats could not be evaluated for behavior; these rats showed clear eyeblink to periorbital electrical stimulation US and should demonstrate classical conditioning similar to other rats. Therefore, all rats were included in the mRNA analysis. Acquisition of classical conditioning was significantly faster and performed to a greater degree in WKY rats compared to SD rats, main effect of strain   = 8.38, p < 0.001]. No interaction between block and strain was observed. Ninety to one hundred and twenty minutes following the conditioning sessions, rats were sacrificed and the hippocampus removed, subdivided, and stored for subsequent analysis by qRT-PCR.Sprague Dawley  = 5.06, p < 0.05]  = 15.4, p < 0.001], and both strains showed learning-induced increases . In CA3, conditioning enhanced BDNF mRNA  with a trend for upregulation in CA1 , but these changes did not differ between strains.In the DG, learning increased BDNF mRNA in SD to a greater extent than WKY, as demonstrated by a strain × conditioning interaction  Figure . BDNF mRF = 8.09, p < 0.01; CA3: F = 10.32, p < 0.005; CA1: F = 6.24, p < 0.05]  = 4.67, p < 0.05]  = 4.94, p < 0.05], strain × conditioning interaction . Arc mRNA did not differ between strains or conditioning groups in the CA1 and CA3 regions.In the DG, classical conditioning upregulated Arc mRNA  Figure . Conditin = 7; SD-BDNF, n = 8; WKY-saline, n = 9; WKY-BDNF, n = 9) were administered and conditioned in two sessions. Only animals that had reliable EMG signals on both days of training were used in the analysis. CRs increased as a consequence of training in all rats for days 1 and 2, main effect of block  and main effect of day   = 12.48, p < 0.001]. Importantly, BDNF infusion into the DG affected WKY but not SD rats, strain × treatment interaction . Neither the main effect of treatment nor the block × strain × treatment interaction was significant.Following CCER, up-regulation of BDNF and Arc mRNA in the DG was blunted in the WKY rats compared to SD rats. Therefore, the effects of administering BDNF into the DG at the time of CCER were evaluated in both strains. Rats  = 8.7, p < 0.01] and treatment × day × block interaction .Given the significant strain × treatment interaction, further analysis was conducted on the effects of BDNF in each strain. In SD rats, BDNF treatment did not alter the acquisition of delay CCER, as neither the main effect nor interactions involving treatment were significant. By contrast, WKY rats were significantly slowed in acquisition by BDNF administration, main effect of treatment [The present study utilized the WKY rat to investigate the role of hippocampal BDNF in the facilitated associative learning that is observed in behaviorally inhibited individuals. The hippocampus was the focus of this study because it contains a high amount of BDNF –8, and dBrain-derived neurotrophic factor is important for hippocampal-dependent learning , 62. WitAn increase in BDNF causes somatodendritic expression of Arc mRNA in the dentate gyrus . Arc, anAlthough WKY rats acquired delay CCER faster and to a greater extent than SD rats, they had smaller increases in BDNF and Arc mRNA than SD rats. Blunted changes in BDNF and Arc mRNA observed in WKY rats can be interpreted as poorer hippocampal function, and is supported by impaired hippocampal synaptic plasticity in WKY rats . Thus, oBrain-derived neurotrophic factor administration enhances various forms of learning and memory . InfusioThe results of the present study provide a potential link between three anxiety vulnerabilities: BDNF dysfunction, small hippocampal volume and impaired function, and behavioral inhibition. BDNF dysfunction can lead to reduced hippocampal volume and impaired hippocampal-dependent learning. In humans, abnormally low levels of BDNF are associated with a smaller hippocampal volume . Howeverand childhood maltreatment (While there is an abundance of evidence associating BDNF and hippocampus volume and function, links between inhibited temperament and BDNF or hippocampal dysfunction has been sparse. Individuals with inhibited temperament have abnormal hippocampal processing of novel stimuli in humans , 81. Intreatment . As descreatment , impairereatment , and pooreatment , 57. Thereatment , 58. ThuIn summary, BDNF dysfunction in the hippocampus was observed in an animal model of behavioral inhibition, the WKY rat. This dysfunction was related to facilitated acquisition of hippocampal-independent associative learning. Gain of function experiments by administering BDNF into the hippocampus of WKY rats “normalized” associative learning. The results suggest a possible mechanism by which hippocampal dysfunction and behavioral inhibition leads to pathological associative learning and vulnerability to develop anxiety disorders.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus aureus. Four combinations  showed SN, SNP + TET showed PS and Indifferent effect (ID) were observed for SNP + NEO against Pseudomonas aeruginosa. SN was observed for SNP + CEF, SNP + GEN, SNP + NEO, SNP + TET and SNP + MUP showed ID, SNP + VAN showed PS against Escherichia coli. In addition, we elucidated the possible mechanism involved in the pharmacodynamic interaction between SNP-topical antibiotics by increased ROS level, membrane damage following protein release, K+ leakage and biofilm inhibition. Thus, our findings support that conjugation of the SNP with topical antibiotics have great potential in the topical formulation when treating complex resistant bacterial infections and where there is a need of more concentration to kill pathogenic bacteria.In order to exploit the potential benefits of antimicrobial combination therapy, we need a better understanding of the circumstances under which pharmacodynamic interactions expected. In this study, Pharmacodynamic interactions between silver nanoparticle (SNP) and topical antibiotics such as Cefazolin (CEF), Mupirocin (MUP), Gentamycin (GEN), Neomycin (NEO), Tetracycline (TET), Vancomycin (VAN) were investigated using the MIC test, Combination assay followed by Fractional Inhibitory concentration Index and Agar well diffusion method. SNP + MUP, SNP + NEO, SNP + VAN combinations showed Synergism (SN) and SNP + CEF, SNP + GEN, SNP + TET showed Partial synergism (PS) against  Pharmacodynamic interactions are those where the effects of one drug are changed by the presence of another drug at its site of action. An interaction is said to occur when the effects of one drug are altered by the co-administration of another drug, herbal medicine, food, drink or other environmental chemical agentsThe use of Silver nanoparticles can be exploited in various fields, particularly medical and pharmaceutical due to their low toxicity to human cells, high thermal stability and low volatility+ leakage and biofilm inhibition could allow for a more careful application of antibiotics that maintain the clinical capability and does not sacrifice the future usefulness of these drugs. To the best of the authors’ knowledge, this is the first report on the possible pharmacodynamic interaction of several topical antibiotics and SNPs.The present study aims to evaluate any pharmacodynamic interactions such as synergistic, additive, antagonistic effect of SNPs when conjugate with the commonly used topical deliverable antibiotics such as Cefazolin (CEF), Mupirocin (MUP), Gentamycin (GEN), Neomycin (NEO), Tetracycline (TET), Vancomycin (VAN) against major causes of wound, burn bacterial infections. In order to exploit the potential benefits of antimicrobial combination therapy, we need a better understanding of the circumstances under which pharmacodynamic interactions expected. In this study, we elucidated pharmacodynamic interactions by increased ROS level, membrane damage following protein release, KPhytophthora infestans. The results of the SNP synthesis and characterization were described in our previous studyThe current study utilizes the SNP synthesized biologically using potato plant pathogenic fungus Staphylococcus aureus ATCC 25922, Pseudomonas aeruginosa ATCC 25619, Escherichia coli ATCC 10536 was evaluated by MIC test  and Gentamycin, Tetracycline activity were also found to be similar level, 1.25 μg/ml against S. aureus ATCC 25922. Mupirocin, Vancomycin found to be more susceptible than other antibiotics tested and neomycin showed no activity against P. aeruginosa ATCC 25619. Cefazolin and Gentamycin showed similar activity, 0.3125 μg/ml followed by Tetracycline and Vancomycin, 0.625 μg/ml against E. coli ATCC 10536 but this strain showed resistant against Mupirocin.Antibacterial susceptibility pattern of SNP and various topical antibiotics against major causes of wound, burn bacterial infection such as MIC test . The MICS. aureus, P. aeruginosa, E. coli were shown in the S. aureus; SNP + MUP, SNP + NEO, SNP + VAN showed synergistic (SN) effect and partially synergism (PS) were observed with SNP + CEF, SNP + GEN and SNP + TET. Interestingly, four combinations  showed synergistic efficacy and one SNP + TET showed PS but the FICI of SNP + NEO were found to be 1.75 μg/ml showed Indifferent (ID) efficacy against P. aeruginosa. Similarly, four combinations such as SNP + CEF, SNP + GEN, SNP + TET, SNP + NEO showed SN efficacy, one combination (SNP + VAN) showed PS and SNP + MUP showed an ID effect against E. coli.Combination assay followed by FIC Index was measured to determine the pharmacodynamic interactions such as synergistic, partial synergistic and antagonistic or additive effects of SNP combined with topical antibiotics. Fractional Inhibitory concentration Index of SNPs and topical antibiotics against S. aureus and ZOI comparison was depicted in the S. aureus and highest ZOI 33, 32 and 31 mm were observed for the SNP + MUP, SNP + VAN and SNP + CEF respectively. In a case of individual or combinations treated against P. aeruginosa, Neomycin (concentration 2.5 μg/ml) alone showed no activity. However, SNP + NEO combination showed 21 mm ZOI and highest ZOI 35, 32 and 31 mm were observed for the SNP + MUP, SNP + GEN and SNP + VAN respectively. The inhibitory effect of individual substance and combination against P. aeruginosa were shown in the E. coli also showed resistance against Mupirocin (concentration 0.625 μg/ml) but the combination SNP + MUP showed 26 mm ZOI and other combinations such as SNP + NEO, SNP + GEN showed ZOI about 36, 33 mm respectively. The results of the individual substance and their combinations against E. coli were depicted in the Pharmacodynamic interactions of SNP combined with topical antibiotics were also evaluated by agar well plate method, Zone of inhibition (ZOI) of SNP or antibiotics alone and the combination were measured and compared with the control. Measurement of ROS generation was utilized to elucidate the pharmacodynamic interaction in this study since the elevated ROS formation leads to the imbalance redox system and can cause oxidative stress, the results of the comparative ROS formation assay were depicted in the + release was carried out to confirm bacterial membrane damage by individual substances and combination tested in this study. All individual substances, including SNP showed significant levels of intracellular K+ generation as compared to the negative and positive control against the strains S. aureus and P. aeruginosa  for the stain E. coli.Measurement of intracellular Kruginosa . But theE. coli followed by 40.33 ± 5.033 for P. aeruginosa and 34.33 ± 9.71 against the strain S. aureus. The inhibitory effect of combinations with antibiotics on the biofilm formation was compared with the SNP alone, the results depicted that all combinations tested showed significant antibiofilm activity (P < 0.05) except the SNP combined with Neomycin (SNP + NEO) when compared to the SNP alone. Interestingly, the equal inhibitory effect was observed for SNP + CEF and SNP + MUP combinations against the strain P. aeruginosa.Antibiofilm assay was performed for SNP alone and combined with topical deliverable antibiotics against three bacterial strains . The resP. aeruginosa cells were observed by SDS-PAGE assay. Lane 1, 10 was loaded with marker, 2 were loaded with SNP and 3, 5, 7, 9, 12 were loaded with individaul antibiotics, lane 4, 6, 8, 11, 13 were loaded with combinations. The results were visualized after SDS-PAGE and staining with coomassie as in P. aeruginosa cells.Membrane damage following intracellular protein caused by SNP, individual antibiotics and combination on P. aeruginosa cells were treated with SNP or combination and control for 4 hrs and visualized in the fluorescence microscopy. P. aeruginosa cells with a damaged membrane showing red color fluorescence that considered to be dead, whereas cells with an intact membrane showing green color fluorescence. The results of LIVE/DEAD BacLight bacterial viability were shown in the The discovery rate of new antibiotics is in decline, while antibiotic resistance in pathogens is rapidly increasing. Drug combinations offer potential strategies for controlling the evolution of drug resistanceS. aureus, P. aeruginosa, E. coli. Shrivastava et al.E. coli showed resistance against SNPS. aureus, Neomycin found to be not effective against P. aeruginosa and Mupiracin also not showed efficacy against E. coli strain. The previous studies stated that, antibacterial activities of few antibiotics were increased in the presence of SNPE. coliThis study elucidates for the first time, pharmacodynamic interactions of SNP – topically deliverable antibiotics against major causes of wound, burn bacterial infections. To examine susceptibility patterns of the SNP, topical antibiotics alone and conjugation with SNP, MIC test was performed against The current study evaluated the entire pharmacodynamic interaction between SNP and topical antibiotics, results of combined assay followed by FIC Index and agar well diffusion assay showed that various degrees of pharmacodynamic interactions such as SN, PS and ID against a common cause of infections in wound, burns. Interestingly, antagonistic (AN) effect was not observed among the combinations against all tested bacterial strains in this study. However, no report was found in the literature showing the antagonistic effect of SNP with antiinfectives. Our study results suggested that the SNP could be conjugated with an anti-infective agents having less or no efficacy, an agents need more concentration to kill pathogenic bacteria and to target both gram-positive/negative resistant bacterial strains causing complex infections.et al.et al.et al.S. aureus, P. aeruginosa, E. coli.In order to elucidate the pharmacodynamic interactions observed in this study, we conducted the comparative ROS formation assay, Comparative measurement of intracellular potassium release, anti-biofilm assay, SDS-PAGE and Microscopic comparison of live/dead cells. Kohanski M. A. et al. have shown that alginate, a polysaccharide of P. aeruginosa biolfim matrix has an important role in P. aeruginosa resistanceBiofilms are microbial communities consisting of cells attached to biotic or abiotic surfaces, embedded in an exopolymeric matrix. These structures are well known for their remarkable resistance to diverse chemical, physical, and biological antimicrobial agents and are one of the main causes of persistent infectionsThis study presented pharmacodynamic interaction between SNP and topical antibiotics such as synergism, partial synergism and indifferent effect against the major cause of wound infections. Moreover, we elucidated the possible mechanism involved in the pharmacodynamic interaction caused by increased ROS level, membrane damage following protein release, K+ leakage and biofilm inhibition. Thus, our findings support that conjugation of the SNP with topical antibiotics have great potential in the topical formulation when treating complex resistance, bacterial infections and where there is a need of more concentration to kill pathogenic bacteria.Staphylococcus aureus ATCC 25922, Pseudomonas aeruginosa ATCC 25619, Escherichia coli ATCC 10536 were obtained from the American Type Culture Collection. LB medium purchased from Oxoid, UK was used for the bacterial cell growth at 37 °C and growth was monitored by measuring the optical density at 620 nm.All antibiotics used in this study  were purchased from Sigma Chem. Co., USA. Phytophthora infestans. Method of synthesis and characterization of the SNP is described in our previous studyThe current study utilizes the SNP synthesized biologically using potato plant pathogenic fungus 6 ml−1; 10 ml each of the serially diluted solutions of the compounds was then added to the bacterial cells. The MIC was defined as the lowest concentration of the agent/drug inhibiting visible growth after overnight incubation at 37 °C.Susceptibility tests with SNPs, Cefazolin, Mupirocin, Gentamycin, Neomycin, Tetracycline, Vancomycin were carried out in 96-well microtitre plates using a standard twofold broth micro dilution method in Mueller Hinton (MH) broth (HiMedia), referring Clinical and Laboratory Standards Institute guidelinesThe MICs of each antibiotic substance alone or in combination were determined by a broth microdilution method in accordance with CLSI standards using cation-adjusted MH broth, modified for a broth microdilution checkerboard procedureTo determine the effectiveness of test substances for synergistic, antagonistic or additive effects, FIC index was measured, using the following formulas6 ml−1 density were treated with each substance or combination so that the final concentration was the MIC or FIC, respectively. Pharmacodynamic interaction, such as synergism, additive, indifferent and antagonistic effect were defined as comparing the zone of inhibition surroundings of well containing topical antibiotic, antibiotic + SNPs and control.Pharmacodynamic interaction was confirmed by agar well plate diffusion method6 ml−1 were treated with each substance or combination so that the final concentration was the MIC or FIC, respectively. Samples were incubated for 1 h at 37 °C. The negative and positive controls were maintained with untreated and 70% ethanol (50 μL/mL) treated cells, respectively. To detect the ROS formation, we used the fluorescent reporter dye 2,7-dichlorodihydrofluorescein diacetate  at a concentration of 5 mM and used a spectrofluorometer (Shimadzu) to measure excitation at a wavelength of 490 nm and emission at 515 nm. The level of reactive oxygen species increase was calculated using the equation: Bacterial cells at a density of 1 × 10Experiments were performed in triplicate and means ± SD were calculatedThe assessment of the potassium ions present in medium was carried out using a luminescence spectrometer (Perkin-Elmer) at an excitation wavelength of 346 nm and an emission wavelength of 505 nm. The cells in exponential phase were obtained, the potassium sensitive probe  was added and treated with a substance or combination followed by incubation at 37 °C for 4 h. The negative and positive controls were maintained with untreated and 70% ethanol (50 μL/mL) treated cells, respectively. The cells were measured following separation of cellular debris by centrifugation at 4000 rpm.6 ml−1 were added in the 96- well polystyrene sterile flat bottom tissue culture plates and incubated for 18 hrs. 10 μl SNP alone or combination so that the final concentration was the MIC or FIC, respectively, and incubated at 37 °C for 6 hrs. The tissue culture plates were washed with sterile phosphate-buffered saline for the removal of floating bacteria and sessile adherent bacteria were fixed using 2% sodium acetate, 0.1% crystal violet stain. Tissue culture plates were washed with deionized water and 95% ethanol was added to the wells after drying. The absorbance of stained adherent bacteria was recorded at 595 nm ELISA reader (Molecular Devices). The % of antibiofilm activity was calculated using the equation: 190 μl bacterial cells at a density of 1 × 10P. aeruginosa cells were observed by SDS-PAGE assay according to the methodThe pharmacodynamic effect of SNP and combination on P. aeruginosa cells were treated with SNP or combination and control for 4 h. Later, the cells were washed, resuspended in sterile phosphate-buffered saline and stained with LIVE/DEAD BacLight bacterial viability kit as per manufacturer’s instructions.The pharmacodynamic effect of the SNP or combination and control was also observed microscopically using Live/dead assay kit (Thermo Fisher Scientific). Three independent experiments were carried out and the results are represented as mean ± SD. The Student’s T test was applied to calculate the statistical significance of the experimental data.How to cite this article: Thirumurugan, G. et al. Elucidating pharmacodynamic interaction of silver nanoparticle - topical deliverable antibiotics. Sci. Rep.6, 29982; doi: 10.1038/srep29982 (2016).


It is widely believed that contact with the natural environment can improve physical and mental health. Urban green spaces may provide city residents with these benefits; however, there is a lack of empirical field research on the health benefits of urban parks.This field experiment was performed in May. Seventeen males aged 21.2 ± 1.7 years (mean ± standard deviation) were instructed to walk predetermined 15-minute courses in an urban park and a nearby city area (control). Heart rate and heart rate variability (HRV) were measured to assess physiological responses. The semantic differential (SD) method, Profile of Mood States (POMS), and State-Trait Anxiety Inventory (STAI) were used to measure psychological responses.Heart rate was significantly lower while walking in the urban park than while walking in the city street. Furthermore, the urban park walk led to higher parasympathetic nervous activity and lower sympathetic nervous activity compared with the walk through the city street. Subjective evaluations were generally in accordance with physiological reactions, and significantly higher scores were observed for the ‘comfortable’, ‘natural’, and ‘relaxed’ parameters following the urban park walk. After the urban park walk, the score for the ‘vigor’ subscale of the POMS was significantly higher, whereas that for negative feelings such as ‘tension-anxiety’ and ‘fatigue’ was significantly lower. The score for the anxiety dimension of the STAI was also significantly lower after the urban park walk.Physiological and psychological results from this field experiment provide evidence for the physiological and psychological benefits of urban green spaces. A brief spring-time walk in an urban park shifted sympathetic/parasympathetic balance and improved mood state. In recent years, the primary focus of healthcare has been shifting from the treatment of disease to health promotion, disease prevention, and improved quality of life. Natural environments such as urban green spaces may provide such benefits, promoting human health and well being. Indeed, many studies have demonstrated a significant positive relationship between exposure to natural environments and physical and mental health. Several questionnaire-based studies reported restorative effects against psychological stressors or mental fatigue -4 and imIn our modern urbanized society, however, opportunities for such interactions with nature are limited. Of late, the potential health benefits of urban green spaces have been studied. Urban green space can enhance the city environment by influencing temperature, wind, humidity, rainfall, soil erosion, flooding, air quality, scenic quality, and plant and animal diversity . In addiMost individuals in industrialized countries live in urban areas and will continue to do so for the foreseeable future ; therefoWe have conducted such a field experiment during winter , but theThis field experiment was conducted in May 2013 in Kashiwanoha Park (hereafter referred to as the urban park) in Kashiwa City, Chiba Prefecture, Japan. A city area around the urban park (hereafter referred to as the city area) was selected as a control site.Seventeen Japanese male university students aged 21.2 ± 1.7 years (mean ± standard deviation) participated in this experiment. Before the experiment, the subjects were fully informed about the aims and procedures involved. After receiving a description of the experiment, the subjects signed an agreement to participate. On the experimental day, alcohol and tobacco were prohibited. This study was performed according to the regulations of the Ethics Committee of the Center for Environment, Health and Field Sciences, Chiba University, Japan.The subjects were instructed to walk predetermined courses through the urban park and the city area. Each course took about 15 minutes to complete. Subjects walked both courses on the same day (at 10 to 12 am and 1 to 3 pm), and the site order was counterbalanced across subjects. On the day of the experiment, the weather was sunny. A 15-minute walk was taken under direct sunlight in the city area at a temperature of 27.0 ± 1.7°C (mean ± standard deviation) and relative humidity of 37.3 ± 8.4% (mean ± standard deviation). In the urban park, the walk was taken under both direct sunlight and shade at a temperature of 24.7 ± 1.6°C and relative humidity of 39.2 ± 5.3%. The trees in the park had light green leaves, and the azaleas were in full bloom  were measured to assess cardiovascular and autonomic nervous system responses. The HRV was measured using a portable electrocardiograph , and frequency spectra were generated using an HRV software tool . For real-time HRV analysis using the maximum entropy method, interbeat (R-R) intervals were obtained continuously. In this study, two broad HRV spectral components were calculated: low frequency  and high frequency . The HF component is an estimate of parasympathetic nervous activity, while the LF/HF ratio is an estimate of sympathetic nervous activity ,30. To nThree different questionnaires were used to investigate psychological responses after walking at each experimental site. The semantic differential (SD) method  used thrt-test was used to compare the mean physiological parameters between the two walking sites. The Wilcoxon signed-rank test was used to analyze differences in psychological indices reported after walking in the two environments. All statistical analyses were performed using SPSS 20.0 . For all cases, P < 0.05 (one sided) was considered statistically significant.A paired P > 0.05); however, all mean heart rate values within one-minute epochs were lower during the urban park walk compared with those during the city walk , city area: 86.1 ± 2.9 bpm; 2, city area: 4.3 ± 0.3 msec2; P > 0.05), while most mean ln(HF) values within one-minute epochs were higher during the urban park walk than during the city walk .In addition, there were significant differences in HRV between the two sites. The mean normalized high-frequency (HF) component (ln(HF)), an estimate of parasympathetic nervous activity, was not significantly different at the start of the walk . Conversely, the positive mood state ‘vigor’ was significantly higher after the urban park walk (P < 0.05). There were no significant differences in the scores for ‘depression’ , ‘anger-hostility’ , and ‘confusion’. Finally, the total STAI score was 14.3% lower after the urban park walk compared with that after the city area walk (urban park: 41.6. ± 7.0 (mean ± standard deviation), city area: 48.6 ± 6.3; P < 0.05; Figure Analysis of responses to three questionnaires completed after the urban park and city area walks, the SD method, the POMS scores, and the STAI scores revealed differences in psychological responses between the two environments. Significantly higher SD scores were observed following the urban park walk compared with those following the city walk for the following three adjectives: ‘comfortable’ , ‘natural’ , and ‘relaxed’ , parasympathetic nervous activity was enhanced (17.1%), and sympathetic nervous activity was suppressed (-20.3%) during a brief walk in the urban park. These results are consistent with those from previous studies on physiological responses to forest settings ,11-15, sAccording to the analysis of the three questionnaires, the subjects in this study felt more ‘comfortable’ , ‘natural’ , ‘relaxed’ , and ‘vigorous’ after a walk in the urban park. In addition, negative emotions and anxiety were significantly lower after the urban park walk. These results on the psychological benefits of walking in an urban park are partly consistent with previous findings ,9-12,15.In a previous study, we examined the effects of urban green space in winter using the same experimental design and locations . The curAlthough we are now living in largely artificial urban environments, our physiological functions evolved in the natural environment ,20,41. TThese findings provide empirical evidence for the physiological and psychological benefits of brief walks in an urban park. However, these results cannot be extrapolated to the female population or to other age groups because only 17 young male adults participated in this study. To generalize the findings, further studies based on larger and more heterogeneous cohorts are required.Identification of differences in physiological states between natural and artificial environments is a critical issue in physiological anthropology, given the increasing disconnect between our evolutionary history and the modern living environment.The physiological and psychological responses elicited by this field experiment provide evidence for the physiological and psychological benefits of urban green space. A brief spring-time walk in an urban park shifted sympathetic/parasympathetic balance and improved mood state.HF: high frequency; HRV: heart rate variability; LF: low frequency; POMS: Profile of Mood States; SD: semantic differential; STAI: State-Trait Anxiety Inventory.The authors declare that they have no competing interests.CS contributed to the experimental design, data acquisition, statistical analysis, interpretation of results, and manuscript preparation. HI and MI contributed to data acquisition and statistical analysis. MM and MT participated in study design and data interpretation. YM contributed to the study design, interpretation of results, and manuscript preparation. All authors contributed to the preparation of this manuscript and are responsible for the final version.


R = 0.17) or body mass index (R = 0.05). The intraclass correlation coefficient for α-angle measurements was 0.84. Sixty-four percent of females in this cohort had an alpha angle >42°. Subtle cam deformity plays a significant role in the pathoanatomy of female patients with symptomatic FAI. As the majority of revision hip arthroscopies are performed due to incomplete cam correction, hip arthroscopists need to be cognizant of and potentially surgically address these subtle lesions.Cam and pincer are two common morphologies responsible for femoroacetabular impingement (FAI). Previous literature has reported that cam deformity is predominantly a male morphology, while being significantly less common in females. Cam morphology is commonly assessed with the alpha angle, measured on radiographs. The purpose of this study is to determine the prevalence of cam morphology utilizing the alpha angle in female subjects diagnosed with symptomatic FAI. All females presenting to the senior author’s clinic diagnosed with symptomatic FAI between December 2006 and January 2013 were retrospectively reviewed. Alpha (α) angles were measured on anteroposterior and lateral  plain radiographs by two blinded physicians, and the largest measured angle was used. Using Gosvig et al.’s classification, alpha angle was characterized as (pathologic > 57°), borderline (51–56°), subtle (46–50°), very subtle (43–45°), or normal (≤42°). Three hundred and ninety-one patients (438 hips) were analyzed (age 36.2 ± 12.3 years). Among the hips included, 35.6% were normal, 14.6% pathologic, 15.1% borderline, 14.6% subtle, and 20.1% very subtle. There was no correlation between alpha angle and patient age ( Femoroacetabular impingement (FAI) is a pathologic condition described by Ganz et al.  in whichCam and pincer morphologies are thought to predominate in men and women, respectively –14. The The prevalence and characterization of cam morphology is increasingly recognized in males. However, it is underrepresented and potentially unrecognized in females. The purpose of this study is to determine the prevalence of cam morphology in non-arthritic females with symptomatic intra-articular hip pain. The study hypothesis is that the prevalence of female cam impingement is higher than typically reported in the orthopedic literature.New female patients presenting to the senior author’s office with a chief complaint of “hip pain” between December 2006 and January 2013 were considered. Inclusion criteria included age under 65 years, Tönnis arthritis grade . The. The9). t-test was performed to compare α-angle and ethnicity and to compare measurements between different radiographic views. Measurements were performed by two senior resident physicians. An intraclass correlation coefficient (ICC) was found between the two sets of measurements. p-Values of <0.05 were considered significant. All statistical tests were performed using SPSS software for Windows, version 13.0 .Pearson’s correlation was used between α-angle measurements, age, and BMI. Student’s 2. Ninety-eight percent were Caucasian. One hundred and ninety-three patients (49.4%) had isolated impingement of the right hip, 151 (38.6%) had isolated left hip impingement, and 47 (12.0%) had bilateral impingement.A total of 969 females were presented to the senior author’s clinic between December 2006 and January 2013 with a chief complaint of “hip pain.” Three hundred and ninety-one patients (438 hips) were eventually diagnosed with FAI and had adequate radiographs for inclusion. The mean age was 36.2 ± 12.3 years . The mean height was 65.3 ± 8.7 in, weight 145.9 ± 37.1 lbs, and BMI 24.0 ± 5.0 kg/mR = 0.17) between patients’ age and size of the cam lesion nor was there a correlation (R = 0.05) between patients’ BMI and size of the cam lesion. There was no difference between the ethnicity of the patient and size of the cam lesion (p = 0.10). Interobserver correlation coefficient for α-angle measurements was 0.84.Table p < 0.001, p = 0.02, and p < 0.001, respectively). The mean angle measured on AP view was less than that of the cross-table lateral (p = 0.09) and significantly less than that of the 90°-Dunn lateral view (p < 0.001). There was no significant difference between the cross-table lateral and 90°-Dunn lateral views (p = 0.36).Table Cam deformities have traditionally been associated with young male athletes, while pincer impingement has been described as a disease of middle-aged women –14. The The notion that cam lesions occur predominantly in young males is supported by recent literature –14, 22. p < 0.001). Beaule et al. also reported smaller cam lesions in symptomatic females compared to males   .The current study’s findings indicate that symptomatic cam FAI may not be restricted to young males. Moreover, we feel that cam impingement should be thoroughly evaluated in all symptomatic females given the consequences of a missed cam deformity, including continued pain and the possibility of additional surgeries. The leading cause of revision FAI surgery is an inadequate cam resection , 29. It The α-angle cut-off of 42° for normal female morphology is based on the classification by Gosvig et al. and Notzli et al. , 21. ThiThis study also highlights significant differences in the α-angle measurements depending on the radiographic view. The frog-leg lateral view detected significantly larger cam deformities than each of the other three radiographic views. Clohisy et al. conducted a level II diagnostic study showing that the frog-leg lateral view provides accurate visualization of the femoral head–neck offset when distinguishing symptomatic FAI patients from asymptomatic controls . Barton To our knowledge, this study represents the largest cohort of symptomatic females evaluated for cam impingement. Radiographs were assessed using a validated system as demonstrated by our high interobserver correlation. Our findings are based on the largest α angles measured from all available radiographs, which minimizes the risk of having missed subtle deformities in different planes. If MRI were available for review, we would have had a greater sensitivity for detecting cam lesions and the prevalence of abnormal α angles may have been even higher than reported.The limitations of this study are related to its retrospective and cross-sectional design. Therefore, no firm causal inferences can be made. Prospectively collected data from long-term follow-up of cohorts with both genders could clarify the clinical relevance of our findings and whether different degrees of cam deformities are associated with an increased risk of symptomatic hip arthritis. This study also lacks a formal evaluation for pincer lesions, so we cannot make an assessment of the prevalence and clinical relevance of mixed FAI presentations.In conclusion, we have found that female patients with symptomatic FAI have a higher prevalence of cam lesions compared to prior reports of asymptomatic females. This may require lower gender-specific radiographic α-angle thresholds to diagnose cam deformities in females. Future studies are required to assess this prospectively and help establish the clinical relevance of these findings.DL: lead author on the study, contributed to all stages of study development. MH: second author, contributed to data analysis and manuscript drafting and editing. JH: third author, contributed to idea of study as well as preliminary data generation and editing of the manuscript. BH: fourth author, contributed to data acquisition ad drafting of manuscript. RF: fifth author, contributed to data analysis and editing of manuscript. SN: sixth author, contributed to study generation and drafting and editing of manuscript.The authors did not receive any outside funding or grants in support of their research for or preparation of this work. Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity.


RT) in a single institute in Taiwan. The complete medical records, medical images, and RT notes of 391 pediatric patients with primary brain tumors treated with cranial RT between January 1975 and December 2005 in Taipei Veterans General Hospital (TVGH), Taiwan, were entered into an electronic registry and reviewed. Eight (2%) cases of post‐RT moyamoya syndrome were identified in the sample of 391 patients. The median latency was 3 years post‐RT. Among the eight patients, three had craniopharyngioma, two had optic glioma, two had medulloblastoma, and one had a suprasellar astrocytoma. The prescribed physical doses of RT were in the range of 40–54 Gy. The incidence was highest in those with optic glioma (0.039/person‐year), followed by craniopharyngioma (0.013/person‐year), astrocytoma (0.003/person‐year), and medulloblastoma (0.002/person‐year). No patients died of vasculopathy. No difference in crude incidence was found between our results and those of other series. The incidence of moyamoya syndrome was diagnosis dependent, with the highest incidence among patients with optic glioma. No regional difference in incidence was found. Long‐term, stable neurological function may be achieved following timely surgical intervention.We aimed to investigate the incidence and long‐term outcome of moyamoya syndrome in pediatric patients with primary brain tumors after receiving cranial radiotherapy ( Radiotherapy (RT) improves local control and overall survival in pediatric patients with brain tumors. However, cranial irradiation also increases the risk of stroke in cancer survivors. The estimated relative risk of stroke or transient ischemic attack (TIA) in pediatric cancer survivors who have received cranial RT was 8, with an incidence of 548/100,000 person‐years Taipei Veterans General Hospital (TVGH) has been dedicated to the treatment of pediatric patients with brain tumors for more than 30 years P‐values less than 0.05 were considered significant. This study was approved by the hospital's institutional review board.A post‐RT stroke or TIA occurred in 13 of the 391 patients listed in Table Among the 13 patients with post‐RT stroke or TIA, nine were found to have moyamoya vasculopathy. One patient, a 5‐year‐old girl with optic glioma and neurofibromatosis type‐1 (NF1) was found with moyamoya vasculopathy before radiotherapy based on a postoperative MRI and was thus excluded. The remaining eight patients had received angiography for suspected moyamoya syndrome. Their clinical characteristics are listed in Table The total follow‐up person‐years and number of patients with moyamoya vasculopathy by disease were, respectively, optic glioma , craniopharyngioma , astrocytoma , and medulloblastoma . The incidence for each type of brain tumor after receiving RT, in person‐years, was thus 0.039 for optic glioma, 0.013 for craniopharyngioma, 0.003 for astrocytoma, and 0.002 for medulloblastoma.P = 0.002) in cumulative incidence of moyamoya vasculopathy in patients with different types of brain tumor after RT. The cumulative incidence of this condition by disease at 7 years was: optic glioma, 40% (standard error: 21.9%); craniopharyngioma, 14.8% (7.9%); astrocytoma, 6.2% (6.1%); and medulloblastoma, 0  for optic glioma, 1/23 (4.3%) for craniopharyngioma, and 1/65 (1.5%) for medulloblastoma The crude incidence of post‐RT moyamoya syndrome in pediatric patients with craniopharyngioma was found to be 2/15 (13.3%) in a Korean series Published data suggests that surgical revascularization is a safe intervention for pediatric moyamoya syndrome, and most treated patients derive some symptomatic benefit Only 69.3% of patients receiving radiation for brain tumors had complete notes and follow‐up imaging, potentially compromising the results. Only those patients with stroke or TIA were assessed for moyamoya syndrome, potentially omitting early cases, though one case was identified on routine imaging. Radiotherapy dose to the vasculature was not estimated, as we could not reliably estimate the scattered radiation dose from the two‐dimensional Cobalt‐60 plan.The incidence of post‐RT moyamoya syndrome in pediatric patients in Taiwan with each type of primary brain tumor was estimated. The incidence differed among diagnoses, with the highest incidence noted in optic glioma. The crude incidence was not different from those reported in two other series, from the United States and Korea. The clinical outcome is usually favorable. Stable long‐term neurological function may be achieved after timely surgical intervention.None declared.


P = 0.055). A HRP–2/pLDH positive RDT was associated with an increased risk of severe malaria–related anemia compared to a negative RDT (adjusted odds ratio (AOR) 18.8, 95% CI 4.32 to 82.0, P < 0.001) and to a HRP–2 only RDT . There was no significant association between RDT result and the administration of parenteral quinine. These results offer preliminary evidence that specific patterns of antigen positivity on RDTs could be utilized to identify patients at an increased risk for complications of severe malaria.The detection of severe malaria in resource–constrained settings is often difficult because of requirements for laboratory infrastructure and/or clinical expertise. The aim of this study, therefore, was to explore the utility of a multiple antigen (HRP–2/pLDH) rapid diagnostic test (RDT) as a low–cost, surrogate marker of patients at high risk for complications of severe malaria. We reviewed programmatic data at a peripheral health center in Western Uganda. Available demographic and clinical data on all individuals presenting to the center who underwent an RDT for suspected malaria infection were reviewed. We fit logistic regression models to identify correlates of two outcomes of interest: 1) severe malaria–related anemia, defined here as hemoglobin ≤7g/dL and 2) receipt of parenteral quinine. 1509 patients underwent malaria testing with an SD FK60 RDT during the observation period. A total of 637 (42%) RDTs were positive for at least one species of malaria, of which 326 (51%) exhibited a single HRP–2 band and 307 (48%) exhibited both HRP–2 and pLDH bands, while 4 exhibited only a single pLDH band. There was a trend towards more severe anemia in patients with a HRP–2/pLDH positive RDT compared to a HRP–2 only RDT (β = –0.99 g/dl, 95% CI –1.99 to 0.02,  P. falciparum malaria, and approximately 550 000 die from manifestations of severe disease [Each year millions of African children are infected with  disease . In area disease . While d disease -5, effec disease ,7. Morta disease -11, and  disease ,13.A major challenge to the identification of severe malaria stems from the rigorous laboratory–based diagnostic criteria ,14,15. HPlasmodium species infections. Additionally, these RDTs have shown some ability to provide a quantitative estimate of parasite density due to the differing sensitivities of the respective antigens [Rapid diagnostic tests (RDTs) are low–cost, simple tools for the diagnosis of malaria and are particularly valuable in settings where microscopy is not readily available -21. Neweantigens -24. BecaWe evaluated programmatic data during the period January–March 2013, collected from the Bugoye Level III Health Center (BHC), in the Kasese District of Western Uganda . The health center functions as a referral center for the Bugoye sub–county, and serves a rural population of approximately 50 000 residents. The center houses a small laboratory with staff trained to perform light microscopy and RDTs, as well other basic tests, such as hemoglobin estimation.The objective of this retrospective, observational pilot study was to assess the diagnostic utility of a three–band (HRP–2/pLDH) RDT at a resource–limited, rural health center in malaria–endemic Western Uganda.We reviewed data on all individuals with fever who underwent an RDT for suspected malaria infection. We abstracted data on patient age, sex, village of residence, patient disposition, laboratory results, and medication prescriptions from clinic registers.P. falciparum/Pan RDT . The RDT is a validated antigen detection test with three individual bands signifying the control, the HRP–2, and the pLDH antigens [P. falciparum, whereas a unique pLDH line indicates infection with one or more of the other Plasmodium species. The presence of a positive HRP–2 line together with a pLDH line indicates an infection with either P. falciparum or a mixed–species infection.Laboratory diagnosis of malaria was made using the Standard Diagnostics FK60 Malaria Ag antigens ,25,26. TRDTs (Lot #090192) for the study were obtained from Kampala Pharmaceutical Industries , and stored in their original packaging at room temperature. RDTs were performed and interpreted by two members of the BHC laboratory staff in accordance with the manufacturer’s instructions. In cases in which the control line did not appear, the result was considered invalid and the test was repeated. Faint HRP–2 and pLDH test lines were considered positive.When available, we reviewed matched thick and thin smears for specimens with three–band positive tests. Smears were prepared using a modified Field’s Stain and examined by light microscopy . AsexualData were entered into Microsoft Excel  and analyzed with Stata 12.1 . We summarized patient characteristics and compared those with two– and three–band positive RDTs using Pearson chi–squared testing. We fit univariable logistic regression models to identify correlates for two outcomes of interest: 1) severe anemia, defined as a hemoglobin <7 g/deciliter (g/dL) and 2) receipt of parenteral quinine. While the WHO traditionally define severe malaria-related anemia a normocytic anemia of Hb <5 g/dL in children and <7 g/dL in adults, we used hemoglobin values of <7 g/dL as the clinically relevant outcome as it represents the transfusion threshold for severe malaria in adults and approaches the threshold at which transfusion should be considered in children when present with other clinical features .P–value of <0.25 [P–value of <0.05 was considered statistically significant in the final models.Our primary explanatory variable of interest was RDT test result, defined as HRP–2 vs HRP–2/pLDH positive. Secondary explanatory variables included sex, age, village distance from the health center, village elevation, and transmission season, defined as high (January) or low . We fit multivariable models including all variables that were significant in univariable models with a pre–specified of <0.25 . A resulEthical approval for study procedures and data collection was provided by the institutional review boards of Partners Healthcare and the Mbarara University of Science and Technology. Informed consent was not required by the ethical review committees due to the programmatic nature of the project.During the observation period, 1509 patients underwent malaria testing with an RDT. A total of 637 RDTs (42%) were positive for malaria. Of the positive RDTs, 326 (51.2%) exhibited a single HRP–2 band, 307 RDTs (48.2%) exhibited both HRP–2 and pLDH bands, while only 4 RDTs (0.6%) exhibited a single pLDH band. Characteristics of the study sample, stratified by RDT result, are summarized in Notably, the prevalence rate of HRP–2/pLDH positive results was twice as high among patients <15 years compared to those ≥15 years. The absolute number and proportion of HRP–2/pLDH positive RDTs declined during the transition from high to low transmission seasons, while that of HRP–2 only positive RDTs remained relatively stable.P. falciparum mono–infections, although there were 4 mixed–species infections . In addition, there were two non–P. falciparum mono–infections , which may have represented sub–patent P. falciparum co–infection not seen on microscopy. All four of the pLDH–only positive RDTs were identified as P. ovale mono–infections. Parasite density was reported for 89 of 90 of the three–band positive individuals. The mean density was 73 105 parasites/μL with a median density of 40 200 parasites/μL (IQR = 12 100–103 238). Parasite densities were only available for three patients with HRP–2 positive RDTs.Of the 307 patients with HRP–2/pLDH positive RDTs, 90 (29.3%) had corresponding blood smears available for interpretation. The majority  of smears from patients with HRP–2/pLDH positive RDT results demonstrated P. falciparum infections (including mixed–species infections) was 99.1% (95% CI 94.5% to 99.9%). The specificity was 75% (95% CI 53.0% to 89.4%), although only a small number of smears were prepared in patients with paired HRP–2 positive (n = 19) and negative (n = 19) RDTs. If, however, we assume that the two RDTs that were three–band positive but negative on microscopy represented true P. falciparum infections, as seen when confirmed by PCR in other studies [The sensitivity for  studies ,32, the P = 0.055, During the observation period, a total of 85 paired hemoglobin (Hb) levels were assessed in patients also undergoing an SD FK60 RDT. Children <5 years of age were more likely to have a Hb performed and had a higher proportion of values <7 g/dL (56.4% vs 26.1%) compared to those individuals ≥5 years of age. The mean Hb was 7.3g/dL (95% CI 6.6 to 8.1) in patients with a HRP–2 only positive RDT, and 6.3g/dL (95% CI 5.6 to 7.1) in patients with a three–band positive RDT  with a trend toward statistical significance.In multivariate logistic regression models, a HRP–2/pLDH positive RDT was the strongest predictor of severe anemia  of these patients. There were no differences in rates of inpatient admission between those with HRP–2 only and HRP–2/pLDH positive RDTs. Patients with HRP–2/pLDH positive results were more likely to receive treatment with parenteral quinine and anti–pyretics. In the multivariate analysis for correlates of receipt of parenteral quinine, the strongest associations were seen with three–band positive RDT and infection during high transmission season, although neither of these reached statistical significance (Our results show a trend towards patients with HRP–2/pLDH positive RDTs having more severe malaria–related anemia that those patients with an HRP–2 only positive RDT. While previous work with the SD FK60 in a reference laboratory found a correlation between parasite density and the presence of the pLDH band , our finWe postulate that the positive pLDH band, which is less sensitive than HRP–2, is a marker of higher parasite density -35. FindAn alternate explanation for the relationship could be HRP–2 antibody persistence leading to false–positive HRP–2 positive RDTs. Prior studies in Uganda have reported HRP–2 band specificities as low as 62% . These fP. falciparum malaria exceeding 95% [However, our clinical experience in Bugoye suggests that very few patients seek treatment for malaria outside of the government health center, given difficult terrain and high costs associated with private facilities. In settings where there is a low probability of prior treatment, such as our site, the specificity of the HRP–2 antigen is typically higher than that associated with studies conducted at urban referral centers ,41,42. Ading 95% .Our study, which was hypothesis generating in nature and primarily involved a retrospective review of routinely collected data, has a number of limitations. Foremost among these is the lack of available paired slides for all specimens. The available slides and Hb results were prepared in a non–random manner at the direction of the supervising clinical officer. We did, however, review smears for nearly 30% of those patients with three–band positive RDTs and Hb values for nearly 15% of patients with positive RDTs. The second major limitation of our study was that the use of the Sahlis method for Hb estimation, which while simple and inexpensive, can be prone to dilution and reading errors. We believe there should be minimal bias stemming from this method because errors would be stochastic and a single staff member interpreted all results. Finally, we performed this study at a single site and with a relatively small sample size, which reinforces the need for corroboration of these findings in larger, and more diverse patient populations. A follow up, prospective study is now under way.In summary, our findings suggest that patients with HRP–2/pLDH positive RDTs have more severe malaria–related anemia than patients with HRP–2 only positive RDTs. We postulate that the positive pLDH band, which is less sensitive than HRP–2, is a marker of higher parasite density and correspondingly, more severe anemia. These results offer preliminary evidence that multiple antigen RDTs could be utilized to identify patients at an increased risk for complications of severe malaria. If confirmed, multiple antigen RDTs may play a role in case management algorithms at peripheral health facilities. Further studies with more comprehensive data collection methods, larger sample sizes, and longer timeframes should be pursued before this approach is adopted.


Lipid droplets (LDs) act as intracellular storage organelles in most types of cells and are principally involved in energy homeostasis and lipid metabolism. However, the role of LDs in resistance to toxins in fungi remains largely unknown. Here, we show that the trapping of endogenous toxins by LDs is a self-resistance mechanism in the toxin producer, while absorbing external lipophilic toxins is a resistance mechanism in the toxin recipient that acts to quench the production of reactive oxygen species. We found that an endolichenic fungus that generates phototoxic perylenequinones (PQs) trapped the PQs inside LDs. Using a model that incorporates the fungicidal action of hypocrellin A (HA), a PQ derivative, we showed that yeast cells escaped killing by trapping toxins inside LDs. Furthermore, LD-deficient mutants were hypersusceptible to HA-mediated phototoxins and other fungicides. Our study identified a previously unrecognised function of LDs in fungi that has implications for our understanding of environmental adaptation strategies for fungi and antifungal drug discovery. Microorganisms have evolved diverse strategies to adapt to various environmental stresses1245678101314th century18192021Lipid droplets (LDs) are dynamic intracellular organelles that contain neutral lipids, including triacylglycerols (TAGs) and sterol esters (SEs), as their main constituents. These are surrounded by a phospholipid monolayer and specific proteins, and they are ubiquitously present in eukaryotic and prokaryotic cells16Phaeosphaeria sp., are taken up into LDs following exposure to light irradiation. This confers resistance in the producer to these phototoxins. Using a PQ derivative, hypocrellin A (HA), as a molecular probe, we found that trapping HA inside LDs resulted in reduced phototoxicity against both the pathogenic fungus Candida albicans and the yeast model Saccharomyces cerevisiae, which suggests a function for this exquisite organelle in drug resistance. In addition, mutants deficient in LDs were found to be more hypersusceptible to HA-mediated phototoxins and other fungicidal agents. We believe that this LD-mediated resistance mechanism may provide valuable insights into how self-resistance to endogenous toxins is conferred to toxin producers and how resistance to external toxins occurs in other recipients.Here, we show that phototoxic perylenequinones (PQs), which are biosynthesised by the endolichenic fungus Phaeosphaeria sp., from the lichen Heterodermia obscurata (Nyl.) Trevis  Trevis , led to Phaeosphaeria sp., which produces the phototoxins, was isolated and purified (1O2), which results in phototoxicity2526The perylenequinonoid skeleton of PQs makes fluorescent microscopic analysis a convenient method by which to study these compounds. Examination by confocal laser scanning microscopy (CLSM) showed that the thallus of the lichen was composed of filaments and algae cells . Stereospurified . When thpurified . We obsepurified . By contpurified . BecausePhaeosphaeria sp. and displays potent antifungal, antibacterial and antivirus activities25272829in vitro study. Because it is a hydrophobic agent, HA has a very high octanol-water partition coefficient (KOW) with a Log P greater than 4, and it is easily dissolved in lipophilic solvents. When HA was dissolved in different solvents of variable polarity or in solvents containing triglycerides to mimic LD components, light-driven 1O2 production by HA was proportional to the polarity of the solvents , LRO1 (orf19.6018) and DGA2 (orf19.6941) in C. albicans, which share sequence identity with their S. cerevisiae orthologues of 45.54%, 55.00% and 32.99%, respectively, were analysed to determine their roles in LD formation. The mRNA levels of ARE2, DGA1 and LRO1 were upregulated in response to oleate stimulation, suggesting a possible function for these genes in driving LD formation  and sterol esters (SEs)—are known to be the major constituents of LDsormation . We thenbination . CLSM obform LDs , consistadiation .S. cerevisiaeare1Δ are2Δ) or SE synthesis (dga1Δ lro1Δ) had a smaller effect on sensitivity to HA-mediated phototoxicity. However, the quadruple mutant strain (dga1Δ lro1 Δare1Δ are2Δ), which does not form neutral lipids, was highly susceptible to the photodynamic inactivation of HA   . Based oC. albicans cells cultured in YPD or YPDO medium to clinically-used antifungal agents37C. albicans cells with large LDs were resistant to farnesol compared with cells with small LDs, possibly due to differences in intracellular distribution  or neutral lipids (RSY3077) (Further testing of er drugs . MoreoveRSY3077) . These dPhaeosphaeria sp., that biosynthesizes phototoxic PQs, we found that the toxins were trapped inside LDs in the producer cells  trevis) was collected from Yunnan province, People’s Republic of China. The lichen thallus was sterilised and cut transversely or longitudinally into sections for CLSM observation. Longitudinal z-stack observations were acquired using 30 confocal sections that covered the entire depth of the lichen. PQs produced by the endolichenic fungus identified as Phaeosphaeria spPhaeosphaeria sp. was cultured on potato dextrose agar (PDA) plates in dark or under light irradiation. Each day, the growth of the colonies was recorded by imaging. After 7 days, a sample of the organisms was scraped from the colonies under light irradiation and suspended in PBS solution for CLSM observation with a Zeiss LSM700 confocal microscope. The samples were also stained with BODIPY at a final concentration of 1 μg/ml for confocal microscopic observations. BODIPY and PQs were excited by 488 nm and 555 nm lasers, respectively, and recorded in an emission spectrum of 490–550 nm for green fluorescence and 570–700 nm for red fluorescence.Lichen . The decreased percentage was calculated to indicate the production of 1O2 generated by HA.The amount of The octanol-water partition coefficients of HA were determined according to a previously described methodSaccharomyces cerevisiae wild-type strain BY4742 and the LD-defective strains RSY3290, RSY1826, and RSY3077 were cultured in YPD medium  at 30 °C with rotational shaking at 200 rpm. C. albicans wild type strains were cultured in YPD, YPDO medium ) or SD medium . C. albicans mutant strains were grown in YPD medium supplemented with the necessary auxotrophic requirements, including 80 mg/L uridine, 20 mg/L histidine, and 40 mg/L arginine. The genotypes of the yeast strains used are listed in The The mutant strains deficient in LDs formation were generated in BWP17 using PCR deletion cassettes, as described previouslyC. albicans or S. cerevisiae strains were cultured in YPD medium at 30 °C overnight. Then, 1 ml aliquots of yeast suspensions (2 × 106 CFUs/ml) in YPD medium were incubated in glass tubes with HA at final concentrations ranging from 0.125 to 4 μg/ml. The samples were placed under a 9 W fluorescent lamp with an irradiation intensity of 10 W/m2, which was detected using a photometer. Aliquots of 100 μl were removed at the indicated times and spread on YPD plates to determine the surviving CFUs.C. albicans SC5314 was grown in SD medium overnight. Cells were collected and stained with BODIPY 493/503 (5 μM) or CMAC (5 μM) for 30 minutes at room temperature (RT) to determine the localisation of LDs and vacuoles. Prestained cells were washed and incubated with HA (2 μg/ml) in the dark at room temperature (RT) for 10 min. To stain the nucleus, cells were pre-incubated with HA (2 μg/ml) in the dark at RT for 10 min and then fixed with 70% ethanol. Fixed cells were stained with 2.5 μg/ml of 4′, 6-diamidino-2-phenylindole (DAPI) to indicate the location of nuclei. Stained cells were washed and then observed by CLSM. Argon lasers at 405 nm, 488 nm and 555 nm were used to excite CMAC/DAPI, BODIPY, and HA, respectively.C. albicans cells, dansyl-labelled amphotericin B (AmB-Ds) was synthesised according to a previously reported methodC. albicans SC5314 cells were treated with 30 μg/ml of AmB-Ds for 12 h. Cells were then collected and washed following with PI (10 μg/ml) staining for CLSM observation. AmB-Ds and PI were excited by 405 nm and 555 nm, respectively, and recorded in an emission spectrum of 430–530 nm for blue fluorescence and 590–700 nm for red fluorescence.To observe the distribution of amphotericin B in C. albicans SC5314 was cultured overnight in YPD or YPDO medium. The collected cells were incubated with HA (2 μg/ml) for 2 min in YPD medium containing SYTOX at a final concentration of 5 μM to observe cell death. Then, cells were exposed to light irradiation with a maximum intensity under a Zeiss Confocal Laser Scanning Microscope LSM700. We manually imaged cell states at 1 or 2 min intervals.C. albicans SC5314 cells were cultured in YPD medium or YPDO medium at 30 °C overnight. Cells were then collected and adjusted to 2 × 105 CFUs/ml with YPD or YPDO medium. C. albicans mutant strains and LD formation-defective S. cerevisiae strains were cultured in YPD medium. They were tested for their susceptibility to antifungal agents including fluconazole (FLC), caspofungin (CAS), terbinafine (Ter), miconazole (Mic), amphotericin B (AmB) and farnesol. After 6 or 12 h of treatment, the cells were serially diluted and spotted onto YPD agar plates. After 48 h of incubation at 30 °C, the plates were imaged, and the inhibitory effect was determined by colony counting.C. albicans SC5314 was cultured in YPD or YPDO medium overnight for RNA extractions. Measurement of the relative quantitative expression of target genes was conducted using an Eppendorf Mastercycler Real-time PCR System−ΔΔCT. The primers used for ARE2, DGA2, LRO1 and ACT1 in this test are listed in ACT1 was used as an internal reference gene.Cells were stained with BODIPY (5 μM) for 30 min or with Nile Red  for 5 minute, two probes of indicating LDs, at room temperature and washed twice with PBS for confocal microscopic observation. The cells were also subjected to a FACScan flow cytometer  with excitation at 488 nm to measure fluorescence intensity, which was indicative of LD size. The resultant data were processed with WinMDI 2.9 software.C. albicans SC5314 or S. cerevisiae BY4742 was cultured overnight in YPD or YPDO medium. For LD mutants, cells were cultured in YPD overnight. Then, cells were collected and diluted to 106 cells/ml and incubated with HA in YPD medium for 2 min. The C. albicans cells were exposed to 10 min of light irradiation, and the S. cerevisiae cells were exposed to 20 min of light irradiation. Then, cells were collected by centrifugation and stained with 40 μg/ml DCFHDA. The loaded DCFHDA was converted into the membrane-impermeable agent DCFH by intracellular esterase. DCFH is rapidly oxidised by ROS into its fluorescent derivative, DCF. The green fluorescence intensity of DCF was used to indicate the amount of ROS production. The stained samples were subject to flow cytometry detection or confocal microscopic observation with excitation at 488 nm and emission in the range of 510–560 nm to measure the ROS levels.C. albicans cells cultured in YPD or YPDO medium. Other data were analysed by Student’s t test. Statistical results are presented as the means ± SD. Asterisks indicate critical levels of significance .One-way ANOVA with the Bonferroni test was used to compare the survival time of How to cite this article: Chang, W. et al. Trapping toxins within lipid droplets is a resistance mechanism in fungi. Sci. Rep.5, 15133; doi: 10.1038/srep15133 (2015).


Progne subis). By attaching altitude loggers to nesting Purple Martins and collecting prey delivered to their nestlings, we determined the flight altitudes of ants and other insects. We then tested hypotheses relating ant body size and reproductive ecology to flight altitude. Purple Martins flew up to 1,889 meters above ground, and nestling provisioning trips ranged up to 922 meters. Insect communities were structured by body size such that species of all sizes flew near the ground but only light insects flew to the highest altitudes. Ant maximum flight altitudes decreased by 60% from the lightest to the heaviest species. Winged sexuals of social insects  dominated the Purple Martin diet, making up 88% of prey individuals and 45% of prey biomass. By transferring energy from terrestrial to aerial food webs, mating swarms of social insects play a substantial role in aerial ecosystems. Although we focus on Purple Martins and ants, our combined logger and diet method could be applied to a range of aerial organisms.The atmosphere is populated by a diverse array of dispersing insects and their predators. We studied aerial insect communities by tracking the foraging altitudes of an avian insectivore, the Purple Martin ( Most terrestrial animal species15789101213Ants, for example, are one of the most abundant and influential animal groups in terrestrial environments1516182015222426272829The abundance and diversity of ants, their value as prey for aerial predators, and their occasional spread across non-native ranges as exotic species78921121Progne subis), North America’s largest swallow, is an ideal predator for sampling aerial insect communities. Purple Martins are abundant and widespread in the United States and Canada, and eat a wide variety of insect prey, including ants, which they capture during flight37383943The Purple Martin , from 27 May to 15 June 2014. These dates fall within the mating seasons of Purple MartinsTo measure the foraging altitudes of Purple Martins, we attached altitude loggers to 25 nesting adults . Each logger consisted of a BMP180 barometric pressure and temperature sensor , a PIC12F683 microprocessor , an RX8564 real time clock , a 24AA256 EEPROM memory module , and an MS621FE manganese lithium rechargeable battery . To protect the barometric pressure sensor from moisture and light, we covered the aperture in the package with adhesive-backed Gore-tex. The loggers were coated with epoxy, leaving the Gore-tex and two programming tabs exposed. The programming tabs were used to set the clock and initiate data logging, and were then cut off with ceramic shears just prior to attaching the logger to a bird. We trapped birds at their nest and mounted the loggers on them using a harness made of 0.7 mm ©Stretch Magic elastic cord and crimp beads. A combined logger and harness weighed ~1.2 grams, about 2.4% of the average breeding Purple Martin body weighth = Purple Martin altitude (m), hb = airport altitude (m), R* = the universal gas constant (8.31432 N · m/(K·mol)), P = logger pressure (Pa), Pb = airport pressure (Pa), g = standard gravitational acceleration (9.80665 m/s2), and M = the molar mass of air (0.0289644 kg/mol).where We thus obtained for each Purple Martin a continuous profile of altitude by time throughout the logging period . Peaks iTenebrio molitor) in place of their original prey, re-fastened their neck collars and returned them to the nest. Collected prey items were later sorted and counted. Voucher specimens of each prey type were pinned and identified. We treated male and female ants separately because they are sexually dimorphic, the females weighing from 4 to 26 times as much as males  to 315,690 s (87.7 hrs), with an average of 156,167  s or 43.4 (±20.4) hrs. We collected prey from 311 nestling provisioning trips, 86 of which were matched to altitude data. Fifteen of the 25 birds yielded no altitudinal foraging data, 9 because of partial or complete logger failure and 6 because they did not provision their nestlings during the observation period. The 10 birds with both altitude and foraging data provided a median of 3.5 altitude-logged foraging trips per bird (interquartile range 1.5 to 12.0), with a maximum of 26 trips.P = 0.91, n = 4 females and 6 males, female median 3.5 trips, interquartile range 2.5 to 9.5, male 6.5 trips, interquartile range 1.5 to 12.0), maximum flight heights , maximum foraging heights , or median foraging heights .We detected no sex differences in data collection or flight behavior and pooled the data. Males and females did not differ in the number of altitude-logged foraging trips (K-W n = 83). As would be expected, foraging trips to higher altitudes lasted longer (duration (s) = 410.8 + 1.3462 * max height (m), n = 83, r2 = 0.32, P = 2.1 × 10−8).Purple Martins flew to an average maximum height per bird of 849 ± 362 m, with an overall maximum of 1,889 m in one male. The maximum heights of nestling provisioning trips ranged from 7 to 922 m above ground . One outlier, however, the cornfield ant Lasius neoniger, is likely a female-calling species and does not engage in high altitude mating flights. Queens of two closely related species, L. alienus and L. niger, fly short distances only a few meters above ground and land to choose mates, copulate, and walk to new nest sites49Lasius neoniger probably exhibits similar behavior and was unique in our study in being both light and low-flying, found only below ~22 m. All other species included in the analysis belong to species or genera known to engage in aerial mating flights  and Temnothorax sp. queens—flew over 100 m above ground whereas heavy carpenter ant queens (Camponotus pennsylvanicus) flew under 40 m. A similar relationship held for median flight altitudes . The pattern was not just driven by the outlying weight of the heaviest species (C. pennsylvanicus), as we got similar results after ln-transforming body weights so they were normally distributed .The 86 altitude-logged foraging trips yielded 56 prey species  and 153 prey by altitude records, captured at up to 922 m above ground. We weighed vouchers of 43 of those 56 prey types. As predicted, flight altitudes of prey species were constrained by body weight, such that low altitudes were populated by species of any weight, but only light species were captured at the highest altitudes . Ants weWe collected 83 prey species  were collected rarely or within a single altitude .