We advocate that antibiotic resistance be reframed as a disaster risk management problem. Antibiotic-resistant infections represent a risk to life as significant as other commonly occurring natural disasters . Despite efforts by global health authorities, antibiotic resistance continues to escalate. Therefore, new approaches and expertise are needed to manage the issue. In this perspective we: (1) make a call for the emergency management community to recognize the antibiotic resistance risk and join in addressing this problem; (2) suggest using the risk management process to help tackle antibiotic resistance; (3) show why this approach has value and why it is different to existing approaches; and (4) identify public perception of antibiotic resistance as an important issue that warrants exploration. On the 1st May 2014 the World Health Organization (WHO)  programs, thereby addressing this issue from a “service” model perspective more akin to disease management or quality improvement processes  to manage the risk associated with bacterial epidemics and pandemics is antibiotics  to get everybody on to the aircraft and safely away as quickly as possible. However, for the Indigenous people, complex socio-cultural relationships meant that it was not possible for “this person” and “that person” to be sat next to each other openly in the small space of an aircraft interior. This would “poison” relationships within the community that would have long lasting and devastating impacts. Consequently, the Indigenous people refused to comply with evacuation orders. As a result, a “bottom-up,” negotiated arrangement was developed that satisfied the complex socio-cultural needs of the target stakeholder community. The answer was as simple as hanging a sheet inside the aircraft that divided space into safe and respected compartments (Veland et al., Examples like the one just described show how when policies and procedures are negotiated from the bottom-up they are owned by the communities to which they are targeted. More often than not, they are successful. The reverse is also the case and policy failure frequently occurs due to poor community consultation. If we consider that antibiotic resistance is a slow-onset disaster then to prevent future policy failures around antibiotic prescription and use, consultation with diverse communities and tailoring solutions specific to those communities will be crucial. For example, education programs about antibiotic use may need to be tailored to specific communities socio-cultural and behavioral contexts, and in languages other than English, since the factors driving antibiotic demand are likely to be diverse.Another key defining element of the ERMP is that emphasis is placed on empowering individuals, families, communities or any other stakeholder, to take responsibility for the risks they face. In this context, once again, the emergency management approach offers a novel perspective where rather than having governments and health professionals take full responsibility, the general public will need to take personal responsibility for the risk they face from antibiotic resistance—once of course they understand that risk and their own role in enabling, or limiting that risk. Microbiologists understand that selection pressure from antibiotic overuse results in increased resistance and that minimizing use is crucial to slowing down the rate of resistance spread. However, how to implement this change in stakeholders is the challenge. Antibiotic resistance is not just a microbial problem—it is also a socio-cultural and behavioral problem. In the context of the issue of the emergence of antibiotic resistance and its effect on the risk profile of epidemics and pandemics, poor stakeholder risk perception underpins inappropriate behaviors, such as patients pressuring clinicians by demanding antibiotics for viral infections, or patients' non-adherence to courses of antibiotics (Michael et al., The academic and practitioner fields of disaster risk management have developed an extensive body of literature on how poor or low stakeholder perceptions of risks and disasters led to inapproriate and poor behavior Bird, . DetermiIn light of this, we propose shifting the discourse to one of increased multiple stakeholder engagement and awareness, thereby rebranding of the issue of antibiotic resistance away from one exclusively of “general health, medicine, and molecular microbiology” to one of “disaster risk reduction” (Dominey-Howes et al., 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.


This study aimed to explore patients’ utilization of repeat prescriptions for antibiotics indicated in upper respiratory tract infections (URTI). An emphasis was placed on whether the current system of repeat prescriptions contributes to patients self-diagnosing infections and if so, identify the common reasons for this.This is a prospective study of self-reported use of repeat antibiotic prescriptions by pharmacy consumers presenting with repeat prescriptions for antibiotics commonly indicated in URTIs. Data were collected via self-completed surveys in Perth metropolitan pharmacies.A total of 123 respondents participated in this study from 19 Perth metropolitan pharmacies. Of the respondents, approximately a third of them (33.9%) presented to the pharmacy to fill their antibiotic repeat prescription one month or more from the time the original prescription was written . Over two thirds of respondents indicated to not have consulted their doctor prior to presenting to the pharmacy to have their antibiotic repeat prescription dispensed (i.e. 68.3%). The most common reasons for this were that their ‘doctor had told them to take the second course’ (38%), followed by potential self-diagnosis (29%), i.e. ‘they had the same symptoms as the last time they took the antibiotics’. Approximately one third (33.1%) of respondents indicated they ‘were not told what the repeat prescription was needed for’ when they were originally prescribed the antibiotic. Respondents who presented to fill their repeat prescription more than 2 weeks after the original prescription written were more likely not have consulted their doctor  and not to know why their repeat was needed .Findings of this study suggested that the current 12 month validity of antibiotics repeat prescriptions is potentially contributing to patients’ self-diagnosis of URTIs and therefore potential misuse of antibiotics. This may be contributing to the rise of antimicrobial resistance. The study also outlines some common reasons for patients potentially self-diagnosing URTIs when using repeat prescriptions. Larger Australian studies are needed to confirm these findings. The emergence of antibiotic resistance poses a local and global threat, with significant repercussions for public health . There iAmongst developed countries, Australia has one of the highest levels of antibiotic use in the world . Under tElectronic prescribing systems in the Australian general practice setting are often defaulted to automatically generate repeats on prescription medications, including antibiotics ,7. The iThere is currently a paucity of literature data surrounding patient utilization of antibiotic repeat prescriptions. It is also not clear what happens in cases when patients delay filling of their antibiotic repeats originally intended for finishing their treatment course following initial diagnosis. Having this in mind, the primary objective of this study was to explore whether the current system of repeat prescribing for antibiotics commonly used in upper respiratory tract infections (URTI) contributes to patients self-diagnosing. Secondary objectives included identifying common reasons for patients self-diagnosing when choosing to use a repeat prescription.Ethics approval was obtained from Curtin University Human Research Ethics Committee.www.randomizer.org), a 20% random sample of pharmacies were selected. Random selection was utilized to maximize representativeness and validity of the study and to allow a wider representation of the population of metropolitan pharmacies in Australia. A total of 67  were invited to take part in the study via telephone calls and posted letters. Pharmacist managers, who were provided with the information surrounding this study, gave verbal consent to take part in the study. The consenting pharmacist provided an information sheet, as well as a self-completed questionnaire to consenting respondents who presented at participating pharmacies with an antibiotic repeat prescription. Data was collected between February and May 2012.This was a prospective study in which data was collected using a questionnaire administered in randomly selected pharmacies in Perth. A list of all Perth Metropolitan pharmacies was generated using the Yellow pages and, using an electronic randomizer  at any time of the day, seven days a week. This was specified in order to minimize the potential for inconsistencies with data collection between pharmacies and minimize the risk of extraneous variables skewing results, such as certain populations (e.g. elderly patients) presenting at particular times of the day/week compared to other patient groups.Data collected was entered into SPSS® Vs. 19. Frequency distributions were obtained and data was compared for differences and associations. Relationships between categorical variables were explored using Chi-square test and a p-value of <0.05 was regarded statistically significant. In cases when numbers in cross-tabulation cells were less than five a Fisher’s exact test was used. Confidence intervals were calculated for prevalence estimates.A total of 25 pharmacies were recruited out of the 67 contacted pharmacies. Out of 25 pharmacies who initially accepted to participate, six of them did not collect any data. Contacted pharmacies that chose not to take part in the study claimed not to have had the time (41.2%), staff (35.3%) or prescription volume (23.5%) required to participate in the research project.A total of 123 surveys were completed throughout the collection period. The majority of respondents were female (67%), working full time (41.8%) and with a general concessional PBS status. The majority of respondents were filling a repeat prescription for themselves or their child (84.4%). More detailed demographic data for the respondents are presented in Table The majority of prescriptions were computer generated 82.6%) and the most common antibiotic prescribed was amoxycillin with clavulanic acid (40.4%) followed by amoxicillin alone (21.1%). The vast majority of antibiotics (88%) were prescribed with one repeat. Of the respondents, 40% of them presented to the pharmacy to fill antibiotic prescription more than 2 weeks after the initial fill date and 33.9% filled their repeat one month or more from the time diagnosis was made. More details about the repeat prescriptions are available in Table .6% and tRespondents indicated that the most common reasons for which the antibiotic repeats were being used (as perceived by respondents) were ‘cough’ (34.5%), followed by ‘not feeling better after the first course’ (19.0%) ‘earache’ (17.2%) and ‘sore throat’ (16.4%). These were followed by ‘blocked nose’ (12.1%), ‘runny nose’ (9.5%) and ‘starting to get sick and not wanting to get worse’ (7.8%).The most common reported original indications (as perceived by respondents) for the antibiotic prescriptions were ‘chest infection’ 37.8%), followed by ‘ear infection’ (20.2%), and ‘tonsillitis’ (14.3%). More details in relation to antibiotics original indications as reported by respondents are provided in Table 7.8%, folThe prevalence of respondents that indicated that no doctor consultation was made prior to presenting to the pharmacy to fill their antibiotic repeat prescription was 68.3% (95% CI: 60.0 - 76.6%). Most respondents indicated that they did not consult a doctor prior to having the antibiotic repeat dispensed because their ‘doctor had told them to take the second course’. This was followed by ‘having the same symptoms as the last time they took the antibiotics’. For more details on reasons for having their repeat filled refer to Figure A significant association was identified between respondents who reported original indications for ‘the flu’ and ‘ear infection’ and their doctor consultation. In this regard, the majority of respondents did not consult a doctor prior to having the repeat dispensed when the antibiotic was prescribed for ‘the flu’  or for an ‘ear infection’ . In regards to educational level, respondents with a university level education were more likely not to have consulted a doctor prior to presenting to have the repeat dispensed (Fisher’s exact test: p = 0.019). No significance differences were found between the above demographic factors and whether respondents presented to fill their prescription within 2 weeks of original prescription written (p > 0.05).Nearly 40% of respondents indicated they did not to know why the repeat prescription was needed ( i.e. 33.1%) of respondents indicated they were ‘not told by their doctor what the repeat prescription was needed for’ when they were originally prescribed the antibiotics and a further 5.8% responded they ‘could not remember’ why the repeat was given).A significant association was identified between timing of original prescriptions prescribed and repeat prescriptions dispensed, in relation to respondents’ knowledge regarding use of their repeat. In this regard, respondents who presented to fill the repeat more than 2 weeks after the original prescription was prescribed, were significantly more likely not to know why their repeat was needed ).This study found that the majority of respondents did not consult their doctor prior to presenting to the pharmacy to have their antibiotic repeat dispensed, despite a significant proportion not knowing what the repeat prescription had been provided. Further, 40% of respondents presented to fill the repeat prescription more than two weeks after the original prescription written (1 in 3 patients presented more than one month and 1 in 5 more than 3 months). These observations are consistent with the findings of a Newcastle study conducted in 2003 [All respondents who claimed to have been prescribed their antibiotics for ‘the flu’ did not consult their doctor prior to presenting to have the repeat dispensed and compared to other patient-reported indications this was a statistically significant finding. This should be viewed also having in mind that the most common reported reason for respondents presenting to fill the repeats was ‘cough’. The above suggests that in these cases, antibiotic repeat prescriptions could have potentially been used by patients for non-bacterial infections. Antibiotics for URTI are most frequently prescribed antibiotics despite many of such infections being viral in etiology ,11. The Approximately a third of respondents indicated their doctor did not tell them why a repeat prescription was needed. This finding suggests potential inadequate communication between the prescriber and patients about their antibiotic use and emphasizes the need for further education focused on addressing this communication gap. This also highlights the significance of pharmacists’ counseling when antibiotic prescriptions are filled, including their repeats. A further exploration of how to better utilize pharmacists skills in improving patients’ knowledge about their repeat prescription should be considered. To assist with this, a better use of dispensing software solutions could also be designed.It appears that the current system of antibiotic repeat prescriptions where they are valid for 12 months may contribute to patients’ lack of knowledge of the need for their antibiotic repeat prescription. In this regard, respondents who presented to fill their repeat prescriptions ≥ 2 weeks  were significantly more likely to not know why their repeat was prescribed. It should be noted that current guidelines limit the course of antibiotic use for most URTI for up to 2 weeks . The curThis study had a number of important limitations. These limitations firstly pertain to a small sample size. Lack of time and staff were the main reasons for pharmacies failing to recruit more patients. Findings of this study should also be interpreted with caution as some of the key questions were answered based on respondents’ perceived knowledge or awareness of symptoms and current condition. Furthermore, potential recall bias should be recognized, given that respondents may not remember what the doctor had originally told them. In addition, it should be acknowledged that other antibiotics not included in this study are used in URTI, such as erythromycin, azithromycin, doxycycline and dicloxacillin. Our study is also limited by the fact that it did not include rural populations in Australia. This was however done to limit confounding variables such as differing trends in infections and antibiotic use in rural and regional parts of Western Australia. Furthermore, our study was conducted in summer and autumn and there may be seasonal variations in antibiotic use during other times of the year. It should also be noted that in terms of wider interpretation of these findings, not all countries utilize a repeat prescription system for antibiotics.In spite of these limitations, it is clear that there are issues with the current system of repeats for antibiotic prescriptions especially around their 12 months validity. Given the failure of previous educational campaigns directed to prescribers in order to reduce default repeat generation for antibiotics  other meSystems allowing automatic generation of antibiotic repeats at the point of prescribing and repeat validity beyond completion of treatment course have potential to contribute to patients self-diagnosing URTI infections and hence inappropriately using antibiotics. Larger scale studies are needed in order to get a clearer picture of the extent of patient self-diagnosis of URTI infections and its impact on antibiotic resistance.


The orientation of guest molecules inside the cavities, have different terms: inside the open cavities of the polymer, or inside the cavities formed by packing different polymers. The host backbone shows responsive dynamic behavior in order to accommodate the size and shape of incoming guest molecule or guest aggregates. Simulations show that the host-guest binding of conjugated polymers is stronger than that of non-conjugated polymers. This detailed study could provide a clear picture for the host-guest interaction for dynamic conjugated microporous polymers. The mechanism obtained could guide designing new conjugated microporous polymers.The host-guest complexes of conjugated microporous polymers encapsulating C The guest encapsulation behavior of conjugated microporous organic polymers as a host have attracted a lot of attention by researchers all over the world123468et al. reported guest-responsive reversible swelling in a dynamic microporous polymer network poly-tetraphenyl pyrene (Py-PP). It encapsulates C60, dye molecules red-emitting 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DMDP) and Nile red (NR) dyes at room temperature2Rao 60, dye molecule DMDP, and dye molecule NR. We first obtained stable structure of building units of the host-guest system. Here, we used density functional theory to optimize the geometry of building units. 60, NR, DMDP, and monomer divinylbenzene (DVB) for control simulations. The right column shows the optimized geometry of PyPP monomer, C60, NR, DMDP and DVB, by using DFT method B3LYP/6-31G(d)1112We chose PyPP as host materials to encapsulate CBased on the PyPP monomer, we constructed PyPP oligomers and PDVB oligomer for next-step construction of host-guest complex. Here, we used Dreiding60, NR, and DMDP were loaded respectively within Amorphous Cell module in Materials Studio 7.0171860, PyPP-NR, and PyPP-DMDP complex respectively. Then geometry optimization was performed in Forcite module in Materials Studio 7.060 complex, PyPP-NR complex, and PyPP-DMDP complex are shown in 2 binding energy based on small molecule fragment is not accurate, because it does not include the entire frameworkIn order to study the molecule orientation of single guest molecule inside host framework, three PyPP oligomers with one guest molecule C60 was investigated. 60 encapsulated in the host framework. It indicates that C60 could stay inside the open pore of PyPP (a1), or stays within the cavities formed from packed oligomers (a2 and a3). The binding energies were computed to be −59.7, −40.2 and −57.2 kcal/mol respectively for a1, a2, and a3 orientations. Since the binding energy is calculated from the equation ∆E = E(complex) − E(host) − E(guest), the negative value of binding energy means that the system is stabilized after guest binding. The host guest interaction is mainly noncovalent π-π interaction.Firstly, the encapsulation of C1, b2, and b3 orientations respectively. The negative value of binding energies indicates that binding process of NR is energetically favorable. The main host-guest interaction is π-π stacking with different orientations.Secondly, we studied NR encapsulation in PyPP framework. 1, c2, and c3 respectively. The negative value also indicates that binding of DMDP molecule to the host framework is energetically favorable. The main host-guest interaction is dipolar interaction between DMDP and aromatic rings in the host frameworkLastly, the host-guest interaction between PyPP and DMDP was explored. 60, NR, and DMDP respectively. We used the same computational methodologies as used for PyPP oligomers. Interestingly, the binding energy of PDVB is much smaller than that of PyPP, indicating that the host guest interaction for PDVB is weaker. For C60 molecule, it mainly stays inside the cavity formed by packing multiple oligomers, such as a1 and a3. C60 could also stay on top of the framework such as a2. Next, host-guest interaction for NR and DMDP were also explored. The simulations also show that NR and DMDP reside in the cavity formed from packing oligomers such as b3 and c3. They could also be parallel to the surface of the framework, such as b1 and c2. Interestingly, we also found configurations where NR or DMDP is inside the closed pore of the PDVB framework. However, the binding is not energetically favorable in this case, since the binding energies were calculated to be positive .et al. mentioned that when the loading of guest molecules is increased, they detected the existence of aggregate guest molecules60, NR, and DMDP. Similarly, multiple configurations were obtained by Configurational bias Monte Carlo method1718Rao 60, NR, and DMDP guest aggregates. The right column of 60 aggregates assembled on top of pyrene arrays. This matches previous experiment well260 molecules will reside layer-by-layer with respect to the pyrene plane. From the comparison between PyPP oligomer pore and C60 guest-encapsulated pores in red, the width of three open pores changes. One increases from 8.6 Å to 9.6 Å, the second decreases from 7.7 Å to 6.7 Å. The third open pore decreases from 11.2 Å to 10.8 Å. This is because the aromatic arms of the open pore will adjust its size in order to enhance the host-guest π-π interaction with C60 molecule.Then the host-guest interaction between PyPP and NR aggregates was investigated. 60, PyPP-NR, and PyPP-DMDP complexes, it follows π-π stacking and C-H-π interaction.Lastly the encapsulation of DMDP aggregates was studied. 60 system. When C60 molecule is inside the open pore of the system, the host-guest interaction is strong, such as 3a1. When C60 molecule is inside the cavity formed from packing oligomers, depending on the packing density, the host-guest interaction varies. If the oligomers are densely packed, the host-guest binding is strong, such as 3a3. However, if cavity formed from packing is large, the host-guest interaction will be weaker, such as 3a2.When the guest orientation changes within the host framework, the host-guest interaction will also change. 1. However, if the contact area between NR and host framework is large, leading to larger host-guest interaction, such as 3b2. When NR molecule is parallel with the host plane, such as NR molecule interacts with host framework with various orientations. NR could be parallel with the framework, or inside the open pore. When the NR molecule is inside the open pore, depending on the orientation of NR, the binding energy varies. If the interaction occurs between only a portion of NR molecule and the host framework, binding is weak, such as 3b3), the binding energy is also large, indicating that the interaction between DMDP and edge of the polymer is very strong.In terms of the interaction between DMDP and PyPP framework, it has several different types as well. In From the trend of Besides conjugated polymer PyPP, we also performed control simulations of host guest interaction for PDVB, which is non-conjugated polymer. As shown in 60 molecule was inside the open pore, and the open pore will close the arm in order to catch C60. The open pore shrinks in order to catch guest molecule and keep it locked within the pore. Secondly, if the size of the guest aggregates is larger than the pore size, the pore will expand in order to encapsulate the aggregates, such as the right pore of For the pore size change after the guest aggregate as shown in From the simulations, we discovered that there are two types of cavities within the PyPP host framework. In conclusion, we explored guest encapsulation for conjugated microporous polymers by theoretical calculations for the first time, discovered different orientations of guest molecules inside the host framework, and confirmed the responsive dynamic behaviour of the polymer backbone by simulations. We also performed control simulations for non-conjugated polymers. The host guest interaction for conjugated polymers is stronger than that of non-conjugated system. This work provides detailed picture of guest encapsulation and could be helpful guidance for future design of dynamic porous materials. For example, future design could focus on new conjugated microporous polymers with pore size appropriate to the size of guest. We found two types of pore and get new insights of open pore, and pore formed from packing oligomers. In order to increase the host-guest interaction, we could adjust the monomer size, or adjust the compactness of the backbone, and ultimately increase the interaction area between the host and guest molecule or guest aggregates.60, NR, DMDP and PDVB monomer, were optimized by using DFT method B3LYP/6-31G(d)1112The structure of PyPP monomer, C60, NR, and DMDP were loaded respectively. Multiple stable configurations were generated from Configurational Bias Monte Carlo method171860, PyPP-NR, and PyPP-DMDP complex respectively. Then geometry optimization was performed in Forcite module in Materials Studio 7.0The guest encapsulation inside PyPP host framework, was studied by Amorphous Cell module of Material Studio 7.060, NR, and DMDP, was also studied by the same methods mentioned above.The host guest interaction between PDVB host framework and guest molecule CHow to cite this article: Xu, L. and Li, Y. Responsive Guest Encapsulation of Dynamic Conjugated Microporous Polymers. Sci. Rep.6, 28784; doi: 10.1038/srep28784 (2016).


Stigma, Discrimination and Living with HIV/AIDS: A Cross-Cultural Perspective,” edited by Pranee Liamputtong with 43 contributors, is the first book, which attempts to collate in one volume, results from empirical research on the subject matter from a cross-cultural and global perspective. Pranee Liamputtong, who holds a personal chair in public health at the School of Public Health at La Trobe University in Melbourne, VIC, Australia, builds on her extensive research work on the cultural and social influences on reproductive health to address HIV/AIDS-related stigma and discrimination from broad and diversified cultural, geographic, and sub-group perspectives.Stigma, a socially constructed process that categorizes people into “them” and “us” on the basis of race, gender, sexual orientation, and disease, among others, leads to discrimination against the stigmatized individuals or groups who are devalued in comparison to those in the mainstream. HIV/AIDS-related stigma and discrimination are issues of great concern because of their ramifications on access to HIV/AIDS prevention, treatment, care, and support for people living with HIV/AIDS (PLWHA). HIV/AIDS-related stigma and discrimination have been recognized globally and many reports of these have appeared in the biomedical literature. However, “Stigma, Discrimination and Living with HIV/AIDS: A Cross-Cultural Perspective,” focuses on the lived stigma and discrimination experiences of PLWHA and how they attempt to deal with these in countries, such as Australia, China, Colombia, India, South Africa, Thailand, the United Kingdom, and the United States. Within the context of these country cultures, layered stigma as experienced by PLWHA already stigmatized on account of their gender, race/ethnicity, sexual orientation, and other infections, such as Hepatitis C, are highlighted in the book.“HIV/AIDS stigma arises from society’s perception of the affected individuals’ deviation from socially accepted standards of “normality.” PLWHA are labeled as being “immoral,” “promiscuous,” or “perverted.” They are, therefore, discriminated against in these settings because they are perceived to be “contagious,” and associated with “death.”Stigma, Discrimination and Living with HIV/AIDS: A Cross-Cultural Perspective,” is divided into 3 parts preceded by an introductory chapter by the editor, which provides a synopsis of each of the 23 chapters of the book. Each of these chapters is written by one or more researchers who have used mainly qualitative research methods to gather real life data of the lived stigma and discrimination experiences of PLWHA in different countries and geographic regions of the world.The 415 page book “Part 1 of the book (chapters 1–6) provides a theoretical basis for understanding HIV/AIDS-related stigma and discrimination from individual, family, and institutional perspectives, and how they interact with other social prejudices, including homophobia, sexism, and racism. Of particular concern and interest here is the stigma and discrimination experienced by PLWHA at the hands of healthcare providers in a variety of healthcare settings. As reported by PLWHA, these take the form of delayed, inadequate, or differential treatment, which directly impacts their health outcomes and quality of life.Part II (chapters 7–18) addresses the lived HIV/AIDS-related stigma and discrimination experiences across social and cultural settings and beliefs around the globe. This part of the book especially highlights the fact that people who before the advent of the HIV/AIDS epidemic were already socially marginalized, stigmatized, or discriminated against by their societies, have turned out to be those that are bearing the brunt of the global HIV/AIDS pandemic. These groups include women, sex workers, the poor, men who have sex with men (MSM), and intravenous drug users (IDUs). The way in which HIV/AIDS stigma is expressed and experienced by PLWHA has been found to be contextual in nature and therefore varies from place to place and from culture to culture.Stigma, Discrimination and Living with HIV/AIDS: A Cross-Cultural Perspective” make up Part 3 of the book. This part of the book describes how PLWHA deal with and attempt to manage stigma and discrimination. Strategies described here include membership of support groups, such as associations of PLWHA.Chapters 19–23 of “Stigma, Discrimination and Living with HIV/AIDS: A Cross-Cultural Perspective” would be useful for a wide variety of readers, including healthcare providers taking care of PLWHA, students, and teachers in health professional schools, and also in courses, such as anthropology, sociology, social work, and public health.“The real life data of the lived stigma and discrimination experienced by PLWHA around the globe contained in the book makes it an invaluable source of first-hand information on these issues for HIV/AIDS policy makers and program managers for policy and advocacy purposes. The data also inform and equip PLWHA, enabling them to better cope with stigma and discrimination. In particular, it will assist health workers in community health centers and hospitals in understanding issues related to HIV/AIDS and allow them to provide culturally appropriate health care to PLWHA from different social and cultural backgrounds. The book is useful for anyone who is interested in HIV/AIDS-related stigma and discrimination in diverse social and cultural settings.Stigma, Discrimination and Living with HIV/AIDS: A Cross-Cultural Perspective,” edited by Pranee Liamputtong and published by Springer, is an easy to read text, which ensures that not only those in the social and health sciences can easily read and understand the text. Terminology used is well-defined throughout the book, which is also thoroughly referenced. Chapter summaries and suggestions for further research complete each chapter. The book does not need to be read cover-to-cover; the systematic division of the text into parts, chapters, sub-chapters, and descriptive headings allow the reader to quickly locate required information from any part of the book.“JA-A identified this book as occupying a unique position in the available literature on HIV/AIDS-related stigma and hence worthy of review. She read the book and crafted out the structure for the review manuscript. WL read the book and crafted the initial draft which was updated by JA-A.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.


Nowadays, novel applications for polyaniline (PANI) make new demands on its morphology controlling, and designing novel PANI or PANI composite polymeric materials has been more and more attractive. In this work, octaaminophenyl polyhedral oligomeric silsesquioxane (POSS) was employed to prepare nanostructured PANI composites via controlled fabrication. By interfacial copolymerization, fibrous nanostructure was obtained. The size and morphology of this structure was adjusted by changing POSS to OAPS ratio: the size increased from about 20 to 200 nm when the molar ratio of POSS in the composites increased from 0.5 to 2.0 mol %. More importantly, further hydrothermal treatment for the samples with higher POSS concentration resulted in mesoporous structure on a more microscopic scale, which helps to improve the thermal stability. In the total synthesis, POSS played an important role in the morphology controlling of the composites. As one kind of conjugated polymers, PANI can be easily obtained via oxidative polymerization in various organic solvents or aqueous system. Because of its good environmental stability under normal processing conditions, excellent physical and chemical properties, adequate level of electrical conductivity and unique doping mechanism, it has been incorporated more and more widely in many applications n, whose organic substituents are connected with a silicon-oxygen core. The sizes POSS cages range from 1 to 3 nm, which can be regarded as the smallest possible units of silica SiO−1) with KBr powder: 3072 (H-Ar), 1594 (C–C), 1112 (Si–O–Si); 29Si NMR (ppm): −76.5, MALDI-TOF (m/z): 77, 399, 400, 401, 876, 877, 878, 954, 955, 956, 1031, 1032, 1033 [C48H40Si8O12 = 1032 amu].FTIR, NMR and MALDI-TOF characterizations and for OPS are listed as followings: FTIR (cm−1) with KBr powder: 1134 (Si–O–Si), 3383 (N–H), 1488 (C–N), 29Si NMR (ppm): −73.3, −77.4; 1H NMR (ppm): 7.8–6.2 (4.0H), 5.2–3.7 (2.0H).FTIR and NMR characterizations for OAPS are listed as followings: FTIR , phenyltrichlorosilane, triethylamine, ethylacetate, toluene, ammonium peroxydisulfate and fuming nitric acid were bought from Shanghai Experiment Reagent Co., Ltd. and they were of analytical grade.Aniline, tetramethylammonium hydroxide  at the scanning rate of 0.02°/s; Infrared spectra were obtained by using a MAGNA-IR 750 spectrometer. Thermal gravimetric analysis (TGA) was performed on a Netzsch STA-409c Thermal Analyzer under a 50 × 10OPS) and octa(aminophenyl)silsesquioxane (OAPS) were started from phenyltrichlorosilane (PhSiCl3) according to the published ways  were collected by filtration and dried under vacuum for the next preparation. The final yields of OAPS were about 35 %.The synthesis and fabrication strategies of POSS-PANI nanofibre are illustrated in Fig. POPA-4 with higher POSS concentration, further hydrothermal treatments  were carried out after 20-min ultrasonic dispersion. Detailed composite samples are listed in Table Interfacial polymerization was performed in an aqueous/organic biphasic system containing aniline and OAPS mixture dissolved in toluene together with ammonium peroxydisulfate dissolved in an aqueous acid solution. The experiments for interfacial polymerization were carried out in a room with constant temperature and humidity, where the temperature was controlled at 25 ± 1 °C). For sample  °C. For


Bacillus cereus sensu lato (B. cereus sl) which includes foodborne pathogenic strains has been extensively studied in relation to its various animal hosts. The aim of this environmental study was to investigate the water compartments  closely linked to the primary B. cereus sl reservoir, for which available data are limited. B. cereus sl was present, primarily as spores, in all of the tested compartments of an agricultural site, including water from rain to groundwater through soil. During rain events, leachates collected after transfer through the soil eventually reached the groundwater and were loaded with B. cereus sl. In groundwater samples, newly introduced spores of a B. cereus model strain were able to germinate, and vegetative cells arising from this event were detected for up to 50 days. This first B. cereus sl investigation in the various types of interrelated environments suggests that the consideration of the aquatic compartment linked to soil and to climatic events should provide a better understanding of B. cereus sl ecology and thus be relevant for a more accurate risk assessment of food poisoning caused by B. cereus sl pathogenic strains.The behaviour of the sporulating soil-dwelling  The life cycles of many human pathogens, including foodborne pathogens, comprise stages in the environment outside the eukaryotic host, for which data are sometimes limited but are required to monitor adverse effects on human health –3. Ready B. cereus group of bacteria, also called Bacillus cereus sensu lato (B. cereus sl), displays a broad diversity of phylogenetically related strains [ B. anthracis, the entomopathogenic bacterium B. thuringiensis, or the human pathogen B. cereus sensu stricto (B. cereus ss), which is responsible for various human infections  [ B. cereus ss is a common source of foodborne poisoning, displaying emetic or diarrheal syndromes [ B. cereus ss food poisoning outbreaks in France revealed the emergence of particularly virulent strains and emphasized the danger for public health caused by these bacteria [ B. cereus sl allows these bacteria to survive in various stressful environments, which contributes to their ubiquity [ B. cereus sl contamination in processed food [The ubiquitous strains , 6, most strains –9. This s, etc.) . More imyndromes , 11 and yndromes . A studybacteria , particuubiquity . Spores sed food . B. cereus sl occurrence has been widely reported in a broad range of environments, such as many types of soils, sediments, dust, and plants , by sprinkling , or by drip irrigation . If groundwater were contaminated with human pathogens, this practice could disseminate them to cultivated plants.Moreover,nts  or whether B. cereus ss survives in the soil only as dormant spores until particularly favourable microenvironments (niches) are encountered to allow its growth [ B. cereus sl in such niches has previously been described, for example, in rhizosphere [ B. cereus sl strains with the arrival of new strains originating from elsewhere, whereas others disappear by death or by leaching. Soil is considered a reservoir for B. cereus sl; however, soil is not a static medium. Soil is permanently affected by water movement, which redistributes solutes, nutrients, and the geochemical background [ B. cereus sl strains in soil or concerning the putative transfer of these bacteria to deeper environments and in their fate in groundwater.Despite its distribution in a broad range of natural habitats, little is known concerning how control , 22 or ozoonosis . Howeverronments , 25. Fors growth . Growth zosphere , insect zosphere , nematodzosphere , arthropzosphere , and earzosphere , 31. In ckground . Little  B. cereus sl ecology should be useful for uncovering the still largely unknown determinants enabling the dispersal and fate of such populations in environmental conditions and might be relevant for a more accurate risk assessment of foodborne poisoning caused by B. cereus sl pathogenic strains. The aim of this study was to determine the occurrence and the fate of B. cereus sl from rain to groundwater through soil and to investigate a possible bacterial transfer from soil to water compartments. For this purpose, we sampled and isolated B. cereus sl in different soil and water compartments of the atmosphere-soil-groundwater continuum in an agroecosystem and performed B. cereus ss inoculation survival experiments of soil and water sampled in the same site.A better understanding ofThe experimental area was in south-eastern France at INRA-Avignon  2 km north of the Durance River. The climate is Mediterranean, with a primary wet period in autumn (high intensity storm events) and with severe drought and high temperatures in summer. The various crops grown in this agricultural area require much irrigation water. The soil is composed of clayey silt (2.5 m maximum thickness) overlying an alluvial aquifer consisting of gravels and of sands (7 m depth) on marl bedrock. The water table depth ranges from 4 to 5.5 m . The expThe bulk soil used in this study was collected from the experimental field, as previously described , during  B. cereus sl occurrence studies, the soil samples were collected in October 2011 from the 0–2, 2–10, 10–20, and 20–30 cm layers of the field and in April 2012 from the 0–2, 2–10, 10–20, 20–30, 30–60, and 60–90 cm layers of the field ( B. cereus sl measurements (performed within 7 days). Substrate-induced respiration (SIR) with glucose was performed using the MicroResp technique [ B. cereus sl occurrence in soil were analysed using Student's t-test to determine P values for differences in expected values versus actual values.Forhe field . Surfaceechnique , which iechnique . The dat B. cereus ss inoculation experiments, the soil samples were collected in March 2012 from the 0–10 cm layer of the field, air-dried, and sieved into a size fraction between 2 and 3 mm. After sampling, drying, and sieving, the soil was slowly moistened and maintained at 21% of the gravimetric water content  and stored for seven days at room temperature before inoculation experiments [Foreriments .2). The bucket was held on a stick 2 m above the ground to avoid any risk of contamination by rain splashing on the soil. The bucket was in the middle of the experimental field, 25 m from the closest hedge to limit the risk of contamination by neighbouring vegetation. The rainwater was collected 3–18 h later and immediately filtered on a 0.2 μm pore size membrane. Then, the particles, including putative bacteria located on the filter, were suspended in 2 mL of filtered rainwater, giving a 100–200-fold concentration of the rain sample, depending on the volume of rainwater collected. The rain samples used for B. cereus sl occurrence studies were collected in October 2011, November 2011, March 2012, and April 2012. During all of these rainfall events, the dominant winds were south-easterly.Rainwater was collected on the site as follows: when rain was about to fall or when the rainfall event had recently begun, an autoclaved plastic bag (50 L) was placed in a circular 21 L bucket with a diameter of 0.30 m  permanently exposed to natural climatic conditions was at the north-west end of the field-site. Cultivated identical to the nearby field, the lysimeter is dedicated to the measurement and calculation of soil water fluxes at the plot scale, with a continuous monitoring of hydrographs of water drainage events . The botμS/cm. Previous analyses of groundwater (~60 samples) in several boreholes near the study area from 2009 to 2011 highlighted the predominance of Ca2+ and HCO3− in solution. The concentration of organic carbon in groundwater, which may be present as a particulate or dissolved, ranged between 0.4 and 9.6 mg/L, with a mean concentration equal to 1.3 mg/L.Groundwater was collected as follows: a piezometer, which tapped the upper part of the saturated zone (depth 6 m) and was in the south-east of the area, was hand-sampled using an immerged pump after the complete renewal of the water in the piezometer. Groundwater, which was collected in a sterile plastic bag, was concentrated as described above for rainwater . The bor B. cereus sl CFU in these environmental samples with the presence of background microbiota, we used a selective medium recommended by food authorities. The Mossel medium , which was purchased from Biokar Diagnostics , allows the growth of B. cereus sl bacteria [ B. cereus sl strains that were discarded in these conditions, our bacterial counts might be slightly underestimated [For the isolation, identification, and counts ofbacteria  and theistimated . We usedμL of each dilution was plated in triplicate on Mossel medium and incubated for 24 h at 30°C. The variability between triplicates did not exceed 0.26 log CFU, indicating that the bacterial counts were reproducible. For determining the B. cereus spore density, the samples were pasteurised for 20 min at 70°C to eliminate vegetative cells and then incubated on selective medium. These thermoresistant bacteria were counted as spore forms because only spores of B. cereus sl survive at this temperature.Aliquots of 0.25 g of soil were supplemented with 1 mL of sterile saline water and vortexed vigorously for 30 s. The suspension was serially diluted 10-fold in sterile saline water, and 100  B. cereus ss strains was used. This strain is a spectinomycin- (Spc-) resistant strain derived from the ATCC 14579 strain [μg/mL) was used for counting this strain after the inoculation of natural samples.For the inoculation of environmental samples, the R2SK model strain for human pathogenic9 strain . The min9 strain . Growth B. cereus R2SK spores, which were prepared on the FNA sporulation medium [n medium  for 7 daB. cereus R2SK vegetative cells used for the inoculation of natural samples were grown in Gordon's medium containing soil extracts [600 nm = 3.0. Then, the cells were washed twice in sterile saline water at 20°C before soil or water inoculation.extracts ; howeverμL of B. cereus R2SK vegetative cells to reach approximately 4 log CFU/g  and incubated at 25°C in the dark. Counting was performed over time as described above for the enumeration of the natural B. cereus sl population  introduced into sterile 2 mL screw-cap microtubes were inoculated with 20 pulation ; howeverpulation . B. cereus R2SK spores or of vegetative cells, which were prepared as described above, and incubated at 12°C in the dark without shaking.Groundwater aliquots (100 mL) were inoculated with 1 mL of B. cereus R2SK that were below the detection threshold (10 CFU/mL) were arbitrarily set at 9 CFU/mL, as previously described [When required, populations ofescribed .n = 3) originating from rainwater that were defined as B. cereus sl bacteria after growth on Mossel medium were characterised, as previously described [ B. cereus sl. In addition, sequencing of the rrs gene was performed after a PCR amplification on 5 randomly selected strains (n = 3 from rain and n = 2 from groundwater), as previously described [ rrs genes were amplified by PCR using the primers 5-AGA GTT TGA TC TGG CTC AG 3-(forward primer) and 5-GG TAC CTT GTT ACG AT TC 3-(reverse primer), using the following temperature profile: 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min, followed by a final extension at 72°C for 5 min. The amplification products were purified and DNA-sequenced by Eurogentec. A BLASTN search was performed against nr/nt database to confirm that best hits (with 100% query coverage and 97–100% identity) were obtained with B. cereus sl sequences. These methods are powerful tools for identifying B. cereus sl but are not able to discriminate isolates at the species level.Some isolates , as previously described [n = 40) and from percolated water (n = 40). The images of the PCR fragments were analysed using the Biogen 99.04 software  for band detection, binary matrix generation, similarity coefficient calculation, and dendrogram construction. Images were also sight-checked to clear unspecific bands detected by the software. Similar visual patterns were obtained between duplicates. A control strain (ATCC14579) was always used, which showed >85% identity in 15 independent analyses. Thus, genuine distinct isolates were distinguished from putative clones when the percentage similarities were <85%.The diversity of theescribed , 47, on  B. cereus sl ubiquitous bacteria has never been studied in rain and was investigated in this study.The occurrence of B. cereus sl at densities ranging from 1.16 to 2.50 log CFU/L (mean ± SEM 1.96 ± 0.28 B. cereus sl log CFU/L), 2 to 4 log lower than the total culturable bacteria. Both the phenotypic characterisation (using an API50CH kit) and sequencing of the rrs gene, which were performed on a few isolates, confirmed that the isolates were genuine B. cereus sl isolates.Our results  indicateB. cereus sl bacteria are commonly isolated from soils. The level of the natural culturable B. cereus sl population was investigated in the soil used during our experiments. B. cereus sl bacteria were present in all of the soil samples, with a population ranging between 4.37 and 5.23 log B. cereus sl/g of soil  were not significantly different , indicating that B. cereus sl was almost exclusively present as spores in the soil tested.As expected, of soil . The mea B. cereus sl population of this soil; the mean density ± SEM per gram of soil displayed no significant difference between samples collected in October and in April .The different seasons did not seem to modify the occurrence of the natural B. cereus sl isolated did not seem to be influenced by the depth of soil in our sampling conditions. For instance, the mean B. cereus sl densities ± SEM per gram of soil were not significantly different between the superficial horizon (0–20 cm: 5.01 ± 0.06 log CFU/g) and the deeper depths .In addition, the amount ofThe decrease of values obtained using SIR measurements with the depth of soil indicates a higher active microbial biomass content in the upper layers of soil investigated . B. cereus sl population consisted nearly exclusively of dormant spores in the tested soil and was not affected by the season or by the soil depth (down to 90 cm)  .B. cereus sl was isolated from all of the samples of water that percolated through the soil in the lysimeter; however, the density of its population and its estimated flux (mean during sampling period) varied  in the three leaching water samples from 2012, and their densities were similar to those densities of the total B. cereus sl CFU , suggesting that B. cereus sl was almost exclusively present as spores in water soil leachates.) varied . We inve B. cereus sl isolated during the beginning of the leaching event were significantly higher than during the end of the same leaching event   . At the  B. cereus sl . To determine whether some groups of strains could be preferentially leached out from soil, the method was also applied on 40 isolates collected from percolated water . The dendrogram did not reveal any specific group of isolates from soil or from percolated water.High diversity was observed among the isolates of B. cereus ss population evolved over time in aliquots of soil and of groundwater that contained their natural microbiota. The water samples originating from rain and from percolated water were not investigated in this study because they were considered transient environments for B. cereus sl.We monitored how a newly introduced model B. cereus sl population of the soil used for inoculation experiments was 4.89 log CFU/g . Soil samples were inoculated by B. cereus ss vegetative cells of the Spc-resistant R2SK strain at a density between 3.66 and 4.16 log CFU/g, slightly below that of the natural B. cereus sl population level in this soil. A noninoculated control soil sample displayed no detectable Spc-resistant CFUs. Counts performed over time showed a rapid decrease of approximately 0.5–1 log in the newly introduced population during the first three days following inoculation .The naturalculation . This de B. cereus R2SK population was represented only by spores. This population reached a steady state, with no further changes in the level of B. cereus R2SK population observed up to 50 days of incubation at 25°C. Soil samples inoculated with vegetative cells (at 7.8 log CFU/g) of B. cereus R2SK still displayed high levels of thermoresistant B. cereus R2SK CFU (6.9 log CFU/g) after a 10-month period of storage in the dark at room temperature (not shown). This result indicates that the spores were extremely stable in this environment.After this period, the remaining newly introduced B. cereus R2SK population was observed over time when the soil was inoculated with spores instead of with vegetative cells (not shown).In addition, no major change in the level of the(1) In Groundwater Inoculated with Vegetative Cells. Aliquots of groundwater were inoculated with B. cereus ss strain R2SK vegetative cells at 8.20 or 8.21 log CFU/L, a density largely above that usually encountered in this groundwater. Counts of the newly introduced population were performed over time. In the tested samples, the natural B. cereus sl population was below the detection threshold (3.48 log CFU/L). B. cereus R2SK showed a high constant death rate during the first 10–15 days  In Groundwater Inoculated with Spores. Groundwater was inoculated with spores of B. cereus strain R2SK at 7.67 or 6.19 log CFU/L , and counts were performed over time  also displayed a rapid decrease during the first days after inoculation. However, the decrease was slower than that observed for the counted spores alone. This result indicates that rapid (but incomplete) germination occurred in the groundwater tested. For up to 20 days of incubation, the total B. cereus R2SK population reached a density of approximately 5–5.5 log CFU/L, which was at least 1 log CFU/L higher than the density of spores, indicating that a significant proportion of the B. cereus R2SK was viable as vegetative cells in this environment. The presence of vegetative cells was confirmed by microscopic observations (data not shown). Then, this total B. cereus ss population decreased slightly over time, up to 40–50 days, but was still at a higher density than spores. Under these conditions, the vegetative cells seemed well adapted to this groundwater environment, because these cells were able to survive for at least 50 days.The total culturableBacillus cereus sl are soil dwelling bacteria, which include foodborne pathogenic strains, that are able to adapt to a wide range of environments. However, little is known concerning the occurrence, fluxes, or fate of B. cereus sl bacteria in various aquatic environments  that are connected to the main reservoir of these bacteria . B. cereus sl has not been described as a regular member of this microbial ecosystem, whereas the spore-formers Bacillus spp. were observed in the atmosphere [ B. cereus sl in each of the four rain samples collected 2 m above ground level. Thus, B. cereus sl bacteria appeared to belong to the normal microbiota of rain. Delort et al. reviewed several studies of microorganisms found in atmospheric water (including rainwater) [ B. cereus sl population is present in our samples. However, whether B. cereus sl occurs in rain drops as free spores or attached to dust particles remains to be investigated. It would be interesting to determine whether B. cereus sl found in raindrops originates from soil dust that has been aerosolized and is potentially associated with long-distance dispersal, as suggested by Kellogg and Griffin [In recent years, an entire microbial ecosystem has been identified in the atmospheric environment, including rain and fog water –18, 48, mosphere  and durimosphere , 50. We inwater) . Among tinwater) . These r Griffin , or dire Griffin . B. cereus sl strains in the investigated agricultural soil, at a level similar to those levels previously described in other soils [ B. cereus sl soil population consisted almost exclusively of dormant spores. Spores do not depend on available organic matter to survive, and this finding may explain why the numbers of B. cereus sl did not decrease with depth similar to total bacteria. The data for B. cereus sl bacteria in relation to soil depth are scant. A previous study showed that a B. thuringiensis population introduced on the surface of a soil by spraying six years before counting significantly decreased at a depth below 10 cm [ B. cereus sl isolates at various depths was not investigated.As hypothesised, we identifieder soils , 53. Thier soils . This poow 10 cm ; however B. cereus ss population from vegetative cells to spores when introduced into a nonfavourable environment, such as the raw soil used in this study, is in agreement with previous studies [ B. cereus ss model population seemed to settle efficiently in the tested soil, with their level remaining highly stable over time, up to ten months. Experimental data concerning the long-term survival of B. cereus sl spores are scant. B. anthracis was shown to stay viable in a slurry of soil for six months under laboratory-controlled conditions [ B. thuringiensis was able to survive for at least thirteen years in the field [The observed shift of a studies , 43, 55.nditions , and B. he field . B. cereus sl spores become active when an excess of easily decomposable organic matter is bioavailable or when the soil presents a high moisture level [ B. cereus ss model population in this bare agricultural soil; however, this soil was not highly moistened, was not enriched with nutrients, and contained its own microbiota. We also investigated the fate of the introduced B. cereus ss population in similar soil samples “cleared” of their own microbiota by treatment with chloroform fumigation, and no change in the level of the newly introduced B. cereus ss population was observed . This finding suggests that a putative competition with the natural microbiota of the tested soil is not responsible for the rapid sporulation of the introduced B. cereus ss population.Previous studies suggest thatre level . In our  B. cereus sl population in the studied soil was constant over time  and because rainwater that permits reinoculation of soil contained a lower density of B. cereus sl population than percolated water, causing its elimination, other sources of bacteria or conditions allowing B. cereus sl multiplication  must sometimes occur. The term “incubator area” describes the hypothesis of Van Ness, whereby certain soil conditions may favour B. cereus sl vegetative growth in “hot spots” of microbial activities, such as the rhizosphere [ Bacillus CFUs counted in different rhizosphere samples were 1 log higher than in the control soil without plants [ B. cereus sl germination [ B. anthracis forms biofilms and persists as a vegetative form in a model rhizosphere system [ B. cereus ss inoculation experiments was collected in March 2012, several months after the last cultivated plant (durum wheat harvested in June 2011). This soil was quite poor in organic matter (organic carbon content 13.2 g kg−1) and roots compared with some grassy meadow (36.1 g kg−1) or forest soils, for instance [ B. cereus ss population. Additionally, the favourable conditions for B. cereus ss growth most likely occur at microspatial and microtemporal scales, which explain the stability and homogeneity of our larger scale in situ observations.Because thezosphere . For inst plants . Roots at plants , providit plants  and drivt plants , 62. At mination  and may e system , 31. Theinstance , providi B. cereus sl growth in soil with different strains , different soils, and different incubation conditions , or in association with a rhizosphere or with soil organisms to test the effect of physicochemical conditions and to provide new sources of organic matter for the growth, germination, survival, and sporulation of these populations.More ecological studies are required to clarify the environmental determinants allowing B. cereus sl density in percolated water was up to 3 log higher than that encountered in rainwater, suggesting that B. cereus sl CFUs were leached out of soil by water seeping through the ground. Given the size of bacteria (a few microns) and the fact that bacteria are most often adsorbed onto larger soil particles, most of the transport process of bacteria in soil would occur in the largest pore sizes of the soil [ B. cereus sl attachment and detachment processes [Thethe soil , 65. Thethe soil ). Such pthe soil , 37. Thethe soil . In thisrocesses , 67 shou B. cereus sl can be transferred from soil to groundwater via leachates. We observed B. cereus sl in some of our groundwater samples but at an extremely low density. A sandy gravel layer of 2-3 m can be found between soil and groundwater. Pore size exclusion phenomena rather than hydrophobic interactions between spores and soil organic content seem to play an important role in the filtration process in soils [In this experimental field context, the aquifer is located only a few meters below the percolated water collection lysimeter equipment. Thus, our data led us to reasonably hypothesize thatin soils , 68. Howin soils . B. cereus sl was sampled in spring 2012 after an extremely dry winter (37 mm between December 2011 and April 2012). The B. cereus sl density in water samples and flows observed from the lysimeter leachates were lower during the April 2012 rain event than during 2011 rain events. Garel et al. [ B. cereus sl occurrence in groundwater could be linked to climatic conditions and seasons .The groundwater sample that did not display anyl et al.  suggestel et al. ; under w B. cereus sl may reach groundwater after being leached out of soil, the survival, germination, or even growth could occur in this aquatic environment.Because B. cereus ss primarily died rapidly; however, a small part remained viable over time. These results suggest that only a minority of the vegetative cells previously grown on rich laboratory media were adapted to survive in a low-nutrient environment such as this groundwater sample. In contrast, a significant part of the B. cereus ss population introduced as spores in these groundwater samples survived. Interestingly, germination occurred, and vegetative cells arising from these spore germination events seemed well adapted to this low-nutrient environment, being able to survive for at least 50 days. Nutritional cues for spore germination may be present as traces in groundwater and could explain the observed B. cereus sl germination in such environment. For instance, several free amino acids are known to trigger B. cereus sl germination [ B. cereus sl can escape ingestion but can also germinate and grow [Introduced in groundwater samples as vegetative cells, the model strain ofmination . A diffeand grow , 71. B. cereus ss counts observed during our experiments and because of a high detection threshold, groundwater samples were inoculated at high densities, . Thus, our results only provide an overview of the possible fate of B. cereus ss in this environment but should require more investigation to decipher what occurs under more natural conditions. Taken together, these data suggest that a proportion of any spore that reaches the groundwater environment may persist there for a long time as both spores and vegetative cells.Because of the rapid decrease in B. cereus sl in groundwater, even at low levels, may not be only temporary. Therefore, risks of the dissemination of B. cereus sl via groundwater should be considered in future ecological studies of these bacteria.Thus, the occurrence of B. cereus sl isolates originating from soil, as observed by a fingerprint method, is consistent with previous results showing much diversity among the B. cereus sl population in temperate soils [ B. thuringiensis (as a pest control agent); however, such utilisation may have occurred in the neighbouring agricultural area, and we cannot exclude the possibility of contamination by B. thuringiensis strains [ B. thuringiensis pest control clonal strain in our soil samples can be ruled out.The high diversity ofte soils –75. The  strains . However B. cereus sl diversity  in environmental samples linked to transport among the unsaturated zones of the soil for a better risk assessment of ground water contamination.No particular group of isolates was identified in our lysimeter percolated samples, suggesting that no particular strains could be leached out of the soil. However, additional investigations should be performed on B. cereus, which is a foodborne pathogen, in rain, soil, soil leachate, and groundwater in the context of a field study. Despite using culturable methods that are expected to underestimate the densities of bacterial populations, the presence of B. cereus sl was detected in various environmental samples, including rain and groundwater compartments, which have rarely been described. In the investigated soil, the B. cereus sl population seemed constant over time; however, after high rain periods, B. cereus sl was leached out of soil suggesting that B. cereus sl may be transferred to groundwater, where it could persist for a long period.To our knowledge, this study provides a first description of the fate and transport of B. cereus sl strains isolated in rain samples, (ii) to clarify the determinants allowing B. cereus sl growth in the main reservoir , (iii) to understand the attachment and detachment processes to/from the soil matrix linked to rain events, (iv) to specify the role of the various ground layers in probable filtration of bacteria before they could reach groundwater, and, finally, (v) to determine the adaptive potential of human pathogenic strains to new environments compared with nonpathogenic ones, given the high diversity of strains observed in environmental samples. The possible fate of B. cereus sl in groundwater should however require more investigation to decipher what occurs under more natural conditions .This descriptive study also opens new fields for future research. This study revealed or confirmed that more ecological studies are required (i) to determine the origin of B. cereus sl through soil is rarely the main or the only one [ B. cereus sl transfer to foods via diverse routes and at diverse steps of food processing requires more investigation, this study brings new insights about B. cereus sl occurrence and behavior in the environment, especially into the water cycle, which may represent yet unstudied routes for food contamination. A proposed model of a possible part of the B. cereus sl life cycle is represented in  B. cereus sl. When particularly favorable conditions are encountered , growth may occur so that the pool of B. cereus sl is maintained in the soil. Therefore, soil may provide a source for the occasional contamination of rainwater (possibly via air dust) and of groundwater (in which B. cereus sl may survive) via leachates and, consequently, could contribute to B. cereus sl propagation in distant environments. Thus, rain and groundwater should be considered when studying the B. cereus sl life cycle in the environment, particularly in the context of agroecosystems submitted to climatic changes . Considering these environmental sources of B. cereus sl may be helpful for the more accurate assessment of the risk of food poisoning, particularly in ready-to-eat food, by pathogenic strains of B. cereus sl.Depending on the type of food, it was proposed that the contamination byonly one . Althoug


Identification of crosstalk between c-MET and other RTKs such as AXL and PDGFR suggest that c-MET network genes (c-MET-AXL-PDGFR) may be clinically relevant to BCa. Here, we examine whether expression of c-MET network genes can be used to identify BCa patients at increased risk of developing aggressive disease. In vitro analysis, c-MET knockdown suppressed cell proliferation, invasion, and migration, and increased sensitivity to cisplatin-induced apoptosis. In addition, c-MET network gene  expression allowed discrimination of BCa tissues from normal control tissues and appeared to predict poor disease progression in non-muscle invasive BCa patients and poor overall survival in muscle invasive BCa patients. These results suggest that c-MET network gene expression is a novel prognostic marker for predicting which BCa patients have an increased risk of developing aggressive disease. These genes might be a useful marker for co-targeting therapy, and are expected to play an important role in improving both response to treatment and survival of BCa patients.Previous studies have shown that  Overexpression of receptor tyrosine kinases (RTKs) occurs in cases of aggressive bladder cancer (BCa); thus RTK-targeting therapies are recommended for such patients , 2. Pharc-MET proto-oncogene, which is located on chromosome 7q21-31 . Response to treatment was assessed by cystoscopy and urinary cytology. Patients who were disease-free within 3 months of treatment were followed-up every 3 months for the first 2 years and then every 6 months thereafter. MIBC patients with clinically localized or locally advanced tumors and good Eastern Cooperative Oncology Group (ECOG) performance status (0 or 1) underwent radical cystectomy and complete pelvic lymph node dissection. Patients not eligible for radical cystectomy due to metastatic disease, poor life expectancy, or poor ECOG performance status (≥2) underwent TUR or biopsy for histopathological diagnosis. Patients with pT3, pT4, or node-positive disease  and those with metastatic disease but good performance status received at least four cycles of cisplatin-based chemotherapy. Patients who refused or did not complete an imaging work-up [computed tomography (CT) scan or magnetic resonance imaging (MRI)] at least once every 3 months to evaluate responses were excluded from further analysis.Primary tumor samples from patients who underwent transurethral resection (TUR) or radical cystectomy at Chungbuk National University in South Korea were histologically verified as urothelial carcinoma. Normal bladder mucosa was harvested from patients with benign diseases such as benign prostatic hyperplasia (BPH), ureter stones, and stress urinary incontinence, after informed consent. All control tissues were histologically confirmed as normal. Patients with concomitant carcinoma European Association of Urology (EAU) guidelines based on the 1973 WHO grading system  at the indicated conditions and centrifuged at 12,500 c-MET network genes and clinical parameters in BCa patients, we examined the expression profiles of these genes in BCa patients using previously obtained microarray data (accession number GSE13507). Microarray data were available for 165 patients. Clinical outcomes, including progression-free survival (PFS), overall survival (OS), and cancer-specific survival (CSS) were obtained from medical records. The correlation between gene expression and disease outcome was examined using Cox proportional hazards regression analysis. Kaplan–Meier (KM) survival curve analysis using a subset of gene expression profiles from patients with “low” and “high” expression of each gene were used to identify the effects of c-MET network gene expression on BCa. The 50th percentile (median) of gene expression was used as the cuff-off value. The log-rank test was performed to assess the significance of differences between two survival curves.To study the association between The study was approved by the Ethics Committee of Chungbuk National University. All subjects provided written informed consent. Sample collection and analysis were approved by The Institutional Review Board of Chungbuk National University.The mean age of the 165 patients in the study cohort was 65.2 ± 12.0 years, and the mean follow-up period was 48.4 months. Of the 165 patients, 62.4% (103/165) had NMIBC and 37.6% (62/165) had MIBC. The mean age of the 34 patients in the normal control cohort was 54.0 ± 10.4 years. The baseline characteristics of the patients and controls are presented in c-MET pathway increases invasion and metastasis of BCa cells [c-MET silencing on proliferation and invasion, and on the apoptotic response to cisplatin (a major chemotherapeutic agent used to treat BCa patients). We found that the BCa cells in which c-MET was knocked down formed fewer  colonies than the negative control cells, suggesting a reduction in cell proliferation in soft agar (c-MET were more invasive than those in which c-MET was knocked down. c-MET-silenced T24 cells were much less invasive than controls cells (non-transfected cells and cells transfected with control siRNA)  . We nextpoptosis .c-MET-knockdown cells migrated less efficiently than control cells  showed that ol cells . We alsoCa cells .c-MET network genes are involved in BCa progression and aggressiveness, we analyzed the expression of mRNA for these genes in a DNA microarray and compared the results with disease characteristics such as tumor grade (G), tumor stage , tumor size, recurrence, progression, and CSS. Further comparisons were then performed after patients were categorized into NMIBC and MIBC groups. The results revealed that c-MET mRNA expression in MIBC patients correlated significantly with OS , 1.110–3.998) .To answer the question of whether 0–3.998) . These d0–3.998) . BCa patc-MET partners, AXL and PDGFR, and BCa progression. The results showed that AXL expression clearly correlated with both NMIBC and MIBC. Bladder tumors (NMIBC and MIBC) showed 0.471-fold higher expression of AXL mRNA than control tissues. AXL mRNA expression by NMIBC  < 0.0001) and MIBC  was significantly higher than that in normal controls . We found that the expression of PDGFR isoforms could be used to distinguish NMIBC and MIBC samples from normal controls. The expression of PDGFRA mRNA clearly discriminated bladder tumors (NMIBC and MIBC) from normal control tissues , with PDGFRA expression in NMIBC and MIBC being approximately 0.274-fold higher than that in controls. The expression of PDGFRB was also clearly different between tumors and normal tissues . PDGFRB expression in NMIBC was significantly greater than that in normal controls (p < 0.0001), but no significant difference was shown between MIBC and normal controls (p = 0.0698). Similarly, PDGFRL was differentially expressed in NMIBC and normal controls, with a modest increase (0.804-fold) (p < 0.0001) in the former. Thus, it is likely that PDGFR expression is increased in all types of BCa. It is noteworthy that the expression of PDGFR isoforms in tissues from NMIBC patients was generally higher than that in tissues from MIBC patients    . KM surv= 0.032) .c-MET network genes, we examined the association between c-MET network gene expression  and BCa prognosis. Expression of c-MET network genes was based on an assessment of the risk score for each patient calculated by combining the expression levels of all three genes. We found that expression of c-MET network genes correlated significantly with disease progression in NMIBC patients  and with OS in MIBC patients   than those with low expression. Similarly, MIBC patients  with high expression of c-MET network genes showed poorer OS than those with low expression  . KM survpression .c-MET makes BCa cells less invasive and more susceptible to cisplatin. Also, the expression pattern of c-MET network genes allows the discrimination of BCa tissues from normal control tissues and appears to predict poor clinical outcomes in a Korean patient population.The results of the present study suggest that loss of c-MET expression occurs in various cancers and is associated with a poor prognosis [c-MET in BCa is associated with poor OS and metastasis-free survival [c-MET is positively associated with muscle invasion and poor long-term survival (p < 0.001) [c-MET expression is closely associated with both tumor aggressiveness and patient survival [c-MET was significantly associated with poor survival, particularly OS in MIBC patients. This suggests that inhibiting c-MET expression may play an important role in preventing BCa progression and improve patient survival.Aberrant rognosis . Overexpsurvival , 7, 10. < 0.001) . Previousurvival , 18. TheIn vitro analysis showed that c-MET knockdown suppressed cell proliferation, invasion, and migration, and reduced the expression of MMP2 and MMP9. This was accompanied by increased sensitivity to cisplatin-induced apoptosis. MMP2 and MMP9 degrade extracellular matrix proteins, thereby facilitating cell invasion and metastasis [c-MET inhibition is likely to reduce cancer invasion and metastasis and to improve the survival of cancer patients. Thus, a more focused understanding of the importance of the c-MET inhibition is needed if we are to develop inhibitors that target c-MET in various tumors. Recently, several c-MET-targeting drugs were tested in clinical trials, and all show promising clinical activity with acceptable side effects [c-MET/VEGFR2 (foretinib) were studied in phase I to II clinical trials in patients with papillary renal cell carcinoma and advanced hepatocellular carcinoma, respectively [MET, VERFR1, AXL, TIE2, KIT, FLT3, and RET, called cabozantinib , inhibits the growth, metastasis, and angiogenesis in pancreatic cancer and glioblastoma, and reduces resistance to gemcitabine. In particular, clinical trials in metastatic castration-resistant prostate cancer (mCRPC) patients reported a promising effect on PFS, bone metastasis, and pain [EGFR T790M mutation), MET amplification, and activated signaling pathways [tastasis , 26. The effects . For exaectively . A noveland pain . Howeverand pain . Acquirepathways . Thus, mpathways .c-MET and other RTKs because crosstalk partners of c-MET may be important biomarkers for co-targeting therapy and help to prevent resistance to individual MET inhibitors. RON, AXL and PDGFR have a crosstalk with c-MET. Overexpression of AXL and PDGFR is associated with aggressiveness and prognosis of a tumor series [PDGFRA and BCa progression, we examined all three PDGFR isoforms: PDGFRA, PDGFRB, and PDGFRL. However, only PDGFRL was associated with NMIBC progression. Most studies that aimed to identify a correlation between BCa progression and PDGFR examined the PDGFRA and PDGFRB isoforms. Therefore, the present study is the first to identify a significant association between PDGFRL expression and BCa prognosis. In addition, Yet et al. only examined the roles of AXL and PDGFR in advanced cases [AXL and PDGFR distinguished NMIBC and MIBC from healthy controls. In particular, expression of both of these genes was higher in NMIBC patients than in MIBC patients. Thus, AXL and PDGFRL may be more specific for NMIBC than MIBC. A large validation is needed to clarify the roles and effects of PDGFR isoforms on BCa (NMIBC and MIBC) prognosis.It is important to examine the crosstalk between r series –19. The ed cases . Here, wc-MET network gene  expression was closely associated with disease progression in NMIBC patients and with poor survival  in MIBC patients. These results suggest that inhibiting the c-MET pathway may prevent disease progression in NMIBC patients and improve the survival of MIBC patients. Also, multi-combination or co-targeting therapies might be needed to prevent acquired drug resistance. Yeh et al. demonstrated that 21.5% (14/65) of patients co-expressing c-MET/AXL/PDGFR showed poor long-term survival (p = 0.015) [c-MET network genes in patients with locally advanced and metastatic BCa. Here, we identified a clinical correlation in patients with NMIBC or MIBC. Thus, the present study suggests that the c-MET network is a promising biomarker and target for co-targeting drugs; this should be tested in clinical trials involving both NMIBC and MIBC patients.We also found that = 0.015) . Howeverc-MET/AXL/PDGFR levels can be used to distinguish cancer patients from normal controls and to distinguish NMIBC from MIBC; and (2) the expression of c-MET network genes is significantly associated with poorer survival rates for BCa patients.Taken together, these data suggest that (1) c-MET network gene expression is a novel prognostic marker for predicting which BCa patients have an increased risk of developing aggressive disease. These genes might be a useful marker for co-targeting therapy, and are expected to play an important role in improving both response to treatment and survival of BCa patients.Identifying the signaling networks involved may provide information that will further our understanding of the mechanisms underlying tumor biology and help to predict potential drug resistance . We beliS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


In this study, we sought to determine the involvement of HGF/c-Met signaling in the anti-metastatic action of quercetin in melanoma.Melanoma is notorious for its propensity to metastasize, which makes treatment extremely difficult. Receptor tyrosine kinase c-Met is activated in human melanoma and is involved in melanoma progression and metastasis. Hepatocyte growth factor (HGF)-mediated activation of c-Met signaling has been suggested as a therapeutic target for melanoma metastasis. Quercetin is a dietary flavonoid that exerts anti-metastatic effect in various types of cancer including melanoma. In a previous report, we demonstrated that quercetin inhibited melanoma cell migration and invasion 3 cross-linked cells to examine the homo-dimerization of c-Met. Quantitative real-time PCR analysis was carried out to evaluate the mRNA expression level of HGF. Transient transfection was used to overexpress PAK or FAK in cell models. Student’s t-test was used in analyzing differences between two groups.Transwell chamber assay was conducted to determine the cell migratory and invasive abilities. Western blotting was performed to determine the expression levels and activities of c-Met and its downstream molecules. And immunoblotting was performed in BSQuercetin dose-dependently suppressed HGF-stimulated melanoma cell migration and invasion. Further study indicated that quercetin inhibited c-Met phosphorylation, reduced c-Met homo-dimerization and decreased c-Met protein expression. The effect of quercetin on c-Met expression was associated with a reduced expression of fatty acid synthase. In addition, quercetin suppressed the phosphorylation of c-Met downstream molecules including Gab1 (GRB2-associated-binding protein 1), FAK  and PAK (p21-activated kinases). More importantly, overexpression of FAK or PAK significantly reduced the inhibitory effect of quercetin on the migration of the melanoma cells.Our findings suggest that suppression of the HGF/c-Met signaling pathway contributes to the anti-metastatic action of quercetin in melanoma.The online version of this article (doi:10.1186/s12943-015-0367-4) contains supplementary material, which is available to authorized users. The incidence and mortality rates of melanoma have increased world-wide in the last 30 years . MelanomRecently, the role of receptor tyrosine kinase c-Met in melanoma pathogenesis has been gaining interest. c-Met is a cell surface receptor consists of a 50-kDa extracellular α chain and a 140-kDa membrane-spanning β chain, and is synthesized from a single-chain 170-kDa precursor . Bindingin vivo [The dietary flavonoid quercetin  is a bioactive compound that wildly distributed in the plant kingdom. It possesses low intrinsic toxicity and does not have carcinogenic activity in vivo . Besidesin vivo . Quercetin vivo . Severalin vivo -24 and min vivo ,26. Morein vivo , and supin vivo  and humain vivo .in vitro and prevented melanoma lung metastasis in vivo. Here, we show that quercetin inhibits HGF/c-Met signaling manifested by suppressing c-Met phosphorylation, interfering c-Met dimerization, reducing c-Met protein expression and attenuating the activities of downstream molecules including Gab1, FAK and PAK, which contributes to the anti-metastatic action of quercetin in melanoma.Our published data  demonstrThe effects of quercetin on HGF-stimulated melanoma cell migration and invasion were determined by the Transwell chamber assays. As shown in Figure It has been reported that activation of the HGF/c-Met pathway promotes cell invasion, migration and allows cancer metastasis . Therefo3 increased c-Met dimerization (lane 3 versus 1), while pretreatment with quercetin reduced the expression level of c-Met dimer (lane 4 versus 3). Besides, stimulation with HGF caused an apparent c-Met dimerization (lane 5 versus 3), which was also inhibited by quercetin pre-incubation (lane 6 versus 5).Next, we determined if quercetin affected c-Met dimerization. As shown in Figure It is reported that most of melanoma cells produce HGF that induces sustained activation of its receptor c-Met . We wondTaken together, these findings suggest that quercetin inhibits c-Met activation probably mainly by inhibiting c-Met phosphorylation and dimerization.To verify the effect of quercetin on c-Met expression, we performed the Western blot analysis in melanoma MeWo and sk-mel-2 cells in addition to A375 and A2058 cells. Whole-cell lysates from the four cells treated with various concentrations of quercetin  for 24 h, or a fixed concentration (60 μM) for various durations  were immuno-blotted with the c-Met antibody. We found that quercetin reduced c-Met expression in both dose- and time-dependent manners in these four cell lines  involvement is essential for maintaining c-Met expression, and inhibition of FAS using inhibitors or the shRNA knockdown greatly reduces c-Met expression . The actAfter phosphorylation, c-Met recruits adaptor protein Gab1 (Grb2-associated binding protein 1), phosphorylates Gab1 at tyrosine site 307 and hence activates downstream FAK  and PAK (p21-activated kinase). Activation of FAK and PAK pathways results in increased cell motility, migration and invasion . To dete#P < 0.05). Quercetin treatment (40 μM) inhibited the cell migratory abilities. This inhibitory effect was reduced from 48% to 33% in PAK overexpressing cells, and from 48% to 30% in FAK overexpressing cells. These data indicate that the inhibitory effect of quercetin on migration is partially reduced by the overexpression of PAK or FAK in melanoma cells.To better understand the involvement of c-Met signaling in quercetin-mediated anti-metastatic effects, we investigated whether overexpression of PAK or FAK reversed the quercetin-mediated inhibitory effect on migration. A2058 cells were transiently transfected with either a FAK-expressing construct (or a PAK-expressing construct) or an empty vector. After 24 h transfection, the expression of FAK and phospho-FAK were increased remarkably, and the quercetin-mediated FAK inhibition was also partially reversed  or the shRNA knockdown approach can down-regulate c-Met expression in human prostate cancer cells, and the production of the 16-carbon fatty acid palmitate by FAS is required for maintaining c-Met expression [et al. [et al. [et al. found that all the flavonoids luteolin, apigenin, and quercetin, which possess a same moiety with a C2-C3 double bond in the C-ring, reduced c-Met expression in human prostate cancer cells [We found that c-Met protein levels were decreased after quercetin treatment in both dose- and time-dependent manners Figure A and B. pression . Similar [et al.  and in b [et al. . Furtherer cells . In thiser cells ,43. Lipier cells . Recent er cells . Querceter cells . TherefoAfter phosphorylation on tyrosine site 1349, c-Met becomes a docking site for recruiting Gab1, which further activates downstream FAK and PAK . BesidesIn summary, our previous  and curr2O  at 70°C at a final concentration of 150 mM, then the solutions were complexed with fatty-acid-free BSA (10% solution in H2O) by stirring for 1 h at 37°C and then diluted in culture medium. The final molar ratio of fatty acid:BSA was 5:1.Antibodies against phospho-Met (Tyr1234/Y1235), phospho-Met (Tyr1349), phospho-Met (Tyr1003), c-Met, phospho-Gab1 (Tyr307), FAK, phospho-FAK (Tyr576/577), phospho-FAK (Tyr925), phospho-FAK (Tyr397), PAK1/2/3, phospho-PAK1 (Ser144)/PAK2 (Ser141), phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197), phospho-PAK1 (Thr423)/PAK2 (Thr402) and FAS were obtained from Cell Signaling Technology . Anti-GAPDH was purchased from Santa Cruz Biotechnology . Goat anti-rabbit IgG, goat anti-mouse IgG and protein markers were supplied by Bio-Rad . Recombinant human HGF was obtained from PeproTech . Other chemicals were obtained from Sigma–Aldrich . Quercetin was obtained from Chromadex (USA). The stock solution of 100 mM quercetin was prepared in dimethyl sulfoxide (DMSO) and stored at −20°C. Palmitate was complexed to bovine serum albumin as previously described . In shor2.A375, A2058, sk-mel-2 and MeWo cell lines were obtained from the American Type Culture Collection , and were incubated in high glucose Dulbecco’s modified Eagle’s medium , supplemented with 10% (v/v) fetal bovine serum  and 1% penicillin/streptomycin  at 37°C in a humidified atmosphere of 5% CO2 incubator. After that, non-invading cells were removed from the upper surface of the membrane by scrubbing. The migrated cells on the underside of the filter were first fixed with 100% methanol and then stained by 0.1% crystal violet solution and counted in five random fields. The relative migration was calculated from the ratio of the migrated cells that quercetin treated versus the vehicle control cells.The cell migratory ability was tested using a commercial Transwell insert . A2058 and A375 cells were suspended in serum-free DMEM medium containing 0.1% BSA. Then 0.1 ml of the cells suspension was added to the top of the Transwell inserts, and 0.5 ml of serum-containing medium with (+) or without (−) HGF (100 ng/ml) was plated in the bottom wells. Quercetin was added to both inserts and wells. The chambers were then assembled and incubated for 24 h or 48 h at 37°C in a 5% CO2 incubator for 2 h. Then A2058 and A375 cells in 0.5 ml serum-free medium containing 0.1% BSA were added to the inserts, while 0.75 ml of serum-containing medium with or without HGF (100 ng/ml) was placed in the lower chambers. Quercetin was added to both the inserts and the lower chambers. Chambers were then assembled and incubated for 24 h or 48 h at 37°C. Subsequent steps were performed in the same manner as described for cell migration assay.For the invasion assay, BD BioCoat™Matrigel™ invasion chamber  were used. 0.5 ml warm (37°C) serum-free medium was added to both the inserts and the wells to allow the chamber rehydrated at 37°C in a 5% COMembrane protein was extracted using Mem-PER™ Plus kit  according to the manufacturer’s protocol. Preparation of total protein lysates and Western blot analysis were performed as described previously . Proteint method and normalized to GAPDH.Total RNA was extracted with Trizol reagent , and reverse-transcripted with oligo-dT using M-MLV reverse transcriptase  according to the manufacturer’s protocol. Quantitative real time PCR was carried out by monitoring the increase in fluorescence of SYBR green with the ViiA 7 Real Time PCR System . The primer sets were synthesized by Invitrogen, HGF primers: forward TCCCCATCGCCATCCCC and reverse CACCATGGCCTCGGCTGG, GAPDH primers: forward CTGCACCACCAACTGCTTAGC and reverse CTTCACCACCTTCTTGATGTC. Each sample was amplified in triplicate for quantification. Data were analyzed by relative quantitation using the ΔΔC3, 0.25 mM, Thermo Scientific, Rockford, IL, USA) was added to cells and reacted at 37°C for 5 min. Cells were then transferred on ice for 10 min. After that, non-reactive BS3 were quenched with 50 mM Tris–HCl (pH 7.4). Cell lysates were separated by 6% SDS-PAGE and immunoblotted with an anti-c-Met antibody.The dimerization of c-Met was analyzed as described previously . Melanomet al. [et al. [Plasmids pCMV6M-Pak1 (Addgene plasmid 12209) was provided by Sells et al.  and myc- [et al. . To overt-test was used to analyze differences between two groups. All data were presented as means ± S.D. from at least three independent experiments. P < 0.05 was considered as statistically significant.The Student’s