Excessive wound healing following glaucoma filtration surgery is the main determinant of surgical failure, resulting from the activation of human Tenon's capsule fibroblasts (HTFs). To mitigate the excessive wound healing, the topicall use of antiproliferative agents, such as mitomycin C (MMC) and 5-fluorouracil (5-FU), has increased the surgery success rate, but the traditional administration of these agents can result in a variety of toxicities with nonspecific damage. However, modulation of the wound healing process to prevent excessive fibroblast proliferation and scar formation can play a major role in improving the outcome of surgery. Therefore, the search for alternative modes of drug delivery and new agents is needed to minimize the ocular complications and improve the success of surgery. We have shown that there is a postoperative overexpression of the LDL receptor (LDLr) in the activated HTFs may provide a novel target for drug delivery systems.We hypothesize that antifibrotic agents (MMC) encapsulated in LDLr targeting drug delivery system (LDL-MMC-chitosan nanoparticles) may be proposed in anti-scarring therapy to increase the safety and effectiveness and to reduce toxicity.A chitosan-based polymeric predrug of MMC was synthesized and its cytotoxicity was proved to be low. In addition, we propose hyaluronic acid film as a container to release LDL-MMC-chitosan nanoparticles gradually at subconjunctival filtering site after glaucoma filtration surgery to eliminate the LDL-MMC-chitosan nanoparticles.This strategy can be applicable to anti-scarring therapy during excessive conjunctival wound healing. This hypothesis integrates advantages of the targeting drug delivery and antifibrotic agents, such as high efficiency, convenience, and lower the toxicity. The most common cause of glaucoma filtration surgery failure is excessive scar formation at the surgical site,2. The ain vivo by an investigation, using the photodynamic therapy (PDT) for anti-scarring after glaucoma filtering surgery [These problems have stimulated the search for alternative modes of drug delivery and new agents to minimize the ocular complications. Numerou surgery . PerhapsLDLr is a single-chain transmembrane glycoprotein that specifically mediates binding and endocytosis of LDL. Receptor-mediated endocytosis is a highly specific, high capacity process that can absorb a large amount of LDL into the cell within a relatively short time. Once internalized, LDLr dissociates from LDL and is recycled back to the cell surface where it is available to interact with many more LDL over its lifetime. Then LDOur previous study have shown that the LDLr are overexpressed in the activated HTFs. As we s2 and -COOH groups in its structure, which is valuable for the drug carrier to readily prepare its conjugates with various drugs to avoid vexatious complications. That is possible to deal with the application of chitosan as a carrier for water-insoluble and water-soluble drug conjugates in anti-scarring therapy. Izume summarized the toxicity of chitosan including a skin sensitization study, temporal skin irritation study, ophthalmic sensitization test, mutagenicity test and patch test for humans; every study and test showed low toxicity of chitosan, which can function well as a drug carrier due to long systemic retention, low toxicity and accumulation in the target tissue[We propose that MMC encapsulated into LDL-chitosan nanoparticles (LDL-MMC-chitosan nanoparticles) will be an effective way to deliver MMC specifically to HTFs. The MMC-chitosan nanoparticles will be made as reports,24. The et tissue.In addition, firstly, nanoparticles increase uptake into activated HTFs that overexpressed LDLr, and steady release MMC. Secondly, chitosan nanoparticles confine and protect the enclosed MMC which may be hydrophilic until they bind to the outer membrane of the targeted HTFs, and lower effective dose of MMC. Thirdly, chitosan nanoparticles have the capability of keeping stability in human body. And finally, the scale dimensions of nanoparticles are between 1 and 100 nm, which favor endocytosis via LDLr.The LDL particle is a naturally occurring nanostructure typically with a diameter of 22 nm. It contains a lipid core of some 1500 esterified cholesterol molecules and triglycerides. A shell of phospholipids and unesterified cholesterol surrounds this highly hydrophobic core. The shell also contains a single copy of apoB-100, which is recognized by the LDLr. As repomCS-CHO/mMMC). When the mCS-CHO/mMMC was 5/1, 10/1 and 25/1, the initial release amount of MMC was 65%, 50% and 45%. There was an obvious initial release within the initial 8 h, and the concentration of MMC in dialysis medium remained unchanged during the following 60 h. Furthermore, the cytotoxicity study on chitosan-based polymeric predrug encapsulated fibroblast indicated that the maximum non-toxic concentration of CS-CHO was 8.3, 42.3 and 54.7 mg/ml in the 24, 48 and 72 h[In vitro, chitosan-based polymeric predrug of MMC was a low cytotoxic controlled delivery system.To test this hypothesis, a chitosan-based polymeric predrug of MMC was synthesized and the drug release rates were controlled by the aldehyde degree of chitosan and the mass ratios of periodate-oxidized chitosan (CS-CHO) to MMC  Figure , which hWe will observe the validity of creating a long-term effective filtering bleb and decrease of the ocular complications, compared with the control. The encapsulation allows constant release rather than a burst of drugs so that a high therapeutic efficiency can be achieved without side effects. The LDL-MMC-chitosan nanoparticles drug delivery system is proposed based on the LDL receptor endocytosis pathway. The receptor-mediated uptake mechanism is an interesting aspect of the LDLr system. Receptor-mediated endocytosis is a highly specific, high capacity process that can absorb a large amount of LDL-MMC-chitosan nanoparticles into the activated HTFs within a relatively short time. Once internalized, LDLr dissociates from LDL-MMC-chitosan nanoparticles and is recycled back to the cell surface where it is available to interact with many more LDL-MMC-chitosan nanoparticles over its lifetime. With a recycle time of approximately 10 min and a lifetime of about 24 h, one can assume that each receptor mediates the transport of about 144 LDL-MMC-chitosan nanoparticles into the activated HTFs per day. Assuming about 1,000 receptors per cell, this provides an extremely efficient system for delivering its MMC. Therefore, LDL-MMC-chitosan nanoparticles accumulate mainly within the activated HTFs instead of bring nonspecific toxicity to both proliferating and non-proliferating cells.In this article, we propose LDL-MMC-nanoparticles as a consistent drug delivery system that specifically bind to LDLr mainly in activated HTFs. This may achieve a long circulation time, low immunogenicity, good biocompatibility, highly selective targeting, lower drug dose, reduced toxicity to normal cells, and increased safety and effectiveness of anti-scarring therapy during excessive conjunctival wound healing.The authors declare that they have no competing interests.TS conceived the hypothesis and drafted the manuscript; XL carried out the study of MMC-chitosan nanoparticles synthesis; JG revised the manuscript critically and gave final approval of the version to be published. All authors read and approved the final manuscript.


HIV counselling and testing (HCT) services can play an important role in HIV prevention by encouraging safe sexual behaviours and linking HIV-infected clients to antiretroviral therapy (ART). However, regular repeat testing by high-risk HIV-negative individuals is important for timely initiation of ART as part of the ‘treatment as prevention’ approach.To investigate HCT use during a round of HIV serological surveillance in northwest Tanzania in 2010, and to explore rates of repeat testing between 2003 and 2010.HCT services were provided during the fourth, fifth and sixth rounds of serological surveillance in 2003–2004 (Sero-4), 2006–2007 (Sero-5) and 2010 (Sero-6). HCT services have also been available at a government-run health centre and at other clinics in the study area since 2005. Questionnaires administered during sero-surveys collected information on socio-demographic characteristics, sexual behaviour and reported previous use of HCT services.The proportion of participants using HCT increased from 9.4% at Sero-4 to 16.6% at Sero-5 and 25.5% at Sero-6. Among participants attending all three sero-survey rounds , the proportions using HCT twice or more were low, with 11.1% using the HCT service offered at sero-surveys twice or more, and 25.3% having tested twice or more if reported use of HCT outside of sero-surveys was taken into account. In multivariable analyses, individuals testing HIV-positive were less likely to repeat test than individuals testing HIV-negative .Although HCT service use increased over time, it was disappointing that the proportions ever testing and ever repeat-testing were not even larger, considering the increasing availability of HCT and ART in the study area. There was some evidence that HIV-negative people with higher risk sexual behaviours were most likely to repeat test, which was encouraging in terms of the potential to pick-up those at greatest risk of HIV-infection. HIV counselling and testing (HCT) services have recently expanded rapidly in sub-Saharan Africa in order to facilitate access to HIV treatment and care Despite increases in the numbers of individuals accessing HIV testing and treatment services, the World Health Organisation reported in 2011 that knowledge of HIV status remains broadly inadequate. In six countries with results from population-based surveys conducted in 2007–2009, the proportion of respondents unaware of their HIV-positive status ranged from about 30% in Kenya to almost 70% in the Congo This paper reports on the uptake of HCT services during the latest round of HIV serological surveillance carried out in 2010 as part of an on-going community cohort study in Kisesa in northwest Tanzania. It also reports on repeat use of HCT services over consecutive rounds of serological surveillance carried out between 2003 and 2010; a period of time which saw a number of changes in terms of the availability of HIV services, including the commencement of a national ART programme at the start of 2005, and the opening of a permanent HCT clinic in the study area later that year. We explore how these changes affected rates of first time and repeat HCT over time.Ethical approval for each survey round of the Kisesa cohort study has been granted by the Tanzanian Medical Research Coordinating Committee and the Ethics Committee of the London School of Hygiene and Tropical Medicine. During the 2003–2004 survey, verbal consent was obtained directly from all study participants including those aged 15–17, due to low literacy rates among the study population. Consent was witnessed and documented for each study participant on their survey questionnaire, by a member of the sero-survey team. During serological surveys in 2006–2007 and 2010, consent was again obtained directly from all study participants including those aged 15–17, however the option of written consent was introduced, for those who were able to provide this. Although data from the subsequent serological survey in 2012–2013 are not used in this paper, it was recommended by the review committee that additional written consent be obtained from parents or guardians for participants aged 15–17 for this round, and this was implemented accordingly.et alThe study setting in Mwanza region has been described in detail by Mwaluko Since 1994, 27 rounds of demographic surveillance and six HIV serological surveys have been completed. Demographic surveillance took place approximately once every six months, collecting information on residence and survival status of all household members, pregnancy among women of reproductive age, and births and migration. Eligibility and invitations for the serological surveys (sero-surveys) were prepared based on having been resident at the last round of demographic surveillance and being aged 15 or older at the time of the sero-survey. Venous blood was collected from all consenting participants during the first sero-survey in 1994/1995, while finger prick blood was collected during all subsequent sero-surveys. Samples were tested for HIV anonymously at the National Institute for Medical Research (NIMR) in Mwanza.A separate and confidential HCT service was provided in temporary village based facilities during the fourth, fifth and sixth rounds of serological surveillance in 2003–2004 (Sero-4), 2006–2007 (Sero-5) and 2010 (Sero-6), for those wishing to know their HIV status (see below for further details). Height and weight measurements were taken during sero-surveys, accompanied by a detailed questionnaire. The questionnaire was administered by same sex interviewers and included items on socio-demographic characteristics, sexual behaviour, health status and previous HCT service use. All study participants were additionally offered free medical treatment for any health problems present at the time of the sero-survey.The study population is served by a government-run health centre located in Kisesa trading centre, by three small government-run dispensaries located in the rural villages, and by a number of private clinics located mainly in the trading centre. HCT services are distinguished as those provided at stand-alone voluntary counselling and testing (VCT) clinics, or via other clinics within a hospital or health centre , referred to as provider initiated testing and counselling (PITC). A stand-alone VCT clinic opened at Kisesa health centre in 2005, while PITC has been offered to all pregnant women attending the health centre antenatal clinic (ANC) since the end of 2008. PITC has also been available at the out-patients department since 2010, where testing may be offered to patients attending the sexually transmitted infection and tuberculosis clinics. Since mid-2009, antenatal PITC is usually offered at the small rural dispensaries, dependant on the availability of test-kit supplies.When HCT services were first provided in Kisesa in 2005, patients testing HIV-positive were referred to Mwanza city hospitals, where free care and treatment services have been available under the national ART programme since the beginning of 2005. At the end of 2008, ART services became available locally at a Care and Treatment Centre (CTC) at Kisesa health centre. At this point, individuals testing HIV-positive were referred to Kisesa CTC for care, while patients already attending Mwanza city hospitals were given the option of transferring to Kisesa CTC if they wished.Since the fourth sero-survey round in 2003–2004, a separate and temporary VCT clinic has been available to all sero-survey participants wishing to know their HIV status. After their questionnaire interview, individuals expressing desire for VCT were directed to a separate purpose-constructed hut for pre-test counselling with a trained counsellor. At Sero-4 venous blood was collected and transported to the NIMR laboratory for HIV testing, and clients were asked to return for their test results and post-test counselling one week later All survey data were double entered. Linking of demographic data, sero-survey interview data, HIV research tests and sero-survey VCT attendance was carried out anonymously using study identification numbers. Sexual behaviour variables collected during sero-survey interviews included age at first sex, number of sexual partners and frequency of condom use with different partner types. Marital change and spouse identification variables were obtained from the demographic data, after which spouses’ HIV-status was anonymously linked in from the serological data. The percentage change in body mass index (BMI) at each sero-survey was calculated using height and weight data collected at the previous sero round, for those who had attended the previous sero-survey.At Seros 4, 5 and 6, the survey questionnaire also captured information on participants’ self-reported prior use of HCT services, including VCT use at various other clinics within and outside the study area , and via PITC at antenatal clinics and/or hospital outpatient departments. At Seros 5 and 6, it was possible to additionally identify those with known prior use of VCT at an earlier sero-survey round, for those who had attended an earlier round.All statistical analyses were done using Stata 12 . In order to ensure that all individuals had an equal period of time during which they might have repeat tested, a repeat tester was defined as somebody who attended both Sero-5 and Sero-6 and who used the VCT service available at each of these rounds. Univariable analyses were used to describe associations between all variables of interest and i) uptake of VCT at the Sero-6 round, ii) repeat use of VCT at the Sero-5 and Sero-6 rounds. Logistic regression models were fitted to identify characteristics independently associated with VCT uptake using a forward-fitting approach and including all variables significant in univariable analyses at the p≤0.10 level. Likelihood ratio tests were used to assess the inclusion of variables in multivariable models.Altogether 8,008 individuals attended the Sero-6 round, of whom 60.9% were women. In total 31.7% of men and 30.5% of women gave consent to use VCT, while 25.9% of men and 25.2% of women subsequently went for VCT. This represents approximately a doubling in the proportion of men using VCT since Sero-4, and a threefold increase amongst women .et alet alless likely to test than HIV-negative women .A full analysis of the associations between socio-demographic, clinical and behavioural characteristics and VCT use at Sero-6 can be seen in less likely to use VCT compared to HIV-negative women  but not if they had previously used HCT .In contrast to earlier rounds, women who were HIV-positive at Sero-6 were significantly and/or reported use of HCT outside sero-surveys). Considering VCT use at sero-surveys only  giving incorrect reports. Furthermore, of 822 instances where HIV-negative individuals had known use of VCT an earlier sero-survey round, in 42.3% of cases (348/822) these individuals failed to report this VCT use when asked at the later sero-survey round. Similar trends were seen among HIV-positive individuals, although in an even larger proportion of cases (49/89 or 55.1%) participants incorrectly reported VCT use at an earlier sero-survey round that did not actually occur. Of 67 instances where HIV-positive individuals had known use of VCT at an earlier sero-survey, in 40.3% of cases (27/67) participants failed to report this VCT use at the later round.When interpreting data which includes reported as well as known or documented use of HCT services, it should be noted that there is a tendency to both over and under report previous HCT service use. The factors most strongly associated with repeat testing in multivariable analyses included area of residence , being of Islamic faith , testing HIV-negative at Sero-5 , reporting using condoms inconsistently  and being married polygamously . Those with no education were also significantly less likely to repeat test than those with a primary level of education .The uptake of HCT services increased steadily in Kisesa between 2003 and 2010, with 9.4% of all participants using VCT at Sero-4, 16.6% at Sero-5 and 25.5% at Sero-6. In spite of this, by the end of the Sero-6 round , a substantial proportion of participants had never used any HCT service – approximately 36% of all HIV-positive individuals and 54% of all HIV-negative individuals. Furthermore, among participants who attended all three sero-survey rounds , almost half (48.0%) had never used any HCT service (at sero-survey and/or elsewhere). This figure rose to almost two-thirds (64.0%) if considering VCT use during sero-survey rounds only.Considering the rapid expansion of HIV testing and treatment services within the study area over the time period in question, it is disappointing that the proportions of individuals who had used an HCT service at least once were not even larger. Previous research has identified numerous barriers to the uptake of HIV testing services in sub-Saharan Africa including prohibitive costs associated with travelling to health clinics, shortages of test-kit supplies, high levels of stigma and discrimination and concerns about confidentiality did not occur, and 378/896 (42.2%) under-reporting previous VCT use that did occur. As a result, the estimate is likely to provide a fairly accurate measure of the proportion of individuals who repeat tested among those who attended all three sero-survey rounds. While repeat HCT use did increase over time, the proportions testing more than once were still low by the end of the Sero-6 round, when 16.7% of all men and 22.9% of all women had ever repeat tested.At each sero-survey round, those who had previously used an HCT service were significantly more likely to use the VCT service offered than those who had never used HCT before. However, among participants who attended all three sero-survey rounds , the proportions repeat testing (i.e. using HCT twice or more) were low, with 11.1% using the VCT service offered at sero twice or more, and 25.3% having tested twice or more if reported use of HCT outside of sero-surveys was taken into account. As the latter estimate includes data on reported use of HCT services, we expect some error around it. However, the numbers of participants over and under reporting previous VCT use were very similar, with 399/917 (43.5%) of all individuals over-reporting previous VCT use that et al in their model The proportions of participants repeat testing in Kisesa are somewhat lower than has been reported elsewhere in sub-Saharan Africa, where estimates ranged from 24.2% in a community cohort study in Uganda In the analysis of factors associated with repeat VCT use (among those attending Seros 5 and 6), there was some evidence that HIV-negative people with higher risk sexual behaviours were among those who were most likely to repeat test. This is encouraging in terms of the potential to pick up those who are most at risk of HIV-infection. However, sero-converters were not significantly more likely to repeat test than those who did not sero-convert. Only 7 individuals sero-converted between the two rounds in this analysis, and so our sample size may have been too small to detect any meaningful difference. Other characteristics which were significantly associated with repeat VCT use included area of residence and level of education, highlighting a persistent inequity among those gaining access to HCT which was first seen in 2003–2004 Outside of sero-surveys, most participants reported having used HCT services at Kisesa health centre, where the VCT clinic was favoured disproportionately by women. This may reflect women who were offered PITC at the antenatal clinic but who were subsequently referred to the VCT clinic for testing . However, it may also reflect a disinclination among men to use the health centre, which may be viewed as a place for women to receive services. Other HCT services such as mobile VCT clinics and the Angaza VCT clinic in Mwanza were used more equally by men and by women, but by smaller numbers of individuals overall, likely as a result of their temporary availability (mobile VCT clinics) or distance from the study area (Angaza clinic). With ever larger numbers of women being tested for HIV antenatally in sub-Saharan Africa This study has a number of strengths including a wealth of data covering an important period of time in terms of the development and increasing availability of HIV testing and treatment services in Kisesa. The study made use of data on both reported and actual use of HCT services, allowing for comparison of rates of testing using the two different data sources. It also allowed for comparison of the accuracy of reported versus actual HCT use, which was seen to be variable. This may have been caused by confusion when answering questions relating to previous HCT use and the exact location of any testing, and/or as a result of reporting biases.et al, unpublished data), which may have affected our results. HIV-positive individuals may have been disproportionately represented among those not participating We have seen declining participation rates in the Kisesa cohort study over time, particularly among younger men living in urban areas (Urassa Although the proportion of individuals using HCT services increased in Kisesa over time, further increases in the rates of first time and repeat HCT use would be required if TasP were to be implemented. Programmes and interventions designed to address the persistence of HIV-related stigma and discrimination are urgently needed, as are interventions designed to increase the uptake of HCT among those living in rural areas and those with least education.


H,1H,2H,2H-perfluorodecyltrichlorosilane (FDTS) and (3-aminopropyl)triethoxysilane (APTES), and at the same time featuring regions of bare SiOx. The patterning process could be applied even on meter-sized substrates with various functional SAM molecules, making this process suitable for the rapid preparation of quasi two-dimensional nanopatterned functional substrates, e.g., for the template-controlled growth of ZnO nanostructures [A rapid and cost-effective lithographic method, polymer blend lithography (PBL), is reported to produce patterned self-assembled monolayers (SAM) on solid substrates featuring two or three different chemical functionalities. For the pattern generation we use the phase separation of two immiscible polymers in a blend solution during a spin-coating process. By controlling the spin-coating parameters and conditions, including the ambient atmosphere (humidity), the molar mass of the polystyrene (PS) and poly(methyl methacrylate) (PMMA), and the mass ratio between the two polymers in the blend solution, the formation of a purely lateral morphology (PS islands standing on the substrate while isolated in the PMMA matrix) can be reproducibly induced. Either of the formed phases (PS or PMMA) can be selectively dissolved afterwards, and the remaining phase can be used as a lift-off mask for the formation of a nanopatterned functional silane monolayer. This “monolayer copy” of the polymer phase morphology has a topographic contrast of about 1.3 nm. A demonstration of tuning of the PS island diameter is given by changing the molar mass of PS. Moreover, polymer blend lithography can provide the possibility of fabricating a surface with three different chemical components: This is demonstrated by inducing breath figures (evaporated condensed entity) at higher humidity during the spin-coating process. Here we demonstrate the formation of a lateral pattern consisting of regions covered with 1 Self-assembled monolayers (SAMs) are well-known and have been intensively studied for many years, partly because of their interesting properties and partly because of interesting perspectives for potential applications as functional, ultrathin coatings –5. Due tElectron beam lithography allows the desorption or destruction of molecules of a SAM layer, line by line –10. AdvaPhase separation of binary polymer blend solutions during a spin-coating process produces nano- and micropatterns on large areas in a fast and scalable fashion. This phase separation has been intensively studied over the past two decades and allows the formation of complex layered or lateral micro- or nanoscale structures –38. ThesHere, we are aiming for a lateral polymer phase morphology that can be completely removed by a selective solvent to make the substrate available for well-defined chemical surface modification. This can be achieved by inserting a silane SAM, which then exposes a functional group. The preparation process of the SAM should not affect the remaining polymer mask, such that it can protect the substrate during the procedure and can be removed afterwards. For the spin-coating of polymer blend films, there are many parameters and conditions, such as the concentration of the polymer solution, the spin rate, and the surface property of the substrate, among others, that affect the final morphology of the polymer blend film. Some examples of both the influence of the substrate –56 and tIn this article we present a method to obtain a polymer-blend film with a purely lateral phase morphology, which means that the blend separates completely into two lateral phases. The introduction of a small amount of water during the spin coating process is crucial for obtaining this purely lateral morphology. Either of the two different polymers can be dissolved independently afterwards by using a selective solvent. The remaining morphology is later on applied directly as a lithographic mask to fabricate nanopatterned self-assembled-monolayer (SAM) templates. Performed at higher humidity, our technique combines polymer-blend phase separation with the breath-figure formation. A three-phase lithographic mask is formed in one process step, giving the opportunity to produce a SAM template with three different chemical functionalities.The polymer-blend lithography method is demonstrated schematically in Here we present a recipe for how to create a purely lateral morphology without this drawback. The morphogenesis of this structure will be the focus of a forthcoming publication. With the structure generated by using the given recipe it is possible to remove one component  and to deposit a SAM on the completely freed silicon oxide substrate areas with very high reproducibility. After the silane molecules have bonded covalently, the remaining polymer phase (PS) is removed. The deposition of the SAM is performed by vapor-phase deposition  in a vacH,1H,2H,2H-perfluorodecyltrichlorosilane (FDTS) SAM, the polymer islands were removed by a snow-jet treatment. In By means of a spin-coating process of a polymer-blend solution at a humidity of 45%, a purely laterally phase-separated film consisting of the two polymer components is produced . In FiguThe dependence of the PS island diameter upon the polymerization degree of PS is shown in For a range of relative humidity from 50 to 65%, the resulting phase morphology is different from the situation shown in 3-terminated FDTS-SAM was deposited in the vapor phase. Next, we removed polystyrene by snow-jet treatment as described before. The FDTS as well as the APTES-SAMs withstand this cleaning procedure without any detectable change at their surface, as can be seen in x regions is in the same range as the one of the original Si wafer. The height of the APTES-SAM was found to be 0.7 nm, measured in contact-mode AFM in liquid. Thus, the APTES regions look like half-filled holes (The (water) holes can directly be filled with a silane monolayer. Here we used the (3-aminopropyl)triethoxysilane (APTES) molecule exposing an amino-functional group. After removal of the PMMA layer with acetic acid, the CFed holes .x) areas. The versatile and fast preparation technique makes this approach attractive for many applications of such ultraflat nanopatterned surfaces.These patterned two-phase or three-phase surfaces, which show a high chemical contrast and at the same time an extremely flat topography, make them an ideal template or platform for constructive lithography , cell adPolymer-blend lithography (PBL) makes use of lateral structure formation during the spin-coating process of a polymer-blend film. The structures are transformed into a patterned SAM with two or three different chemical functionalities by a lift-off process. PBL starts with spin-casting of a polymer blend  onto a substrate at a defined relative humidity. By selecting adequate conditions, a polymer blend film with a purely lateral phase morphology is formed. After the selective dissolution of one of the polymer components, the remaining second polymer component can be directly used as a lithographic mask. This lithographic mask, in turn, can be removed by snow-jet lift-off after deposition of a silane monolayer (SAM) on the unprotected areas in the vapor phase.3-terminated FDTS monolayer and the amino-terminated APTES monolayer, and leave at the same time uncovered regions of bare silicon oxide on the substrates.For the examples demonstrated, the fabricated nanopatterned template shows a chemical contrast between the functional group of the silane SAM and the bare silicon oxide. This quasi two-dimensional pattern has about 1 nm topography. The bare silicon oxide surface can be filled with another silane SAM for specific applications. The lateral structure size within the nanoscale pattern is determined by the diameter of the PS islands formed during the spin-coating process. The mean value of the statistically distributed diameters of PS islands can be varied between 90 and 500 nm by changing the molar mass of the PS moiety. Combined with breath figures, this lithographic method can even be used for the fabrication of three-component templates. Here we use it for the patterning of the CFThe quasi two-dimensional chemical patterns open the potential for their application as templates for the subsequent self-assembly of inorganic materials, for cell-adhesion studies, for laterally controlled dewetting, or for constructive lithography. The extreme flatness (rms roughness below 0.5 nm) allows for a highly sensitive monitoring of growth processes by AFM. Together with the chemical variability, polymer-blend lithography (PBL) can become an important tool for studying surface-initiated processes.Polymer solution: Poly(methyl methacrylate)  and polystyrene  were purchased from Polymer Standards Service GmbH and dissolved directly in methyl ethyl ketone . The total concentration of the two polymers was 15 mg/mL and the mass ratio between PS and PMMA was 3:7. To demonstrate the tuning of the diameter of PS islands, a set of polymer solutions were made with various PS molar masses, i.e., 9.58, 35.6, 96 and 248 kg/mol. All other parameters were kept constant.Cleaning of Si substrates and SAM templates: Silicon substrates were used as delivered with their native oxide layer. The substrates and the SAM templates were cleaned by the snow-jet method [2 ice crystals, which were produced by expanding CO2 through a nozzle . In this way, surface contaminants are removed either by mechanical impact or by dissolution in CO2.t method : The wafPreparation of a polymer-blend lithographic mask: The polymer blend films were spin-cast at a speed of ca. 1500 rpm onto silicon substrates cleaned by snow-jet treatment (at least 20 seconds for a 2 cm × 2 cm substrate). For the two-phase SAM templates, the relative humidity was set to 45% during the spin-coating process and for the three-phase templates to 65%. The humidity was controlled by venting the chamber (about 1 L volume) with a mixture of nitrogen and water-saturated nitrogen . The humidity in the chamber was measured by a hygrometer (Testo 635).Fabrication of SAM templates: For the two-phase template the PMMA was selectively dissolved by acetic acid, as shown in H,1H,2H,2H-perfluorodecyltrichlorosilane  and evacuated to a pressure of 50 mbar. The PS islands were later removed by snow-jet blasts. For sufficient impact it is important that the CO2 gas cylinder is at room temperature and has a proper filling level. The polymer mask can be alternatively dissolved in THF, following the protocol described above for acetic acid. For the three-phase template the (3-aminopropyl)triethoxysilane  SAM was deposited onto the silicon surface inside the holes of the lithographic mask in the gas phase, shown in Sample characterization: The polymer blend masks were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The AFM images were made with a commercial multimode system (DI Multimode IIIa) in tapping mode. The samples were scanned under ambient conditions immediately after they had been removed from the solution. SEM images were taken at 2 kV with a LEO 1530 SEM by using a secondary electron detector. All AFM images of the SAM templates were taken in contact mode in the liquid cell filled with demineralized water (Bruker Dimension Icon-PT).File 1Snow-jet treatment of FDTS-SAM.


Age-related macular degeneration (AMD) is the leading cause of visual loss in older population. Angiogenesis is an important factor associated with the development of CNV due to AMD. Treatment of CNV with intravitreal anti-VEGF monotherapy is currently the standard of care. However, not all patients respond to monotherapy, and modified anti-VEGF treatment regimen and combination therapy may target reducing treatment frequency or improving visual outcome. This paper reviews the many clinical trials that have been performed utilizing several treatment regimens. While many trials have shown that this variable therapy is justifiable, further study is required to determine correct regimens and dosage. Age-related macular degeneration (AMD) is one of the leading causes of substantial and irreversible vision loss. The prevalence of AMD can be expected to increase along with life expectancy, which has risen steadily , 2. WithVEGF is a key mediator involved in the control of angiogenesis and vascular permeability and has been shown to be induced by hypoxia in cultured human RPE . VasculaA MEDLINE search of the English language literature from 1990 to present was conducted. The search strategy was based on combinations of medical subject headings (MeSH) and keywords and was not restricted to specific journals or years of publication. The searches were supplemented by handsearching the bibliographies of included studies and reviews.Three antivascular endothelial growth factor (VEGF) therapies are currently used for the treatment of patients with wet age-related macular degeneration (AMD): pegaptanib , ranibizumab , and bevacizumab . Petaganib is an oligonucleotide aptamer and was the first VEGF antagonist to be approved by the US Food and Drug Administration for use in wet AMD. However, wet AMD patients treated with petaganib still experience visual decline , 6. For Ranibizumab (Lucentis) is also a humanized antibody fragment against VEGF which was specifically designed for intraocular use as a smaller antibody fragment to penetrate through the retina better. The Food and Drug Administration (FDA) approved ranibizumab for treatment of subfoveal neovascular AMD in June, 2006. It was the first treatment for AMD shown to improve visual acuity in a substantial percentage of patients. Bevacizumab (Avastin) is a recombinant humanized monoclonal immunoglobulin antibody that inhibits the activity of VEGF. It has a similar action and is related to the ranibizumab compound with respect to its structure. Bevacizumab was approved by the FDA for the treatment of metastatic colorectal cancer in 2004, but it has not been licensed for the treatment of wet AMD or any other ocular conditions. However, it is recently used off-label worldwide not only for wet AMD but also for other ocular disease entities associated with macular edema and abnormal vessel growth. Since 2009, there have been increasing number of studies that have compared the properties of ranibizumab and bevacizumab and investigated their efficacy on AMD. The pivotal phase III Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD (MARINA)  and the P < 0.001) than those achieved by the control groups.In the ANCHOR trial, ranibizumab was compared with verteporfin photodynamic therapy (PDT) and demonstrated similar findings: 90–96% of the ranibizumab-treated versus 64–66% of the PDT-treated patients maintained vision, whereas 34–41% versus 6% of each group, respectively, gained more than 15 letters. These outcomes were significantly better  , 21. TheThe prospect of indefinitely adhering to the monthly treatment schedules of MARINA and ANCHOR has raised ocular and systemic safety concerns as well as convenience and cost issues for patient and physician alike. The identification of alternative dosing strategies capable of reducing the number of required anti-VEGF injections while still achieving visual acuity outcomes similar to those reached in the pivotal trials has since been a subject of great interest. The observed biphasic treatment effect raised the possibility that, after the initial 3-month loading phase, maintenance of VA gain may be achieved with less frequent treatments. A PIER trial evaluated ranibizumab administered monthly for 3 months, followed by quarterly injections, and compared this with sham treatment. Under this schedule, ranibizumab did provide a significant VA benefit; a significantly greater number of patients achieved VA stabilization at 24 months compared with patients receiving sham treatment. However, subgroup analysis revealed that VA gains observed during the first 3 months of treatment were only maintained in 40% of patients over the duration of the trial, and for the remaining 60% quarterly dosing was not suitable , 23. ResMore recently, the Efficacy and Safety of Ranibizumab in Patients with Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (EXCITE) study directly compared the PIER regimen with a fixed monthly treatment arm (0.3 mg ranibizumab) . Althougμm, or new hemorrhage. The current norm in clinical practice with ranibizumab or bevacizumab is to implement an initiation/induction phase followed by an individualized maintenance phase that is modeled after one of two basic approaches: traditional PRN  or “treaOf the three studies, the PrONTO study demonstrated the best visual acuity results. The PrONTO study evaluated an OCT guided, variable-dosing regimen with ranibizumab (0.5 mg) and showed that mean visual acuity improved by 9.3 ETDRS letters at 12 months. Over a 2-year period, mean BCVA outcomes were similar to MARINA and ANCHOR with a mean of 9.9 injections (5.6 in the first year and 4.3 in the second). In comparison, results from the SAILOR study were not as good. In this study, the mean change in BCVA at 12 months from baseline was 0.5 and 1.7 letters in the treatment-naive and previously treated groups, respectively, at the 0.3 mg dose and 2.3 letters in both groups at 0.5 mg. It is worth noting that participants were not monitored as closely in SAILOR as compared with PrONTO, averaging nine visits through 1 year and a mean of 4.9 injections. The 12-month results from SUSTAIN were slightly better than those from SAILOR (mean BCVA from baseline of 3.6 letters), yet still not as good as the monthly treatment trials. In contrast to SAILOR, participants in the SUSTAIN trial were followed monthly (more like PrONTO) and the mean number of injections over the first year was higher at 5.6. Other relatively large studies using a traditional PRN approach have recently been published –33. An aKang and Roh , 35 receIn addition, Gupta et al. evaluated a treat and extend approach with bevacizumab and found nearly identical results at 12 months following a mean of 7.3 injections in the first year . AlthougThe development and propagation of CNV membranes involve proangiogenic factors, vascular permeability molecules, and inflammatory proteins. Current standard treatment with monthly intravitreal injections of anti-VEGF monotherapy can be limited to the angiogenic component of CNV development and burdensome for both the physician and patient. Patients are subjected to increased risk with monthly treatments that may be lessened with treatment options given with less frequency . CombinaPhotodynamic therapy was approved in 2000 by the FDA for the treatment of CNV secondary to AMD. Treatment involves intravenous administration of a light-sensitive dye called verteporfin followed by laser-guided, location-specific activation within the CNV membrane. Activation of the verteporfin molecules incites a phototoxic event within blood vessels, induces endothelial cell damage, platelet aggregation, and eventually leads to thrombosis of vascular channels. Treatment size is limited by the greatest linear diameter of the CNV lesion being treated , 38. While PDT is intended to specifically target CNV vessels, collateral damage to surrounding blood vessels may lead to ischemia of healthy tissue. Following PDT of a CNV membrane, induced ischemia can lead to production of proangiogenic factors, especially VEGF. Therefore, combining verteporfin PDT and anti-VEGF therapy may be beneficial compared with either modality alone, yielding longer treatment-free intervals and requiring fewer intravitreal injections . The RhuFab V2 Ocular Treatment Combining the Use of Visudyne to Evaluate Safety (FOCUS) study is a multicenter, randomized, single-blind study designed to evaluate the safety and efficacy of standard fluence Photodynamic therapy (sfPDT) in combination with intravitreal ranibizumab , 40. It The most effective dosing regimen and monitoring program for anti-VEGF therapy has yet to be firmly established but new treatments are aimed at extending and improving on the efficacy of ranibizumab. VEGF Trap-Eye is a promising new anti-VEGF drug . Structurally, VEGF Trap-Eye is a fusion protein of key binding domains of human VEGFR-1 and -2 combined with a human IgG Fc fragment. Functionally, VEGF Trap-Eye acts as a receptor decoy with high affinity for all VEGF isoforms, binding more tightly. VEGF Trap-Eye differs from established anti-VEGF therapies in its higher binding affinity for VEGF-A and its blockage of placental growth factors-1 and -2 , 42. P < 0.01) [Recently, the 1-year results of two parallel randomized, double-masked phase 3 clinical trials (VIEW 1 and VIEW 2) on the efficacy and safety of VEGF Trap-Eye for the treatment of neovascular AMD were reported . Phase I < 0.01) . In contrast to current anti-VEGF antibodies, which are rapidly cleared, the VEGF Trap-Eye is relatively degraded more slowly. Due to its high binding affinity and the ability to safely inject high doses into the eye, VEGF Trap-Eye may have longer duration of effect in the eye. Its adoption into clinical practice will depend on efficacy at 4- and 8-week intervals. If effective at 4- and 8-week intervals, VEGF Trap-Eye offers a competitive price advantage over ranibizumab and the opportunity to significantly reduce treatment burden on patients and physicians.AMD is a complex mechanism in which a variety of mediators are likely to be involved. Any of these could serve as a potential target for the treatment of ocular neovascularization. In preclinical and clinical studies, several targets have already been evaluated. For example, treatment regimes of a drug blocking the transduction of the signaling cascade within the cell (tyrosine kinase) and one inhibiting the further intracellular production of VEGF  might achieve a better visual outcome . Yet theAge-related macular degeneration (AMD) affects the many elderly population. Before the development of anti-VEGF, the diagnosis of neovascular AMD meant frequently loss of useful vision. But targeted anti-VEGF therapy has significantly improved the treatment of neovascular AMD. The appropriate method, dose, and types of combination therapy remain undetermined but randomized trials are currently continuing and will provide critical insight into the clinical applicability of new regimens. It hopefully can help in the treatment of resistant CNV with longer duration and less frequency between treatments.


Approximately 70% of all patients with myelodysplastic syndrome (MDS) present with lower-risk disease. Some of these patients will initially respond to treatment with growth factors to improve anemia but will eventually cease to respond, while others will be resistant to growth factor therapy. Eventually, all lower-risk MDS patients require multiple transfusions and long-term therapy. While some patients may respond briefly to hypomethylating agents or lenalidomide, the majority will not, and new therapeutic options are needed for these lower-risk patients. Our previous clinical trials with ezatiostat , a glutathione S-transferase P1-1 inhibitor in clinical development for the treatment of low- to intermediate-risk MDS, have shown significant clinical activity, including multilineage responses as well as durable red-blood-cell transfusion independence. It would be of significant clinical benefit to be able to identify patients most likely to respond to ezatiostat before therapy is initiated. We have previously shown that by using gene expression profiling and grouping by response, it is possible to construct a predictive score that indicates the likelihood that patients without deletion 5q will respond to lenalidomide. The success of that study was based in part on the fact that the profile for response was linked to the biology of the disease.RNA was available on 30 patients enrolled in the trial and analyzed for gene expression on the Illumina HT12v4 whole genome array according to the manufacturer’s protocol. Gene marker analysis was performed. The selection of genes associated with the responders (R) vs. non-responders (NR) phenotype was obtained using a normalized and rescaled mutual information score (NMI).We have shown that an ezatiostat response profile contains two miRNAs that regulate expression of genes known to be implicated in MDS disease pathology. Remarkably, pathway analysis of the response profile revealed that the genes comprising the jun-N-terminal kinase/c-Jun molecular pathway, which is known to be activated by ezatiostat, are under-expressed in patients who respond and over-expressed in patients who were non-responders to the drug, suggesting that both the biology of the disease and the molecular mechanism of action of the drug are positively correlated. Myelodysplastic syndrome (MDS) is a clonal stem cell disorder resulting in bone marrow failure and variable cytopenias. Development of new treatment strategies has greatly improved the outlook for patients with MDS. There are three FDA-approved drugs for therapy of patients who have become transfusion-dependent, including two hypomethylating drugs (HMAs), azacitidine and decitabine, and the thalidomide derivative lenalidomide. Patients with higher-risk disease have been shown to benefit from HMA therapy ,2, whileS-transferase P1-1 (GSTP1-1), causes dissociation of the enzyme from the jun-N-terminal kinase/c-Jun (JNK/JUN) complex, leading to JNK activation by phosphorylation. Activated JNK phosphorylates c-JUN, which ultimately results in the stimulation of all myeloid lineages hematopoietic progenitor’s proliferation and maturation. In addition, subsequent activation of the caspase-dependent apoptotic pathway increases reactive oxygen species in human leukemia blast cells. This cascade can trigger apoptosis. In other words, the therapeutic action of ezatiostat appears to include both proliferation of normal myeloid progenitors as well as apoptosis of the malignant clone.Ezatiostat , a glutathione analog inhibitor of the enzyme glutathione Our previous phase 2 study of ezatiostat demonstrated that this drug can elicit a therapeutic response in a proportion of patients with lower-risk MDS . TrilineGene expression profiling studies can define signatures that are capable of improving existing classification and prognosis of multiple diseases, especially malignancies which tend to be heterogeneous or of unknown or uncertain origin. MDS is a group of hematopoietic stem cell disorders that pose a unique challenge for gene expression profiling by virtue of their inherent heterogeneity. However, we have previously shown that profiling can generate distinct expression signatures based on the uniform grouping of patient response to a specific drug therapy . In an aA separate research protocol was submitted to the institutional review boards (IRBs) at the University of Massachusetts Memorial Medical Center, Worcester, MA, and at Saint Vincent’s Comprehensive Cancer Center, New York, NY, seeking permission to perform the microarray analysis as described below. Once the research protocol was approved by the respective IRBs and informed consent was obtained from each patient, samples from lower-risk MDS patients treated with ezatiostat in the phase 2 clinical trial at those institutions were obtained. Mononuclear cells from pre-therapy bone marrow aspirates were stored in Trizol at °C.All patients had low- or intermediate-1-risk MDS as determined by the International Prognostic Scoring System (IPSS) and had not received growth factors for 4 weeks prior to study enrollment. Hematological improvement response was based on International Working Group (IWG) 2006 criteria ,9.6 mononuclear cells using Trizol (Invitrogen) and analyzed for gene expression on the Illumina HT12v4 whole genome array according to the manufacturer’s protocol. RNA was available on 30 patients enrolled in the trial at the two institutions.Total RNA was purified from 5-10 × 10p-value is computed according to how many times the matching scores of the random permutations are more extreme than the actual score. Because in our study the number of samples is small, we opted for not performing the permutation test and focused instead on analyzing the 100 genes with the highest (50) and lowest (50) NMI scores.The selection of genes associated with the responders (R) vs. non-responders (NR) phenotype was obtained using a normalized and rescaled mutual information (NMI) score. This quantity was obtained using a kernel-density-based estimate of the joint probability and the mutual information distribution between the phenotype and each gene profile. The resulting mutual information  was thenTo project the gene profiles into the space of pathways, we used a single-sample Gene Set Enrichment Analysis (ssGSEA) -17. The http://www.broadinstitute.org/msigdb); ii) an internal database of signatures of oncogene activation containing over 300 gene sets defined from data generated in our laboratory, from GEO datasets, and from the biomedical literature; and iii) gene sets representing hematopoietic cell populations [The selection of gene sets/pathways more associated with the responders vs. non-responders phenotype was obtained using an NMI as was done with the gene profiles (see above paragraph). The sources of gene sets/pathways were: i) the C2 sub-collection of curated and functional gene sets from the Molecular Signatures Database (MSigDB) release 2.5  were identified using a sensitive metric based on the normal mutual information  Figure . A majorReduced expression of miR-129 has been found in a variety of primary solid tumors and has been shown to reduce proliferation by targeting the G → S cell cycle kinase CDK6 in lung epithelial-derived cells . InteresIn contrast, miR-155 is over-expressed in patients who responded to ezatiostat. miR-155 has been previously shown to be over-expressed in bone marrow cells of patients with acute myelogenous leukemia (AML) . RecentlSingle-sample Gene Set Enrichment Analysis was performed to find the most salient differences in terms of pathways and biological processes between responders and non-responders. Most notably, three pathways, mTOR, JAK2 and JNK, were all found to be under-expressed in the responders , which may be activated by receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), and G protein-coupled receptors (GPCRs). It has been shown that the PI3K/Akt/mTOR pathway is activated in high-risk MDS, when compared to lower risk or healthy controls . In addiLastly, and most striking, was the finding that the JNK/JUN pathway, which has been shown to be central to ezatiostat’s molecular mechanism of action, is also under-expressed in responding patients. This gene set, as defined by the GEO dataset GDS2081, was derived from expression studies in primary cultured human epidermal keratinocytes, with activated JNK/JUN exposed to the JNK inhibitor drug SP600125 and analyzed on Affymetrix HGU95Av2 arrays . A heatmIn conclusion, a bedside-to-bench strategy correlating MDS patient pre-treatment genomic data with clinical response to ezatiostat has yielded positive markers for this investigational drug’s clinical efficacy. These signature genes and signaling pathways positively correlate with the known mechanism of action of ezatiostat. The genomic signature reported herein that distinguishes responders from non-responders among MDS patients treated with ezatiostat may enable the future selection of patients who are most likely to positively benefit from ezatiostat treatment. These markers could potentially be developed into a clinical diagnostic test for MDS patient sensitivity to ezatiostat treatment.Gail Brown is an employee of Telik, Inc. Azra Raza has received honoraria from Celgene Corporation to serve on their speakers’ bureau. Naomi Galili, Pablo Tamayo, Olga B Botvinnik, Jill P Mesirov and Margarita Roserika Brooks declare that they have no competing interests.NG conceived the study, obtained the Illumina results, and wrote the manuscript. PT, OBB and JPM did the computational analysis and generated the figures. MRB coolected the clinical characteristics and made Table 1. GB and AR conceived the study, assigned the clinical response criteria and participated in the writing of the manuscript. All authors read and approved the final manuscript.


Problem-solving is an executive function subserved by a network of neural structures of which the dorsolateral prefrontal cortex (DLPFC) is central. Whereas several studies have evaluated the role of the DLPFC in problem-solving, few standardized tasks have been developed specifically for use with functional neuroimaging. The current study adapted a measure with established validity for the assessment of problem-solving abilities to design a test more suitable for functional neuroimaging protocols. The Scarborough adaptation of the Tower of London (S-TOL) was administered to 38 healthy adults while hemodynamic oxygenation of the PFC was measured using 16-channel continuous-wave functional near-infrared spectroscopy (fNIRS). Compared to a baseline condition, problems that required two or three steps to achieve a goal configuration were associated with higher activation in the left DLPFC and deactivation in the medial PFC. Individuals scoring higher in trait deliberation showed consistently higher activation in the left DLPFC regardless of task difficulty, whereas individuals lower in this trait displayed less activation when solving simple problems. Based on these results, the S-TOL may serve as a standardized task to evaluate problem-solving abilities in functional neuroimaging studies. Features which typically differ from one variant of the task to another, however, are the number of allowable operators required to achieve the goal state, and the specific constraints (or “rules”) imposed on the problem-solving task (representation). Therefore, conventional tower tasks do not measure the cognitive processes underlying discovered or created problems . Perhaps due to their narrow problem definition and ease of standardization, tower tasks have enjoyed widespread clinical application  . An experimental task like the S-TOL is said to be valid for measuring an intended cognitive function when it produces measurement outcomes  that are consistent with the theory of response behavior that guided its construction . Importantly, the S-TOL was intentionally designed with an eye toward its potential for translation to clinical samples . Therefore, the task was constructed with the aim of achieving high levels of accuracy, even in individuals with possible central nervous system dysfunction, to reduce the likelihood of frustration on the task and to facilitate comparisons between activation blocks containing lower and higher difficulty problems. This approach in designing the S-TOL was considered essential to ensure that any observed differences in patterns of brain activation between clinical and non-clinical groups are not confounded with differences in accuracy on the task. Accordingly, this task incorporated problems requiring no more than three moves to achieve a target configuration, and utilized a simplified response format , thereby reducing the need for complex response devices and minimizing demands on working memory. The development and validation of a standardized tower task that can be readily incorporated into neuroimaging protocols could increase consistency of methods across studies and facilitate comparisons of results between clinical and non-clinical samples.SD = 1.7) of formal education. The ethno-racial composition of the sample according to 2011 Canadian census categories was as follows: Chinese (34.2%), White (21.1%), South Asian (13.2%), Black (5.3%), Filipino (5.3%), Latin American (5.3%), Japanese (2.6%), Korean (2.6%), Southeast Asian (2.6%), West Asian (2.6%), and Other (5.3%). Prior to commencing the neuroimaging protocol, participants completed a brief screening measure to collect demographic information and to rule out the presence of any serious manual, ophthalmic, neurologic , or psychiatric illness .An initial sample of 43 healthy adults provided informed written consent to participate in this study. Four participants were subsequently excluded because they obtained atypically inaccurate performances (<60%) on zero-move (ZM) trials of the S-TOL , and one participant was excluded due to technical problems with an event-marker file for the neuroimaging task. The final sample comprised 38 adults who were recruited from the University of Toronto Scarborough's undergraduate research participant pool as well as the surrounding University community. Participants were largely right-handed (76.3%) and female (60.5%) with an average 13.8 years  is a continuous-wave fNIRS system described in previous studies conducted by our research group  they could move only one ball at a time, and (2) they could not put more balls on a peg than what it could hold. Participants completed three practice trials and had the opportunity to ask questions about the task. After the practice trials, participants were given the following instruction:The task consisted of two trial types: multiple-move (MM) trials, which included problems that could be completed in a minimum of either two or three moves, and ZM trials, during which participants observed two boards displaying identical configurations   psychometric properties when compared to its predecessor, the NEO-PI-Revised  channels were excluded for each participant primarily because of saturation with ambient light when contact with the skin was not optimal or in lateral channels due to interference from hair shafts. Time synchronization markers denoting the beginning and end of each block were delivered to the fNIRS acquisition device using a serial connection. Based on the markers that separated MM and ZM blocks, data for local baseline segments and activation segments were extracted and compared. The activation segments consisted of blocks  that were 60-s in duration. Each block consisted of 6 trials that began with a 7-s observation period and a 3-s response period. The local baseline segments included the first 10-s of each block, representing 20 observations sampled at 500-ms intervals at the beginning of each block. The primary measure of interest in this study was oxygenated hemoglobin (oxy-Hb), although deoxygenated hemoglobin and total hemoglobin measurements were also collected but not reported because oxy-Hb is more commonly associated with neural activity.Raw fNIRS light intensities were manually screened to exclude channels which had poor signal quality  and subsequently underwent signal processing to exclude physiological artifacts using a low-pass filter with a finite impulse response and a linear phase filter with an order of 20 and a cut-off frequency of 0.1 Hz  that obtained similar performances across MM and ZM conditions, t(23) = 2.01, p = 0.05. Contrasts of activation associated with MM and ZM conditions for these highly accurate participants (n = 24) were visualized separately from the less accurate participants (n = 14) to highlight differences in functional activation between these groups. All data were visualized on a standard MRI template , primary analyses contrasted ZM and MM blocks. Therefore, the main difference between ZM and MM blocks was that the latter required participants to solve problems that required a minimum of two or three moves (rather than none) to achieve the goal state. According to the Related-Samples Wilcoxon Signed Rank Test, participants made more errors on MM as compared to ZM trials, ants n = 4 that obThe fNIRS time-series data were analyzed with multilevel models  using the False Discovery Rate (FDR) approach  in all multilevel analyses. Thus, a two-unit change on this effect-code represents the unstandardized mean difference in oxy-Hb across the ZM and MM conditions.Within the context of the present study, multilevel models take into account that the data points comprising each participant's experimental time-series measurements  are nested within the respective participants and that the number of data points may be unbalanced across participants due to signal processing. Thus, variance in the dependent variable  is partitioned into within-person (Level-1) and between-person (Level-2) components, allowing predictor terms to be represented at both the level of the experimental condition  and at the level of the participant, respectively. In the primary analyses, we examined the Level 1 effect of problem-solving difficulty on the S-TOL across 16 fNIRS channels, controlling for Type I error  attained 97.5% (SD = 3.72) accuracy on ZM problems, 88.9% (SD = 15.8) on two-move problems, and 89.2% (SD = 12.73) on three-move problems. Mean RT was 161 ms (SD = 914) for correct ZM problems, 896 ms (SD = 155) for two-move problems, and 1018 ms (SD = 206) for three-move problems.On the S-TOL, all participants (T-scores (with a normative mean of 50 and SD of 10) for the participants' self-reported levels of deliberation as measured by the NEO-PI-3 was within normal limits . There were no significant correlations (Spearman's rho) between Deliberation scores and accuracy indices from the S-TOL (Table p's > 0.05).n = 38) is presented in Table The results of multilevel analyses comparing oxy-Hb across the MM and ZM conditions for all participants (n = 24). Significant increases in oxy-Hb compared to ZM were observed in 12 channels, encompassing the anterior aspects of the right  and left  DLPFC. Conversely, significant decreases in oxy-Hb compared to ZM were observed in four channels encompassing the medial PFC . The results of these analyses are portrayed in Figure Table Fourteen participants did not meet the 90% accuracy threshold. Table Beyond analyses of accuracy on the S-TOL and its relationship to PFC activity on simple and complex problems, we examined the personality trait deliberation as an external validator of the aforementioned findings. As previously mentioned, deliberation refers to the tendency to think carefully before acting. The left DLPFC is crucial for problem-solving, specifically, the extraction of goal information and the generation of an internal problem representation. Accordingly, we predicted that deliberation would moderate the relationship between problem-solving and left DLPFC activation on the S-TOL. Specifically, we hypothesized that individuals higher in trait deliberation would show consistently high levels of left DLPFC activation across both higher and lower levels of task difficulty. Less deliberate individuals were hypothesized to demonstrate greater left DLPFC activation when solving problems of higher difficulty but less activation when solving simpler problems.For this ancillary analysis, the left DLPFC was defined as our region of interest using channels 1, 2, 3, and 4. The time-marked data points from these channels were aggregated with list-wise deletion because some fNIRS data were missing at random. The aforementioned data screening and filtering yielded a total of 18, 305 data points across 21 participants for analysis of the left DLPFC. To test our hypothesis that Deliberation might moderate activity in the left DLPFC on the S-TOL, we estimated a multilevel model that examined the Level 1 effect of problem-solving, the Level 2 effect of Deliberation, and the problem-solving × Deliberation cross-level interaction in the prediction of oxy-Hb. The last term of this model was of particular interest because it tested whether or not within-person differences in oxy-Hb across the ZM and MM conditions varied as a function of between-person differences in trait Deliberation.b = −0.01, SE = 0.00, t(18282) = −12.51, p < 0.001]. In order to probe the nature of this significant interaction, the effect of problem-solving in the left DLPFC was examined at high (+1 SD) and low (−1 SD) levels of Deliberation  = 18.13, p < 0.0001], as compared to those who reported higher levels of this trait . That is, whereas those participants who reported higher levels of Deliberation did not show a significant difference in oxy-Hb across the ZM and MM conditions, those participants who reported lower levels of this trait showed higher activation in the MM condition relative to the ZM condition. Furthermore, Deliberation was not significantly related to oxy-Hb during the MM condition , but it was marginally associated with increased activation during the ZM condition . This latter result suggested that participants who self-reported higher levels of Deliberation may have been more engaged in problem-solving on the S-TOL even on ZM trials. This significant interaction is illustrated in Figure As expected, this analysis uncovered a significant cross-level interaction between problem-solving and Deliberation on the S-TOL [The present study described a new computerized version of the TOL . When activation on these problems was contrasted with ZM problems, increases in oxy-Hb were observed primarily in the left DLPFC and right medial PFC. Conversely, decreased activation was observed in superior channels in the medial PFC and a single channel in the right IFG. The results of this study partly converge with prior neuroimaging research using other versions of the TOL which found largely bilateral DLPFC activation during the active completion of problems requiring two or more moves to achieve a goal configuration (Baker et al., 9%. When Whereas increased activation was observed in primarily left DLPFC for two- and three-move problems, significant deactivation was also detected in dorsal aspects of the medial PFC and right IFG. Many studies using the TOL did not report on areas which showed significant deactivation on this task; however, Boghi et al.  found prAn extensive body of research has shown that whereas the performance of attention-demanding cognitive tasks is typically associated with increased activation in the DLPFC and decreased activation in the medial PFC (Fox et al., Reduced activity in the right IFG was an unexpected finding given that inhibition and attentional control, functions frequently ascribed to this region (Hampshire et al., As an external validator of the current findings, patterns of left DLPFC activation on the S-TOL were examined in relation to a personality trait descriptor of decision-making, referred to as deliberation. This trait reflects the extent to which individuals think carefully before acting or speaking (McCrae and Costa, A number of limitations should be considered when interpreting the current findings. First, a greater number of females were recruited in this study. There is emerging evidence suggesting that females completing the TOL may show greater activation bilaterally in the DLPFC and right parietal cortex than their male counterparts (Boghi et al., Anthony C. Ruocco designed the study, developed the problem-solving task, and wrote the majority of the manuscript. Achala H. Rodrigo assisted in the development of the problem-solving task, conducted statistical analyses, and wrote portions of the manuscript. Jaeger Lam and Bryanna Graves wrote portions of the manuscript. Stefano I. Di Domenico and Hasan Ayaz assisted with statistical analysis and wrote portions of the manuscript.The optical brain imaging instrumentation utilized in the present research was manufactured by fNIR Devices, LLC. Dr. Hasan Ayaz was involved in the development of the technology and thus offered a minor share in fNIR Devices, LLC. All other 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.


Laryngeal carcinoma is a common head and neck cancer with poor prognosis. Patients with laryngeal carcinoma usually present late leading to the reduced treatment efficacy and high rate of recurrence. Despite the advance in the use of molecular markers for monitoring human cancers in the past decades, there are still no reliable markers for use to screen laryngeal carcinoma and follow the patients after treatment. Epigenetics emerged as an important field in understanding the biology of the human malignancies. Epigenetic alterations refer to the dysregulation of gene, which do not involve the alterations of the DNA sequence. Major epigenetic changes including methylation imbalance, histone modification, and small RNA dysregulation could play a role in the development of human malignancies. Global epigenetic change is now regarded as a molecular signature of cancer. The characteristics and behavior of a cancer could be predicted based on the specific epigenetic pattern. We here provide a review on the understanding of epigenetic dysregulation in laryngeal carcinoma. Further knowledge on the initiation and progression of laryngeal carcinoma at epigenetic level could promote the translation of the knowledge to clinical use. Head and neck squamous carcinoma is estimated to be the sixth most common malignant tumor worldwide. Of which, laryngeal carcinoma is the second most common head and neck squamous carcinoma . AccordiMost of the laryngeal carcinoma patients are male . MoreoveDiagnosis of laryngeal carcinoma depends on the following: symptoms ; signs ; assistant examination  . Among tIn order to find out the definite cause of laryngeal carcinoma and the available index for the diagnosis of this disease, researchers put their attention on the research of molecular markers. Since 1983, the epigenetics of human cancer draw the researchers' attention . It is nWu and Morris defined epigenetics changes as “changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence” . Thus, aIn 1969, Griffith and Mahler proposed that modification of DNA base is a possible way to modulate gene expression . In humaChromodomain-helicase-DNA-binding protein 5 (CHD5) is involved in modifying chromatin structure. CHD5 protein contains chromatin remodeling, helicase, and DNA-binding motifs. Reduced CHD5 expression has been reported in neuroblastoma . MutatioE-cadherin ) is a 97-kDa transmembrane glycoprotein. Functional E-cadherin protein contains five extracellular cadherin repeats, a transmembrane region and a cytoplasmic tail. E-cadherin functions as adhesion protein of epithelial cells and is involved in cell attachment and the cell polarity and tissue architecture . Loss ofP16 or cyclin-dependent kinase inhibitor 2A is a cell cycle regulator controlling G1 to G2 cell cycle arrest. P16 could bind to cyclin-dependent kinase 4 protein (Cdk4) resulting in loss of interaction between Cdk4 and cyclin D1 leading to cell cycle arrest . The expDAPK is one of the most frequently methylated genes in laryngeal carcinoma. Park et al. demonstrated the highest methylation frequencies (87%) in the laryngeal carcinoma tissues . SimilarAs mentioned above, focal methylation of specific tumor suppressor genes is common in cancer cells. The number of methylated tumor suppressor genes is higher in cancer cells in comparison with the analogous normal counterpart. However, if we look at the density of methylated CpG dinucleotides, there is a substantial reduction of 5-methylcytosine content . The gloAn example of hypomethylated genes in laryngeal carcinoma cells is S100A4. S100A4 is an acidic calcium-binding protein and functions as oncogene in a variety of human malignancies. In oral squamous cell carcinoma, S100A4 could suppress expression of the epithelial protein E-cadherin . It coulThe reasons for DNA hypomethylation remain unclear. It is suggested that the global changes in methylation patterns are associated with the dysregulation of the methylation machinery. With a better understanding of the functional role of different DNA methyltransferases, it is now proposed that the loci-specific focal hypermethylation is linked to the dysregulation of de novo methyltransferase 3A and 3B. On the other hand, the global loss in methylcytosine content is resulting from the functional error of DNMT1. Global hypomethylation could lead to activation of oncogenes . It is nIn laryngeal carcinoma, it has been reported that hypomethylation is linked to the polymorphism of the genes regulating DNA methylation including methionine synthase (MTR) and 5, 10-methylenetetrahydrofolate dehydrogenase, 5, 10-methenyltetrahydrofolate cyclohydrolase, 10-formyltetrahydrofolate synthetase (MTHFD1), and methylenetetrahydrofolate reductase (MTHFR). According to Kruszyna et al., patients with 2756AG or GG genotypes on MTR have a higher risk of developing laryngeal carcinoma .Histone is the structural unit of nucleosomes and is important to the packing of DNA. Posttranslational modification of histone  could control the activity of the surrounding DNA . ModificIn human genome, the protein-coding portion is only about 2% . The majPIWI-interacting RNA, transcription initiation RNA, and microRNA are 3 major small noncoding RNA encoded in the human genome. PIWI-interacting RNA (26–31 bp) was discovered in 2006 and functions through interacting with the Argonaute family protein PIWI . PIWI-inIn comparison with the other 2 small noncoding RNAs, the pathway of microRNA biogenesis and function in human cancers is much clearer. MicroRNA is small noncoding RNA that regulates gene expression at posttranscriptional level . Mature Head and neck cancer has a distinctive microRNA expression in comparison with their normal counterpart, and the microRNA expression is suggested to play a significant part in the pathogenesis of head and neck cancer . In laryLet-7 was discovered in 2000 and is one of the well-studied tumor suppressing microRNAs . Let-7 iMicroRNA-7 is a proliferation suppressor . There aDownregulation of microRNA-206 has been reported in breast carcinoma, leiomyoma, lung carcinoma, renal cell carcinoma, and rhabdomyosarcomas –117. In In laryngeal carcinoma, studies on tumor-suppressing microRNA are comparably more than the oncogenic microRNA. One representative example of the oncogenic microRNA identified in laryngeal carcinoma is microRNA-21. MicroRNA-21 upregulation was first reported in human glioblastoma in 2005 . OverexpMicroRNA is a kind of epigenetic regulator. MicroRNA itself however is also controled by epigenetic mechanisms including DNA methylation.http://www.mirbase.org/), microRNA 9 has 3 isoforms. They are hsa-mir-9-1 (MI0000466), hsa-mir-9-2 (MI0000467), and hsa-mir-9-3 (MI0000468). In oral and oral pharyngeal carcinoma, microRNA-9 is a candidate tumor suppressor and could control cell proliferation through regulating the PTEN pathway. MicroRNA-9 methylation has recently been reported in oral pharyngeal carcinoma including laryngeal carcinoma [According to microRNA registry miRBase , we could foresee that epigenetic studies on laryngeal carcinoma will change from candidate gene approach to global high-throughput profiling. This provides an opportunity to explore and dissect the disease from different perspectives. More importantly, suitable epigenetic markers could be identified through the gaining understanding of the disease.


During the imagine-self perspective, participants with high psychopathy showed a typical response within the network involved in empathy for pain, including the anterior insula (aINS), anterior midcingulate cortex (aMCC), supplementary motor area (SMA), inferior frontal gyrus (IFG), somatosensory cortex, and right amygdala. Conversely, during the imagine-other perspective, psychopaths exhibited an atypical pattern of brain activation and effective connectivity seeded in the anterior insula and amygdala with the orbitofrontal cortex (OFC) and ventromedial prefrontal cortex (vmPFC). The response in the amygdala and insula was inversely correlated with PCL-R Factor 1  during the imagine-other perspective. In high psychopaths, scores on PCL-R Factor 1 predicted the neural response in ventral striatum when imagining others in pain. These patterns of brain activation and effective connectivity associated with differential perspective-taking provide a better understanding of empathy dysfunction in psychopathy, and have the potential to inform intervention programs for this complex clinical problem.While it is well established that individuals with psychopathy have a marked deficit in affective arousal, emotional empathy, and caring for the well-being of others, the extent to which perspective taking can elicit an emotional response has not yet been studied despite its potential application in rehabilitation. In healthy individuals, affective perspective taking has proven to be an effective means to elicit empathy and concern for others. To examine neural responses in individuals who vary in psychopathy during affective perspective taking, 121 incarcerated males, classified as high ( Empathy, the social-emotional response that is induced by the perception of another person's affective state, is a fundamental component of emotional experience, and plays a vital role in social interaction . To create the medium- and low-psychopathy groups, two groups of volunteers were matched to high scorers on age, race and ethnicity, IQ (WAIS), comorbidity for DSM-IV Axis II disorders, and past drug abuse and dependence, from pools of incarcerated volunteers scoring between 21 and 29 , and volunteers scoring below 20 on the PCL-R , respectively. Participants were paid for their participation in the study.One hundred twenty-four adult right-handed males between the ages of 18 and 50, incarcerated in a medium-security North American correctional facility, volunteered for the study and provided informed consent to the procedures described here, which were approved by the Institutional Review Boards of the University of New Mexico and the University of Chicago. Participants underwent the PCL-R assessment, including file review and interview, conducted by trained research assistants under the supervision of Dr. Kiehl. Three participants were excluded for excessive movement in the scanner. Participants scoring 30 and above on the PCL-R were assigned to the high-psychopathy group  Axis I diagnosis, lifetime history of a psychotic disorder or psychotic disorder in a first degree relative, or current alcohol or drug use.Participants in the MRI scanner were instructed to adopt either a self-perspective or an other-perspective while viewing visual stimuli depicting right hands and right feet of individuals in painful and non-painful situations [stimuli and procedure similar to Jackson et al. ]. All stImagine that these situations are happening to you”), and for the imaging-other perspective blocks (“Imagine that these situations are happening to someone else”). A colored border (blue or yellow) around the stimuli was used to further cue participants about which perspective to employ. Block order was pseudo-randomized across participants. Painful and non-painful scenarios were randomized within each block. Post-scan debriefings were conducted to make sure that subjects did follow the perspective-taking instructions.A mixed block-event related fMRI design [24 blocks (12 imagine-self and 12 imagine-other) with a total 120 trials] was employed, in which instructions were given to the subjects at the beginning of each block, i.e., for the imagine-self perspective blocks .High-resolution T1-weighted structural MRI scans were acquired using a multiecho MPRAGE pulse sequence  yielding 128 sagittal slices with an in-plane resolution of 1.0 × 1.0 mm.N = 121 .Functional images were processed with SPM8  in Matlab . For each participant, functional data were realigned to the first image acquisition of the series and re-sampled to a voxel size of 2 × 2 × 2 mm3. Structural T1 images were co-registered to the mean functional image and segmented using the “New Segment” routine. A group-level structural template and individual flow fields were created using DARTEL, and the flow fields were in turn were used to spatially normalize functional images to standard MNI space. Data were smoothed with an 8 mm full-width at half maximum (FWHM) isotropic Gaussian kernel. Three participants were eliminated from further analysis due to issues related to movement or image quality, leaving p < 0.001 and with an extent threshold based on Gaussian random fields set to control the whole-brain family-wise error rate (FWE) at p < 0.05.Statistics were calculated at the first level using the general linear model. The design matrix included three regressors for each stimulus category (detailed above), representing the event onsets and their time and dispersion derivatives. Movement parameters from the realignment output were included as regressors of no interest. All participants were entered into a second-level pooled analysis, and full brain activations were thresholded voxelwise at a priori coordinates (FWE-corrected p < 0.05). Beyond existing literature on the processing of empathy-inducing stimuli in healthy populations, there may be additional cortical or subcortical brain regions that contribute to abnormal processing of these regions in psychopathy. For instance, the ventral striatum has been found to be over-reactive in adolescents with conduct disorder as well as sexual sadists  were defined using the MarsBar ROI toolbox. We focused on brain regions that were of maximal importance to the hypotheses under investigation, informed by the existing literature on empathy for pain in particular from a meta-analysis of 32 fMRI studies of empathy for pain . This network includes the anterior insula (aINS), anterior midcingulate cortex (aMCC), supplementary motor area (SMA), inferior frontal gyrus (IFG), dorsomedial prefrontal cortex (dmPFC), mPFC, and somatosensory cortex, in both hemispheres (Table The entire sample of 121 participants (regardless of their psychopathy level) showed significant neuro-hemodynamic increase in the network of regions involved in the actual experience of physical pain under the imagine-self trials , when compared to low-scoring incarcerated controls.During the imagine-other perspective, individuals with low scores on the PCL-R compared with individuals with high scores on the PCL-R, showed greater signal change in the SMA, right mPFC, intraparietal sulcus, precentral gyrus, and parahippocampal gyrus/amygdala, pSTS, dorsal aINS and dorsal ACC. In participants with high scores on the PCL-R, the imagine-other perspective was associated with greater activation in the dlPFC and ventral striatum , whereas it negatively correlated with aINS activity during imagine-other perspective . Factor 1 was negatively correlated with response in aINS during third-person perspective . Activity in the dmPFC was negatively associated with both Factor 1  and Factor 2  during imagine-self perspective. The hemodynamic response in the dlPFC was positively correlated with both Factor 1  and Factor 2  during imagine-other perspective. The response in the ventral striatum during imagine-other perspective significantly correlated with scores on Factor 1  during imagine-other perspective. No significant correlation was found in imagine-self perspective with either Factors 1 and 2. See Table The hemodynamic response in the aINS was significantly greater in individuals scoring high on psychopathy  during imagine-self perspective, and the reverse was found for imagine-other perspective Figure . Factor Functional connectivity analyses seeded in the anterior insula revealed distinct patterns in functional coupling between the low- and high-psychopathy groups. During imagine-self perspective, individuals scoring low on the PCL-R showed a negative connectivity between the aINS and the hippocampus and the OFC Figure . In the Functional connectivity analyses seeded in the right amygdala showed distinct patterns of co-variations depending on the perspective adopted in controls vs. psychopaths. During imagine-self perspective, controls exhibited a significant negative coupling between the amygdala and ventral and mPFC, while participants with high scores on the PCL-R showed a positive coupling with the pSTS/TPJ, ventral and mPFC, and dlPFC Figure . During Perspective taking while observing or imagining other's feelings has been described as an empathic attentional set that facilitates other-oriented emotional and motivational responses congruent with the perceived welfare of that person , both conditions of imagine-self and imagine-other in pain were associated with signal increase in brain regions implicated in the perception of pain and distress, when viewing body parts suffering injuries or facial expressions of pain  was found in participants who scored high on psychopathy. In addition, functional connectivity analyses, seeded in the right aINS during imagine-self perspective negatively co-varied with activation in the hippocampal gyrus and OFC in control participants (low on psychopathy), and was positively coupled with the right pSTS region in psychopaths. During imagine-other perspective, the aINS positively covaried with activity in the right dlPFC and PCC in controls, and negatively with the OFC and PCC in high psychopaths. Altogether, the hemodynamic response in the aINS shows distinct profiles of activation depending on whether participants adopted an imagine-self or imagine-other perspective taking. These results from the imagine-other perspective condition support two recent functional neuroimaging studies in children with conduct disorder (Lockwood et al., Signal change in the right amygdala was detected during imagine-self perspective in all participants, and during imagine-other perspective in controls. The hemodynamic response in the amygdala was inversely correlated with individual scores on PCL-R Factor 1 during imagine-other perspective. This is in line with most neuroimaging studies of psychopathy that documented reduced amygdala response to fearful and aversive stimuli (Marsh and Blair, It is very interesting to note that imagine-self perspective was associated with activity in the amygdala in psychopaths when they focus on their own affective reaction. While most studies report a reduced response in the amygdala in psychopaths, an fMRI study conducted on a small number psychopaths and controls found increased activation in the right amygdala in the psychopath group with respect to controls when viewing negative IAPS pictures (Müller et al., Finally, the increase of activity in the ventral striatum during imagine-other perspective in psychopaths, which was predicted by their scores on Factor 1 of the PCL-R, is an intriguing finding. This could suggest that psychopaths not only experience blunted vicariously arousal to others' pain and reduced feelings of concern when adopting their perspective, but they may in fact find the distress of others pleasurable or positively arousing. The ventral striatum is selectively recruited during reward anticipation in healthy participants (Diekhof et al., There is general consensus among theorists that the ability to adopt and entertain the psychological perspective of others has a number of important consequences, including empathic concern (e.g., Blair, 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.