KS), a cancer which has become prominent in the HIV era and whose incidence may be changing with the rollout of antiretroviral therapy (ART). To achieve this, we evaluated HIV‐infected adults receiving care between 2007 and 2012 at any of three medical centers in Kenya and Uganda that participate in the East Africa International Epidemiologic Databases to Evaluate AIDS (IeDEA) Consortium. Through IeDEA, clinicians received training in KS recognition and biopsy equipment. We found that the overall prevalence of KS among 102,945 HIV‐infected adults upon clinic enrollment was 1.4%; it declined over time at the largest site. Among 140,552 patients followed for 319,632 person‐years, the age‐standardized incidence rate was 334/100,000 person‐years . Incidence decreased over time and was lower in women, persons on ART, and those with higher CD4 counts. The incidence rate among patients on ART with a CD4 count >350 cells/mm3 was 32/100,000 person‐years . Despite reductions over time coincident with the expansion of ART, KS incidence among HIV‐infected adults in East Africa equals or exceeds the most common cancers in resource‐replete settings. In resource‐limited settings, strategic efforts to improve cancer diagnosis in combination with already well‐enumerated at‐risk denominators can make healthcare systems attractive platforms for estimating cancer incidence.In resource‐limited areas, such as sub‐Saharan Africa, problems in accurate cancer case ascertainment and enumeration of the at‐risk population make it difficult to estimate cancer incidence. We took advantage of a large well‐enumerated healthcare system to estimate the incidence of Kaposi sarcoma ( Cancer incidence is one of the most fundamental parameters in cancer epidemiology. Incidence encompasses both the natural history of a malignancy and the effects of interventions to reduce occurrence Kaposi sarcoma (KS) is an example of a malignancy in a resource‐limited setting which would benefit from knowledge about incidence. From a perspective of percentage of all recorded cancers, KS was among the most common cancers in sub‐Saharan Africa even before the human immunodeficiency virus (HIV) epidemic To overcome the challenges inherent in a resource‐limited setting, we used a newly assembled collection of healthcare system‐derived databases, the International Epidemiological Databases to Evaluate AIDS (IeDEA) Consortium in East Africa, to derive a well‐substantiated (in terms of numerator and denominator) estimate of cancer incidence in a large representative population of HIV‐infected adults in sub‐Saharan Africa. We focused on KS, not only because of its ease of measurement and clinical relevance, but also to demonstrate how adding the selective measurements to an already well‐enumerated healthcare system‐based population has the potential to be a powerful platform for the study of other cancers.We performed a cohort analysis among HIV‐infected adults (≥18 years old) receiving care at one of three medical centers in Kenya and Uganda that participate in the East Africa IeDEA Consortium. Established in 2005 by the U.S. National Institutes of Health, the IeDEA Consortium has as its main procedural objective the harmonizing of data collected by geographically disparate, but representative, cohorts of persons infected with HIV or at risk for HIV We included data from HIV‐infected adults who were receiving care at one of three medical care systems: the Immune Suppression Syndrome (ISS) Clinic located at the Mbarara Regional Referral Hospital in Mbarara, Uganda Prior to 2007, at all sites, the diagnosis of KS was largely clinical only  with rare pathologic confirmation. In January 2007, skin punch biopsy capacity for histopathologic diagnosis (provided free to patients) was introduced at IDI, followed in October 2008 at the ISS Clinic and AMPATH. Through support from IeDEA, clinicians received training in the recognition of KS, the importance of early pathologic diagnosis, and how to perform a biopsy. Biopsy interpretations were adjudicated by dermatopathologists at the University of California, San Francisco, who had available to them, at their discretion, the use of immunohistochemical staining against the latency‐associated nuclear antigen of KSHV. For the purpose of this analysis, a KS diagnosis was defined by the presence of a clinical‐only diagnosis of KS or a clinical KS diagnosis accompanied by definitive or indeterminate pathologic confirmation. When there were both a date of diagnosis on clinical grounds and a date of pathologic confirmation, we used the earlier date as the overall date of diagnosis. Patients with a clinical diagnosis and a negative biopsy were not considered to have KS.Age, sex, weight, ART use, hemoglobin, and CD4+ T‐cell count were obtained from the ambient electronic medical record databases. Because ART adherence information was not recorded routinely, we assumed that once started on ART, patients continued on ART.In an analysis of KS prevalence, we first determined the frequency of KS among patients upon enrollment in their respective medical systems, limiting to those newly enrolled on or after 1 January 2007 who had also not yet started ART. Prevalence upon clinic enrollment in ART‐unexposed patients serves as a rough correlate of KS incidence in the community and is one measure of the burden of KS facing HIV care clinics. Because KS might go unnoticed at an administratively demanding initial clinic visit, we assumed that any KS recorded within 30 days of enrollment was present at enrollment and was therefore prevalent.For the incidence analysis, we studied all patients who had no diagnosis of KS, as of the time they became eligible for the analysis, for the subsequent occurrence of KS. Therefore, 1 January 1 2007 was considered time zero for patients enrolled prior to then, and the date of the initial clinic visit was time zero for those enrolled after 1 January 2007. We calculated incidence rates, as total new cases of KS divided by the at‐risk person‐time , for the overall population as well as subgroups defined by age, sex, calendar year, medical center, ART status, and CD4+ T‐cell count. For ART status, we classified the population according to person‐time in which ART was not used (“non‐ART use”) and person‐time in which ART was used (“ART use”). Given the importance of capturing the early time period after ART initiation 3 , but, at the ISS–Uganda, there was no evidence for systematic change in prevalence (P for trend = 0.52).Among 102,945 patients, a total of 1409 KS diagnoses were made at clinic enrollment, of which 63% were clinical only, 36% were biopsy confirmed, and 0.9% were clinical with an indeterminate biopsy. Over time, the frequency of diagnoses accompanied by biopsy increased (to about 50%) and then plateaued  0.4–4.2 years). Among non‐ART users, the total person‐time was 88,566 person‐years , while among ART users, it was 231,038 person‐years  . Observation ended with the development of KS in 1025 patients, death in 8609, transfer to another medical facility in 6214, administrative closure in 76,775, and loss to follow‐up in 47,929.P for trend <0.001; Fig. Of the 1025 incident KS diagnoses , 63% were clinical only, 36% were biopsy confirmed, and 1.1% were biopsy indeterminate. Over time, the frequency of diagnoses accompanied by biopsy steadily increased , KS incidence was 146/100,000 person‐years  among non‐ART users, 32/100,000 person‐years  among all ART users, and 41/100,000 person‐years  among new ART users.When evaluating KS incidence among subgroups, there was no clear pattern associated with age, but incidence was higher among men at 526/100,000 person‐years  compared to women , and higher with decreasing CD4 count  among those not on ART, 245/100,000 person‐years  among all ART users, and 325/100,000 person‐years  among only new ART users.When estimating cancer incidence, resource‐limited settings, in contrast to resource‐rich ones, have difficulties in both the ascertainment of cancer cases  and the true at‐risk population . We have demonstrated how a large healthcare system‐based platform–East Africa IeDEA–can overcome many of the prior limitations of estimating cancer incidence in sub‐Saharan Africa and be used to precisely estimate the incidence of KS, one of the most common cancers in the region. The cornerstone of this approach is the very large population that is enumerated longitudinally at the individual person level in electronic databases with standardized coding at the IeDEA sites. To this large population, we enhanced KS diagnosis through clinician training and provision of skin punch biopsy equipment for histopathologic diagnosis. These strategic efforts increased the specificity of diagnosis compared to the heretofore convention of clinical‐only diagnosis Conventionally, the gold standard approach for estimating cancer incidence in both resource‐rich and resource‐limited settings is to derive it within a defined geographic area. In resource‐rich settings, this is accomplished by combining data from the population‐based cancer registries (numerator) and a relevant geographic population census (denominator). The resultant incidence estimates are both accurate and precise because functional underlying healthcare systems diagnose and record most, if not all, cancers that occur, cancer registries collect these diagnoses, and up‐to‐date census data from large geographic regions enumerate the at‐risk population. In contrast, in sub‐Saharan Africa, the CI5 project deemed that only four cancer registries–each covering only a small region of a single country–had data of sufficient quality Instead of targeting a geographic population, we used healthcare populations to estimate KS incidence in HIV‐infected individuals, an approach that has been attempted by others in Africa Table . Prior t3 stratum can be appropriately evaluated, and when doing so, KS incidence in Southern Africa was still considerably higher  than our estimate . Another potential explanation is that although not described in the report, it is believed, on historical grounds, that a large fraction of the KS diagnoses in Southern Africa were clinical only, which are prone to false positivity 3 group  compared to Bohlius et al.  or Rohner et al. . We speculate–as do the original authors–that this may be because of underascertainment of KS during the 2004 to 2007 period in Southern Africa. In fact, in Rohner et al., when analysis is restricted to 2007 to 2010, overall KS incidence on ART  is much closer to our estimate, and, if children were excluded, it would even be closer. Finally, differences in underlying prevalence of KSHV may also contribute to the differences in KS incidence. Methodologic uncertainty in measuring KSHV infection status precludes clarity on this point, but we earlier found that, when using the same serologic assay in representative samples, KSHV prevalence was higher in Uganda than in Zimbabwe or South Africa An approach more similar to ours was used to estimate KS incidence among HIV‐infected adults attending any one of six HIV clinics in Southern Africa IeDEA As this study was conducted during the heart of the scale‐up of ART in East Africa, it offers insights into the impact of ART on the development of KS. In what we believe is a unique analysis, we found that the prevalence of KS declined at the largest of our sites (AMPATH). Among several potential explanations for this, the most obvious is that more HIV‐infected patients are achieving access to ART at earlier HIV disease stages and hence averting the development of KS while untreated in the community. KS incidence among those in care also declined, which is consistent with the growing proportion of patients on ART and achieving higher CD4 counts. Finally, the impact of ART is most clearly seen in the comparison of CD4 count stratum‐specific KS incidences between ART users and non‐ART users. Yet, none of these findings directly estimates what observers most commonly want to know: What is the reduction in KS incidence caused by ART if it could be tested in a randomized trial? As discussed in detail elsewhere While we believe this work is among the most comprehensive attempts of KS incidence estimation in Africa by virtue of the provision of training and biopsy capacity to optimize the sensitivity and specificity of KS case ascertainment and the large well‐enumerated at‐risk denominator, there are still limitations. The first is that biopsy confirmation did not accompany all diagnoses, thus leaving open the possibility that some of the clinical‐only KS diagnoses were incorrect. We know that during this time period, at these sites, an important fraction of clinically suspected cases of KS concurrently sent for pathologic interpretation did not get confirmed as KS 3), KS incidence was considerably lower  but, again, still comparable to other important cancers  as measured in resource‐rich regions Our findings provide perspective about the impact of KS on the HIV healthcare system both now and into the future in East Africa. Our ASIR  provides a good estimate of the overall burden that KS poses. To provide context, the most common cancer among women in the United States is breast  and among men is prostate ; the ASIR for all cancers combined is 460/100,000/person‐years The same difficulties we described for the numerator and denominator in KS incidence estimation also apply to other cancers in the region, and hence, a healthcare system‐based approach to incidence estimation should be considered for these as well. This is most relevant for HIV‐infected populations for which the ART rollout has rapidly created a large well‐enumerated primary care system but will likely apply to HIV‐uninfected populations in the future as the lessons from the ART rollout get applied more generally. Again, the elegance of this approach rests primarily in the individual‐level enumeration of a large at‐risk population. If highly sensitive and specific diagnosis of a given cancer can be added to the population, accurate and precise cancer incidence estimation follows. We believe that this is most immediately plausible for cancers which feature feasible screening and case ascertainment, such as cervical, breast, and hepatocellular.In summary, we have demonstrated how a strategic investment to improve diagnosis in combination with an already well‐enumerated large denominator allowed us to use a healthcare system‐based population to derive an improved estimate of the incidence of KS in East Africa. We found that, overall, KS incidence exceeds the most common cancers in resource‐rich settings, but it is declining as the ART rollout extends. Yet, KS incidence among patients on ART does not fall to zero, even among those with “normalized” CD4 counts. Attention now must turn to why HIV‐infected patients on ART still develop KS.


The drive towards organic computing is gaining momentum. Interestingly, the building blocks for such architectures is based on molecular ensembles extending from nucleic acids to synthetic molecules. Advancement in this direction requires devising precise nanoscopic experiments and model calculations to decipher the mechanisms governing the integration of a large number of molecules over time at room-temperature. Here, we report on ultrahigh-resolution scanning tunnelling microscopic measurements to register the motion of molecules in the absence of external stimulus in liquid medium. We observe the collective behavior of individual molecules within a swarm which constantly iterate their position to attain an energetically favourable site. Our approach provides a consistent pathway to register molecular self-assembly in sequential steps from visualising thermodynamically driven repair of defects up until the formation of a stable two-dimensional configuration. These elemental findings on molecular surface dynamics, self-repair and intermolecular kinetic pathways rationalised by atom-scale simulations can be explored for developing new models in algorithmic self-assembly to realisation of evolvable hardware. The interaction of simple individual units within an ensemble that leads to advanced collective behavior without any chain of control is generally referred to as swarm intelligence 1341367Similar to the self-organised structures at the macroscale, unsupervised ordering also takes place at molecular length scales, driven by the process of self-assembly141541622232425262829The subject matter that remains to be fully clarified is that of the mechanisms leading to the formation of a self-assembled molecular architecture in real-time under innate conditions at room-temperature. This is of significant research interest as knowing the stepwise evolution of molecular systems and pattern formation can serve in modelling algorithms that can be explored in effectively predicting design of new materials formed through self-assembly. Additionally, such information can have broad implications in sectors where bottom-up self-assembly plays a crucial role such as biomolecular computationin situ probe microscope capable of capturing stunning real-space snaphots and time-elapsed images of fullerene molecular dynamics at the liquid-metal interface at room-temperature. We observe the local dynamic relocation of individual fullerene molecules and subtle differences in molecular motion as a function of their respective position within the swarm. Crucially, we were able to provide direct experimental evidence for defect formation and healing at a molecular scale. We further rely on atomic-scale molecular dynamics (MD) simulations to compute the intermolecular and metal-molecule interaction energies and quantify the differences in molecular motion as a function of their respective position within the matrix. There is a clear thermodynamic benefit for self-assembly into the observed automatically-healed and tightly-packed patterns. These preliminary design rules gleaned from precise nanoscopic measurements and theoretical calculations can be directly applied in designing new algorithms and refining if possible previously reported step-by-step equilibration rules framed for agent-based algorithms32In this article we report on the application of a high resolution 60) molecular layer were performed is shown in 60 molecules with an intermolecular distance (measured between the center of one fullerene cage to the other) of ~1 nm is shown in the zoomed-in three-dimensional in situ STM image (recorded 10 minutes after drop casting the C60 solution on the gold surface). Previously it has been shown that it is possible to trigger a cascading electrical response through a well-ordered molecular layer by applying an input voltage pulse at a specific location over a single molecule within the layer, opening up possibilities for programming molecular circuitsA schematic description of the liquid-cell within which the STM imaging measurements on a fullerene (Cin situ STM snapshot (recorded immediately after deposition on gold thin film supported on mica substrate) of C60 molecules in three dimensional format showing regions which are partially ordered and the majority of the molecules in a disordered phase as a result of competitive adsorption mechanisms. Zooming-in on a selected region shows well resolved single C60 molecular units within local domains  which follows the geometry of the selfish herd60 molecules assembling on Au(111) in bulk solvent medium). From the simulation cells  of 30 ns of room temperature dynamics. 60 coordination number is increased in steps. The RMSF values are computed from the centres of mass of each C60 molecule and the values are averaged over multiple molecules (binned according to C60 coordination number) and temporal structures. The simulation data indicates that the low-coordination species are more mobile than buried species, thereby substantiating the STM findings.To better understand and quantitatively map the location-specific differences in single molecule mobility, we performed molecular dynamics (MD) simulations taking into account the actual experimental conditions olecules  and the n energy . Time- ain situ STM image of a C60 molecular layer recorded immediately after liquid-phase deposition on gold thin film supported on halide substrate (see methods section for sample preparation) is shown in 60 molecules near holes as measured by RMSF fluctuations in computed C60 positions  and data can be recorded closer to video-rates by upgrading the electronics design (bandwidth of power amplifier and feedback control system) of the scanning probe microscope and achieving suitable signal-to-noise ratioCapturing minute structural fluctuations in the cooperative behaviour of molecules and registering features such as molecular error correction is experimentally challenging especially at the liquid-solid interface222939in-situ STM image of a molecular layer showing the arrangement of individual molecules within the matrix and areas adjacent to the molecules that remain empty. The computed free energy profile in 60 molecule to Au(111) plus an additional 1.0 eV that is obtained from intermolecular packing, as the C60 node is connected to six neighbouring nodes. This thermodynamic driver towards pristine SAMs is due purely to C60-C60 van der Waals packing interactions. The plot shows an average benefit of −0.2 ± 0.1 eV obtained for each additional contact made as the C60 goes from an isolated adsorbate (coordination number of zero) to a maximally coordinated (coordination number of six) node in the hexagonally-close packed monolayer. Note the free energy benefit becomes small in the middle of cluster-formation  were purchased from Alfa Aesar in powder form and dispersed in n-tetradecane solvent (Sigma Aldrich) at a concentration of 10−2 M using a low-power sonic bath procedure for ten minutes followed by centrifugation at 1500 rpm for 60 minutes. 10 μL of the as-prepared supernatant C60 solution was gently drop casted onto the gold thin film, placed within a Teflon-based liquid-cell for subsequent STM imaging at room-temperature. For the metal substrate 100 nm of Au (99.99%) was electron-beam evaporated  onto freshly cleaved mica  at a rate of 1 Å s−1. Such metal thin films on mica were found to contain nanoscale defects that impeded molecular self-assembly. For engineering defect-free and ultra flat gold thin films, we employ halide support platforms on which gold was evaporated using identical protocols as reported previouslyThe Cn-tetradecane liquid medium. The in situ STM measurements were performed in state-of-the-art noise free laboratories with excellent noise, vibration, temperature and humidity control. The bias voltage is applied to the sample and the metal tip is grounded through the high-sensitive preamplifier. The pre-amplifier employed in this study is capable of current detection as low as 1pA. Image processing was performed using Gwyddion 2.39. To avoid tip artifacts the STM metal probe was calibrated by measuring standard reference differential conductance spectra and imaging over blank Au(111) surface.The STM measurements were performed in constant-current mode at room-temperature using a Veeco Scanning Tunnelling Microscope, Nanoscope IIIa, Multimode (Scanner model: E- Scanner). A mechanically cut Pt/Ir wire  was used as the STM probe. For real-time tracking of molecular dynamics the STM probe was scanned at a speed of 1 ms/line with a tip drift rate of ~1 nm/min in 60 physisorption energies on Au(111) were calculated using two independent methods, van der Waals corrected DFT60 monolayer assembly was modelled using molecular dynamics simulations. Full details of the methods and models used are given in the supporting information.The CHow to cite this article: Nirmalraj, P. N. et al. Capturing the embryonic stages of self-assembly - design rules for molecular computation. Sci. Rep.5, 10116; doi: 10.1038/srep10116 (2015).


Kaposi sarcoma is a tumour caused by human herpes virus 8, also known as Kaposi sarcoma-associated herpes virus. It usually affects the skin and oral mucosa; however, it can also sometimes affect the lungs, the liver, the stomach, the bowel, and lymph nodes. Several body sites may be affected simultaneously. The involvement of the tonsils is rare. We described an isolated localization of Kaposi's sarcoma of the right tonsil in a HIV-positive patient. HIV/AIDS represents an important clinical and public health issue worldwide. The estimated HIV/AIDS prevalence in 2012 was 35,300,000 worldwide in 2012 . In case of severe immunodeficiency, the probability of occurrence of several opportunistic disorders  can increase. Kaposi's sarcoma (KS) is a frequently reported neoplasia in HIV-positives; it is caused by human herpes virus 8 (HHV-8), also known as KS-associated herpes virus (KSHV) , stomachHere, we present a case of KS of the right tonsil in a HIV-positive patient, surgically treated and exposed to antiretroviral drugs.A 42-year-old man was admitted to the Emergency Department of the S.Francesco Hospital, Nuoro, Italy, because of a painless swelling, moderate bleeding, located in his right tonsil. He referred to its occurrence without any other relevant symptoms 1 month ahead.Oral examination shows a purple swelling  in the uThe histopathological analysis identified a KS Figures , and 5. His past medical history includes a borderline hypertension treated with a diet approach.μL and >100,000/mL, respectively (CDC stage C2). On the basis of the TIS classification (μL) and the level of viral load decreased gradually .After surgical treatment his CD4 lymphocyte count and HIV viral load were 207 cells/fication  17], he, heμL anμL.After 15 months since KS diagnosis the patient is alive and KS-free with a CD4 cell count of 342 cells/KS is an important opportunistic disease, which frequently occurs in HIV-positive patients characterized by a severe immunodeficiency. The KS incidence in the general population is 1 case per 100,000 inhabitants , whereasThe AIDS-related KS lesions, affecting the skin of the upper trunk and face , 3, and The therapeutic choice and the following prognosis are extremely variable, depending on the stage and the KS type.The AIDS Clinical Trials Group Oncology Committee published criteria for the evaluation of the epidemic KS . The staThis classification includes two group of patients: “Good risk category (T0I0S0)” and “Poor risk category (T1I1S1).” The patient that we described was classified as T0I0S0.KS can be treated with radiotherapy, chemotherapy, biologic agent as interferon alfa, and surgery: in HIV-positive patients it can be managed with HAART or with a combination of this therapeutic approach.HAART seems to be indispensable in the treatment of the AIDS-related KS, alone or in combination with systemic or/and local therapy . HAART cRadiotherapy is an effective palliative treatment reducing pain, bleeding, and oedema. Cutaneous KS is highly radiosensitive as described by Donato et al. in a group of 38 AIDS-associated KS lesions . They shChemotherapy (CHT) can be local  or systemic (intravenous). Local CHT, based on bleomycin, cisplatin, and vinblastine, can represent an alternative therapy for limited cutaneous KS with complete or partial response rates (range: 60–92%) , 26. SysInterferon alfa, showing immunomodulatory, antiviral, and antiangiogenetic actions, can be used alone or in combination with HAART. It can favour a remission of the KS lesions in 20 to 60% of the exposed cases , 29. ThePatients with clinical progression despite CHT and/or HAART can be currently approached with the so-called “Target Therapy”  and thalidomide, metalloproteinases (MMPs), and cytokine signalling pathway inhibitors) . MMPs arBiopsy of the lesion is deemed mandatory for the histological diagnosis, but in cases of isolated cutaneous and visceral KS surgical excision is recommended in association with HAART, as showed in our review , 14, 16.The patient we managed underwent tonsillectomy and HAART with complete eradication of the tumours.Isolated KS of the tonsil is very rare and can simulate benign or malignant lesions, such as pyogenic granuloma, carcinomas, and sarcomas. Histological diagnosis, HIV testing and CT scans of the chest and abdomen, and an endoscopy of the upper gastrointestinal tract can be helpful for an appropriate stage of the tumours and, consecutively, to plan a better therapeutic approach.


AAO with pore diameter of ~70 nm and pore length of ~50 μm was used as the template for synthesis of NWs. After deposition of Au as the seed layer, NWs with a structure of Ni/CuOx/Ni were grown with a length of ~12 μm. The lengths of 1st Ni, CuOx, and 2nd Ni were ~4.5 μm, ~3 μm, and ~4.5 μm, respectively. The Ni/CuOx/Ni device exhibits bipolar resistive switching behavior with self-compliance characteristics. Due to the spatial restriction of the current path in NW the Ni/CuOx/Ni NW devices are thought to exhibit self-compliance behaviour. Ni/CuOx/Ni NWs showed bipolar resistive changes possibly due to conducting filaments that are induced by oxygen vacancies. The reliability of the devices was confirmed by data retention measurement. The NW-based resistive switching memory has applications in highly scalable memory devices and neuromorphic devices.We demonstrate synthesis of Ni/CuO They are candidates for next-generation non-volatile memories because of their simple metal/insulator/metal structure, low power consumption, high stacking density, fast switching speed, high scalability, simple fabrication, and multistate behaviors12342O103O42SnO4To improve the device performance and to increase the density of memory, the size of the devices must be reduced. Such reduction can be studied by using low-dimensional nanomaterials such as nanowires (NWs), nanotube, or nanodots. Low-dimensional materials have different characteristics from bulk and thin-film materials due to nanoscale sizes, since the materials are confined in low dimensions and have very high surface-to-volume ratios. These nanomaterials have unique chemical, physical, or optical behavior that makes them candidates for use in nanoscale device applications. The resistive switching characteristics of nanostructure materials, such as Cux NWs have been shown to exhibit RRAM characteristics, but only resistive switching layer was synthesized using AAO templatex/Ni NW devices exhibit self-compliance bipolar resistive switching behavior.Currently, bottom-up approaches have been used to synthesize various nanomaterials, e.g. nanodots, nanowires, and nanotubes1116181921The synthesis process of Ni/CuOx/Ni NWs is schematically illustrated in x/Ni NW RRAM devices. After Ni, CuOx, and Ni NW growth, total NW length was ~12 μm . When current is applied, the Ni+ ions react with electrons (e−) and are changed to metallic Ni at the cathode side. The CuOx consisted of CuO and CuO2 formed simultaneously. The coexistence of Cu2O and CuO can be explained by the charge transfer reactions and diffusion. The reaction of copper ions with OH− can yield Cu2O or CuO during ECD process. Among various materials that can be synthesized by ECD we chose Ni as the electrodes and CuOx as the resistive switching materials. Both Ni and CuOx can be easily deposited by ECD processes. In addition, the process is simple, inexpensive; can be done at low temperatures.In this experiment, Ni and CuOx/Ni NWs were measured with Pt electrodes in voltage sweep mode (DC). The voltage was applied at one Pt electrode and the other Pt electrode was grounded. To compare Pt/Ni/CuOx/Ni/Pt with Pt/SiO2/Pt, Pt electrodes without Ni/CuOx/Ni NWs were fabricated on the SiO2 substrate (x/Ni/Pt and Pt/SiO2/Pt. The current−voltage (I–V) curves of Pt/Ni/CuOx/Ni/Pt . When the set and reset voltages are of opposite polarity, the RRAM device is called a bipolar switching device. The operation voltage is found to be high in this device structure. This is due to the length of CuOx. The device is based on NWs and the length of CuOx is about 3 μm. It is believed that the operation voltage can be reduced if the NW length for resistive switching layer is reduced and/or the properties of CuOx layer is engineered by controlling the process parameters, for example, the current density, pH, deposition temperature, etc. Ni/CuOx/Ni device shows self-compliance behavior in the set part, which means that the devices do not need CC. Data retention by the Ni/CuOx/Ni NW device was measured at RT for 2 h. It maintained high resistance state (HRS) or low resistance state (LRS) for 2 h (f the NW . When a  for 2 h .os)2324x/Ni NW device before set or reset process the CuOx layer contains many Vos. When positive bias is applied, Vos diffuse from anode to cathode (grounded electrode) and form metallic filaments under the electric field during the set process26x layer can increase the diffusion mobility of Vos and increase the local electric field. Furthermore, the one-dimensional nature of NWs in this confined configuration provides a linear path for Vos diffusion, and can give a stable switching characteristic to the device. During the reset process heat released by Joule heating disrupts the conducting paths.Resistive switching in the NWs is thought to be associated with migration of oxygen vacancies (VSET (5.2 to 7.2 V) was wider than the range of VRESET (−4.5 to −4.0 V). The difference is related to the nature of the processes. The set process is affected by the competition among different filamentary paths at randomly uncertain filament sizes, orientations, and locations, so the process has a large random componentRESET distribution is expected.The range of Vx/Ni NW RRAM, both the metal (Ni) and oxide (CuOx) layers were ~70 nm in diameter. This small diameter constrains the filament path along which the current flows. Therefore the device shows the self-compliance current behavior. The CC is essential to keep the RRAM from failing permanently. However these devices show self-compliance current and do not need CC during the operation process. The reason to show self-compliance behavior is related to defect concentration/species in resistive switching layer. Further study is under way to investigate the exact nature to show the self-compliance behavior in our device structure.The device was fabricated with a metal/oxide/metal structure in a single NW using ECD process. In the Ni/CuOWe used ECD process, which is a ‘bottom-up’ technique, to fabricate the NW structure. Bottom-up processing can be used to synthesize smaller geometries. ECD process does not require the vacuum equipment and etching step; process time is short and the process step is simple in comparison to top-down techniques. This experiment shows the feasibility of NW RRAM devices fabricated using ECD process. In this experiment, metal/insulator/metal structured NWs could be scaled down to nanoscale dimension and exhibit RRAM characteristic with self-compliance behavior. The properties of oxide layers can be controlled by adjusting the current density, pH of solution, and/or deposition temperature during ECD process; NW diameter can be controlled by changing the dimension of AAO template. Furthermore NW lengths can be changed by controlling the deposition time. These properties suggest the possibility of controlling NWs for possible application to next-generation RRAMs.x/Ni NWs that show RRAM characteristics were synthesized by ECD process in an AAO template. We demonstrated the feasibility of using ECD to produce Ni/CuOx/Ni NW RRAM devices. The devices showed bipolar RRAM characteristic and self-compliance behavior. Due to the spatial restriction of the current path in NW the Ni/CuOx/Ni NW devices exhibit self-compliance behaviour. Because of the self-compliance behavior, this device did not need CC during the measurement process. The resistive switching mechanism is related to migration of Vos, which form conductive filaments when an electric field is applied, whereas Joule heating may break the filaments. The range of set voltage Vset (5.2 ~ 7.2 V), which is wider than the range of reset voltage VRESET (−4.5 ~ −4.0 V) because the set process, the formation of conducting filaments has a stochastic component. Ni/CuOx/Ni NW devices retained data for more than 2 h. In this study the feasibility Ni/CuOx/Ni NW in RRAM was demonstrated. Since it is possible to control the NW lengths, diameters, and CuOx characteristics by controlling the deposition time, AAO diameters, and ECD process conditions, the technology presented in this work can be a good candidate for NW-based device applications.Ni/CuOx/Ni NWs were fabricated using ECD on AAO nanotemplate with a pore diameter of ~70 nm obtained by two-step anodization2) was electro-polished twice in a perchloric acid HClO4/C2H5OH (1:4 in volume ratio) solution for 300 s at 7 °C. As the first anodization step, the Al sheet was anodized at 40 V in 0.3 M oxalic acid (H2C2O4) solution for 24 h at 7 °C. Then the AAO sheet was etched in a mixed solution of 0.2 M H2CrO4 and 0.4 M H3PO4 for 4 h at 60 °C. Then the second anodization was performed for 12 h. After the second anodization, the pores were widened in 0.1 M H3PO4 for 30 min at 30 °C. The Al sheet was sliced into 1 × 2 cm2 samples, which were immersed in supersaturated HgCl2 solution. To obtain an AAO layer, the barrier layer was removed by immersion in 0.1 M H3PO4 for 40 min at 30 °C. This process yielded AAO with a thickness of 50 μm and a pore diameter of ~70 nm.Ni/CuOx/Ni NWs were formed on the AAO template  sulfate pentahydrate (Aldrich) and 3 M lactic acid (Aldrich). After adding a certain amount of 5 M NaOH, the solution was left overnight with magnetic stirring. The stable solution was then adjusted to desired pH 93132x was deposited by ECD on the Ni layer of the AAO under constant current of 4 mA for 30 min at 60 °C. The sample was rinsed with DI water, then Ni was electrodeposited on the CuOx layer in the AAO under the constant current of 4 mA for 2 h at 20 °C. Finally, the AAO layer was dissolved by immersing the samples in 1 M NaOH aqueous solution for 4 h at RT; Ni/CuOx/Ni NWs were remained thereafter. The samples were rinsed in DI water, then centrifuged for five times to spread the NWs.Ni/CuOtemplate . A 120-nx/Ni NWs were drop-cast on a 100-nm-thick SiO2/Si substrate. For the tip contact in the probe station, first Pt electrodes (15 × 15 μm2) were deposited at the end of the NW and again larger Pt (30 × 30 μm2) pad was deposited at end of the electrode. Focused ion beam was used for Pt electrode deposition.To measure electrical resistive switching properties Ni/CuOHow to cite this article: Park, K. and Lee, J.-S. Controlled synthesis of Ni/CuOx/Ni nanowires by electrochemical deposition with self-compliance bipolar resistive switching. Sci. Rep.6, 23069; doi: 10.1038/srep23069 (2016).


Active learning text classification is a supervised machine learning approach that has been shown to significantly reduce the manual annotation workload by semi-automating the citation screening process of systematic reviews. In this paper, we present a new topic detection method that induces an informative representation of studies, to improve the performance of the underlying active learner. Our proposed topic detection method uses a neural network-based vector space model to capture semantic similarities between documents. We firstly represent documents within the vector space, and cluster the documents into a predefined number of clusters. The centroids of the clusters are treated as latent topics. We then represent each document as a mixture of latent topics. For evaluation purposes, we employ the active learning strategy using both our novel topic detection method and a baseline topic model . Results obtained demonstrate that our method is able to achieve a high sensitivity of eligible studies and a significantly reduced manual annotation cost when compared to the baseline method. This observation is consistent across two clinical and three public health reviews. The tool introduced in this work is available from  Wallace et al. Whilst good results are obtained in the clinical domain, Miwa et al. Z with a set of documents D and a set of words W . The goal is to determine those latent topics that best describe the observed data. In PLSI the probability distribution of latent topics is estimated independently for each document. In practice, this means that the complexity of the model  grows linearly with the size of the collection. A further disadvantage of PLSI is the inability of the underlying model to generalise on new, unseen documents (i.e. the model is not fully generative). Extending upon of PLSI, LDA assumes that topic distributions are drawn from the same prior distribution which allows the model to scale up to large datasets and better generalise to unseen documents.Topic models are machine learning methods that aim to uncover thematic structures hidden in text. One of the earliest topic modelling methods is the probabilistic Latent Semantic Indexing (PLSI) In this article, we present a novel topic detection model to accelerate the performance of the active learning text classification model used for citation screening. Our topic detection method can be used as an alternative approach to the LDA topic model to generate a topic-based feature representation of documents. The proposed method uses a neural network model, i.e., paragraph vectors k-means clustering algorithm and treats the centroids of the clusters as representatives of latent topics, assuming that each cluster corresponds to a latent topic inherent in the texts. After detecting latent topics in a collection of documents, we represent each document as a k-dimensional feature vector by calculating the distance of the document to the k cluster centroids. Additionally, our topic detection model computes the conditional probability that a word is generated by a given topic and thus readily determines a set of representative keywords to describe each topic. The topic-based representation of documents is used to train an active learning text classification model to more efficiently identify eligible studies for inclusion in a review. The contributions that we make in this paper can be summarised in the following points:1.We propose a novel topic detection method that builds upon the paragraph vector model. We introduce various adaptations to the paragraph vector method that enable the underlying model to discover latent topics in a collection of documents and summarise the content of each topic by meaningful and comprehensive text labels.2.We incorporate the new topic detection method with an active learning strategy to support the screening process of systematic reviews.3.We conduct experiments, demonstrating that our topic detection method outperforms an existing topic modelling approach when applied to semi-automatic citation screening of clinical and public health reviews.To our knowledge, our work is the first that utilises the vector representations of documents produced by the paragraph vector model for topic detection. We hypothesise that documents lying close to each other in the vector space form topically coherent clusters. Based on this, our approach clusters the paragraph vector representations of documents by applying the 2In this section, we detail our proposed topic detection method. We then provide an overview of the active learning process used in our experiments and discuss the evaluation protocol that we follow to asses the paragraph vector-based topic detection method.2.12.1.1w has n parameters in its word vector: N context words that occur before Several approaches on representing the meaning of words using mathematical expressions such as vectors and matrices have been proposed, with neural network models recently gaining much attention 2.1.2d, its representation is defined as a parameterised vector d denotes the document including the sequence of words. By modelling and maximising the probability using both word and paragraph vectors, the paragraph vectors are adjusted to capture co-occurrence statistics of words within the documents. Just as word vectors capture similarities between words, paragraph vectors capture similarities between documents. Paragraph vectors representing documents covering the same topic are thus likely to lie close to each other in the vector space. Recent work has used paragraph vectors to detect similarities between Wikipedia articles and research papers More recently, a neural network model has been proposed that is able to induce word vectors and paragraph vectors jointly 2.1.3Topic models assume that a set of documents has a specific number of latent topics, and words in a document are probabilistically generated, given the document’s topics. For example, if a topic assigns high probabilities to the words “alcohol”, “drunk”, and “accidents”, we can infer that the topic is about alcohol-related accidents. Our novel contribution is the development of a topic detection method using the paragraph vector model. To aid the study identification process of systematic reviews, it is useful to capture semantic similarities between articles and group studies according to the latent topics within them. Since typical approaches to topic models are based on bags-of-words, important information that can be used to calculate semantic similarity, e.g., word order, is lost k-means clustering algorithm to obtain K cluster centres of the paragraph vectors. As a distance metric for k-means clustering, we use the cosine similarity between paragraph vectors , previous work has demonstrated that the cosine of the angle between word or paragraph vectors provides robust results K cluster centre vectors K latent topics K-dimensional topic-based representation K cluster centroids. The i-th feature value of the topic-based document vector determines the degree of correlation between the document and the i-th latent topic and is calculated as follows:2.1.4w is generated given a document Given that paragraph vectors are trained by solving word prediction tasks, we can compute the conditional probability w is generated given the i-th i-th topic as follows:Using the cluster centre vectors, we can determine a set of words that best describe a given topic, since the cluster centre vectors are in the same vector space as the paragraph vectors. The probability 2.1.5“… feeding practices for infants and young children…” and “childhood obesity”). Moreover, it can be noted that both models capture synonymous or semantically related words that occur as keywords in the same topic .The automatically assigned topic descriptions show that the two topic detection methods tend to induce thematically coherent topics which are also representatives of the underlying abstract. For example, topics 3 and 4 extracted by the paragraph vector-based topic detection method seem to be related to two of the key points discussed in the abstract  In our experiments, we simulate a human feedback active learning strategy 2.3We report the performance of the active learner when applied to the first stage of the screening process . Cross-validation experiments are performed on two publicly available clinical datasets 2.3.1k-means algorithm to cluster the paragraph vectors into 300 clusters which resulted in a topic-based representation of 300 dimensions. With regard to the baseline LDA topic model, we used the freely available MALLET toolkit In order to maximise the performance of the active learner, we tune the parameters of the topic modelling methods. Specifically, we train the paragraph vector-based topic detection method by setting the dimensionality of word vectors to 300, the dimensionality of document vectors to 1000 and the number of training epochs to 500. We then applied the 2.3.2N is the total number of citations, FN the number of true positive (eligible studies), true negative (ineligible studies), false positive (studies that are incorrectly classified as eligible) and false negative instances (studies that are incorrectly classified as ineligible), where the superscript M and A denote manual and automatic screening decisions, respectively. In the definition of burden, the sum We evaluate the performance of the active learning process, over different learning iterations, using two metrics, namely Yield and Burden As a further evaluation, we use the work saved over sampling at 95% recall (WSS@95%) which shows the percentage of ineligible citations that can be safely and automatically excluded  when the underlying active learner yields a recall performance of 95%. Previous approaches 3We investigate the performance of active learning, in terms of yield and burden, over an increasing number of manually labelled instances that are used for training. During the last iteration of the active learning process, both yield and burden are 100% since the active learner has identified all eligible studies but with the maximum manual annotation cost .In the clinical COPD dataset, the AL_PV method shows approximately the same burden performance with the AL_LDA model. However, our active learning strategy converged faster to a high yield value when compared to the baseline AL_LDA method. The AL_PV method improved the yield performance of the baseline model by approximately 3–7% in the COPD dataset. For a given manual annotation workload of 17% , the AL_PV method automatically identified 91% of relevant studies compared to 87% of relevant instances retrieved by the AL_LDA method. By increasing the manual annotation workload to 20%, the AL_PV method achieved a yield performance of 96% while the baseline AL_LDA a yield performance of 89%. With regard to the Cooking Skills dataset , we observe that during the early learning iterations the performance obtained by the AL_PV model slightly fluctuated and in some cases the model obtained a lower yield and burden performance than the AL_LDA. In subsequent learning iterations, the AL_PV achieved a superior yield and burden performance compared to the baseline.3.1k-means algorithm to generate k cluster centroids. The AL_BoW_K-Means method uses k-means over a standard BoW representation of documents. The AL_Average_WV performs k-means clustering over the mean word representation of documents, i.e., the average of vectors for words that appear in a document. Word vectors are obtained by the word2vec tool In this section, we evaluate the paragraph vector-based topic detection method by computing the work saved over sampling at 95% recall (WSS@95%). We further implement two additional baseline methods, namely the AL_BoW_K-Means and the AL_Average_WV. The two baseline methods follow a similar approach to our proposed method to compute a vector representation of documents. Firstly, they apply the 4The experiments that we conducted demonstrate that the proposed topic detection method can improve upon a state-of-the-art semi-automatic citation screening method In our approach, we followed a retrospective evaluation protocol Moreover, we will investigate alternative uses of topic modelling techniques that can further facilitate the study identification phase in systematic reviews. Specifically, although the literature of some disciplines is indexed using well-organised  bibliographic databases, e.g., MEDLINE 5In this paper, we presented a new topic detection method to support the screening phase of systematic reviews. Our proposed method uses a neural network model to identify clusters of semantically related documents. By treating the cluster centroids as representatives of latent topics, we enable the model to learn an informative and discriminative feature representation of studies. This new topic-based representation of studies is utilised by an active learning text classification model to semi-automatically identify citations for inclusion in a review and thus directly reduce the human workload involved in the screening phase.We evaluated our approach against an active learning strategy that employs topic-based features extracted by Latent Dirichlet Allocation (LDA) in both clinical and public health reviews. Experimental evidence showed that the neural network-based topic detection method obtained an improved yield and burden performance when compared to the baseline method. Additionally, we demonstrated that in four out of five reviews, the proposed method drastically reduced the manual annotation cost while retaining 95% of eligible studies in the final review.The authors declare that they have no conflict of interest.


Kaposi sarcoma (KS) is a malignant disease most commonly diagnosed in the setting of a human immunodeficiency virus (HIV) infection and in patients receiving immunosuppressive treatment. Pulmonary KS has never been reported in association with endogenous Cushing’s syndrome (CS).A 60-year-old woman presented with symptoms and signs of CS. Adrenal CS was confirmed by standard biochemical evaluation. Imaging revealed a right adrenal lesion (diameter 3.5 cm) and multiple pulmonary nodules, suggesting a cortisol-secreting adrenal carcinoma with pulmonary metastases. The patient underwent right adrenalectomy with a pathohistological diagnosis of an adrenal adenoma. Subsequent thoracoscopic wedge resection of one lung lesion revealed pulmonary KS with positive immunostaining for human herpes virus 8 (HHV-8). HIV-serology was negative. Hydrocortisone replacement was initiated for secondary adrenal insufficiency after surgery. Post-operative follow up imaging showed complete remission of all KS-related pulmonary nodules solely after resolution of hypercortisolism.KS may occur in the setting of endogenous CS and may go into remission after cure of hypercortisolism without further specific treatment. Endogenous Cushing’s syndrome (CS) is a rare disorder with an incidence of ca. 1/100.000 population [pulation . The sympulation . The diaKaposi sarcoma (KS) is a vascular, highly malignant tumor involving blood and lymphatic vessels [ vessels  with an  vessels ,6. A dys vessels . For thi vessels ,8. KS ha vessels . Accordi vessels . The the vessels ,12.We recently experienced the extraordinary case of an adrenal CS associated with pulmonary KS, which reached a complete remission after treatment of CS by adrenalectomy. To our knowledge this is the first reported case of pulmonary KS related to endogenous CS.A 60-year-old woman was admitted to our endocrine department with a diagnosis of Cushing’s syndrome (CS) presumably caused by a cortisol-secreting adrenal carcinoma with pulmonary metastases. The patient reported a progressive course of central obesity, moon-shaped face, muscle atrophy and weakness, asthenia and emotional disturbance over the last six years. During this period she had developed arterial hypertension, which was poorly controlled despite five antihypertensive drugs. Five years ago abdominal magnetic resonance (MR) imaging had been performed in the local hospital for further evaluation of a suspected liver hemangioma, and revealed an incidental finding of a 2.5 cm lesion in the right adrenal. No endocrinological evaluation was performed. Three years before admission she underwent abdominal computed tomography (CT) imaging for evaluation of recurrent hypertensive crises. No change of the adrenal tumor size was found. Screening for pheochromocytoma and primary hyperaldosteronism was negative. In the weeks before presentation at our department the patient complained of polyuria and exertional dyspnoe. The family physician diagnosed diabetes mellitus, which was treated with dietary measures. Furthermore, abdominal CT imaging was repeated, which showed an increase of the adrenal tumor size to 3.3 cm. The patient was subsequently admitted to the local hospital for further endocrinological investigation.18 F-FDG for both the adrenal lesion and the pulmonary nodules was observed  levels were suppressed indicating an adrenal origin of the hypercortisolism  testing was negative and testing of the immune status showed normal CD4 cell counts  as well as a normal CD4/CD8 ratio  eight weeks after adrenalectomy. The differential white blood cell count revealed relative lymphopenia before the adrenalectomy which was normalised 8 weeks after adrenalectomy  indicating longer term severe immunosuppression. Clinical symptoms of CS were present for at least 5 years and an incidental adrenal lesion had already been diagnosed in MR imaging 5 years before. CS is associated with immune dysregulation and CS patients have been shown to display impairment in immune regulation, including changes in both the percentage of specific lymphocyte subsets and specific lymphocyte functional activities . The patOur case also emphasizes the importance of hormonal workup in adrenal incidentalomas including screening for cortisol-producing adenoma, hyperaldosteronism and pheochromocytoma which was delayed and only incompletely performed in our patient.The combination of cortisol excess with positive FDG-uptake of both the adrenal tumor and the pulmonary nodules clearly suggested a cortisol-secreting adrenal carcinoma with pulmonary metastases. Malignant adrenal lesions have a higher standardized uptake value (SUV) than benign lesions, but in most cases have a tumor diameter >5 cm . MoreoveCurrently, four different types of KS are distinguished, the classic, endemic, epidemic HIV related and the iatrogenic type which is related to treatment with immunosuppressants. Iatrogenic KS shows similar features as have been observed in the patients with endogenous CS. Based on our case and the two earlier reports of cutaneous KS in ACTH dependent CS, we suggest adding a fifth type of KS with endogenous corticosteroid-excess as cause of KS Table  being, hOur findings demonstrate that endogenous immunosuppression due to Cushing’s syndrome may lead to pulmonary Kaposi sarcoma which may regress after treatment of hypercortisolism. The data emphasize the key role of glucocorticoid excess in the pathogenesis of Kaposi Sarcoma and the high relevance of restoration of immune function for its treatment. The data further emphasize the need of careful evaluation of patients with adrenal lesions.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.The authors declare that they have no competing interests.MB, CLR and SH treated the patient and drafted the article. JP initially treated the patient. VW and SK carried out the immunoassays. BA critically revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/14/63/prepub


It is difficult to synthesize the vast amount of textual data available from social media websites. Capturing real-world discussions via social media could provide insights into individuals’ opinions and the decision-making process.We conducted a sequential mixed methods study to determine the utility of sparse machine learning techniques in summarizing Twitter dialogues. We chose a narrowly defined topic for this approach: cervical cancer discussions over a 6-month time period surrounding a change in Pap smear screening guidelines.We applied statistical methodologies, known as sparse machine learning algorithms, to summarize Twitter messages about cervical cancer before and after the 2012 change in Pap smear screening guidelines by the US Preventive Services Task Force (USPSTF). All messages containing the search terms “cervical cancer,” “Pap smear,” and “Pap test” were analyzed during: (1) January 1–March 13, 2012, and (2) March 14–June 30, 2012. Topic modeling was used to discern the most common topics from each time period, and determine the singular value criterion for each topic. The results were then qualitatively coded from top 10 relevant topics to determine the efficiency of clustering method in grouping distinct ideas, and how the discussion differed before vs. after the change in guidelines .This machine learning method was effective in grouping the relevant discussion topics about cervical cancer during the respective time periods . Qualitative analysis determined that a significant portion of the top discussion topics in the second time period directly reflected the USPSTF guideline change , and many topics in both time periods were addressing basic screening promotion and education It was demonstrated that machine learning tools can be useful in cervical cancer prevention and screening discussions on Twitter. This method allowed us to prove that there is publicly available significant information about cervical cancer screening on social media sites. Moreover, we observed a direct impact of the guideline change within the Twitter messages. Social networking websites have fundamentally changed the way in which individuals and organizations communicate with each other ,2. AlmosThe immense volume of messages on social media sites  , precludFor this paper, we sought to explore the feasibility of these machine learning approaches to analyze Twitter content about cervical cancer and Pap smears. Specifically, we examined Twitter messages about these topics before and after the US Preventive Services Task Force (USPSTF) guideline change on March 15, 2012  Table 1Table 1, For this study, we gathered and stored messages from a specific online social networking site, Twitter, the second largest social networking site after Facebook. Twitter Within the raw dataset, we calculated the raw frequency data for messages related to cervical cancer screening during an entire 6-month time period, that is, we examined the total number of occurrences of the keywords on Twitter from January 2012 to June 2012. We then divided the data into two distinct time periods: before  and during or after  the USPSTF guideline change.As we wanted to establish the face validity of using newer machine learning methods to analyze Twitter data, we used a sequential mixed methods approach for our study by first completing a quantitative summarization of the data using sparse principal component analysis, followed by qualitative content analysis of example messages.After downloading the data, we first used an exploratory quantitative analysis using machine learning algorithms. Machine learning encompasses a set of statistical and computational tools, which assist in extracting meaning and insight from very large collections of data , which split the results into a number of distinct topics. Each topic was characterized by a short list of keywords that were all determined to be correlated. In addition, few Twitter messages that were most strongly and statistically associated with the topic were identified. In practice, SPCA was used both as a topic modeling algorithm (while considering the list of keywords) and clustering algorithm (while considering the example Twitter messages). This form of SPCA was used in our previous studies -16.In order to apply the SPCA approach to Twitter data , we modified the list of stop-words in our algorithm. For example, the text attached to the symbol “@” from the result terms was removed as it represents a username on Twitter rather than a part of the message content itself, and the uniform resource locator (URL) addresses were also removed since they were not considered as intelligible text. Hashtags (“#”) were permitted as they allowed users to indicate that their messages were related to the same topic  but did not change the meaning of the tweet. We restricted our analysis to messages tagged as “English” in the main dataset (but we handled the remaining non-English messages as described in more depth below). Finally, we allowed retweeted (indicated by “RT”) messages within our dataset and only the original message as an individual tweet that could be categorized in the cluster analysis was included, but that message was weighted more heavily in the results based on its appearance in the dataset.As an output of our analysis, the SPCA algorithm required fixed parameters such as number of topics, terms per topic, and tweets per topic for the results. Based on our previous work , we specOn identifying the best model, we recorded the singular value criterion, for which higher values indicate a higher prevalence of the topic within the entire set of messages  and the five keywords most strongly associated with each topic, which were automatically generated from the algorithm. As we used a sparse approximation of principal component analysis, there was a small degree of shuffling in the topic order by singular value criterion.We used the example tweets and keywords from the quantitative machine learning SPCA to conduct a qualitative review of the output. In particular, we read all example tweets from the top 10 relevant topics in both the baseline and follow-up periods to determine the nature and cohesiveness of the topics. By use of open coding ,19 we deIn a few cases, the results included non-English topics despite our attempt to limit the sample to English messages only. This is most likely because the “English” designation within the Twitter data is imperfect for some messages. Non-English messages most often involved the word “Pap” with a different meaning in another language—these were reported below but not analyzed in the qualitative phase of the study. In addition, some topics returned a smaller number of tweets than specified, largely due to the amount of retweeting of an identical message that was weighted more in the output.Next, after dividing the Twitter messages into baseline and follow-up periods surrounding the guideline change, there were 2,549 messages about cervical cancer on Twitter in the baseline period and 4,673 messages in the follow-up period.The clustering analysis revealed that the models gave similar results overall, with marginally better performance of 10 tweets per topic as more topics were generated  and 3, tThe qualitative analysis of the 10 tweets per topic results are shown in In terms of the content of the topics that were generated, In the baseline period, the top topic was about screening promotion, followed by information on anti-vaccination and that for individuals with abnormal test results. In these baseline results, 10–40% of the messages were different from the overall topic. In the follow-up period, three of the top topics were directly about the 2012 USPSTF guidelines, with very strong cohesiveness of these topics . Several of the message categories in the follow-up period expressed gratitude or relief about not having to have annual Pap smears any longer, which is a direct reflection of the updated 2012 USPSTF guideline stating that women with normal results can now wait 3 years between Pap smears. All the remaining top topics in the follow-up period were decidedly mixed without any single discussion topic dominating the messages.We successfully employed a sequential mixed-methods approach to analyze specific cervical cancer prevention and screening discussions on the online social media site “Twitter”—first using a quantitative topic modeling approach to parse the large text dataset, followed by qualitative analysis of the example Twitter messages that were pulled from the topic modeling.The machine learning methods provide statistically relevant terms, and indexing these to specific tweets was insightful, particularly when there is rich information shared through external links. However, while the resulting keywords from the quantitative approach were insightful, the true meaning of the messages was much more evident when we analyzed the full text examples for additional context. In other words, machine learning topic modeling required contextualization and content expertise, and much more significant adaptations to the algorithm would be needed to be able to replace human interpretation of the messages. Although the machine learning approach could not replace qualitative analysis altogether, it did allow for a more focused qualitative analysis, because it allowed us to prioritize the voluminous content available online.Our study demonstrates that social media is a platform in which individuals shared direct information about the new 2012 cervical cancer screening guidelines from the USPSTF. In fact, a large proportion of the top topics in the follow-up period were solely dedicated to this shift in screening recommendations. The content of these topics was so distinct from the baseline topics that we feel confident that a machine learning approach can successfully detect such large public changes in dialogue. In addition, the information shared about the change in guidelines was mostly neutral or positive for eliminating the need to have a Pap smear every year. This finding appears to be more positive than other existing literature on women’s perceptions about the new guidelines, which found that a substantial proportion of women expressed concerns about increasing the intervals between screenings .Our findings also demonstrated that a large portion of the cervical cancer messages on Twitter were about health promotion and education, beyond discussions about the 2012 screening guidelines. This included new science about the effectiveness of Pap smears and information  to encourage women to get Pap smears, which is consistent with our previous qualitative work . We feelOur small exploratory study has limitations. First, we chose one social media site, Twitter, which has a 140-character limit for each post. While Twitter represents a fast-growing platform for Web-based communication , TwitterFinally, from a methodological standpoint, we faced some limitations in applying a novel approach, such as a few irrelevant sets of messages. While this is expected to a certain extent, future studies will be able to quantify the expected error when using these methods and perhaps additional refinements to minimize such error. The topic modeling and clustering approach also relies on a simple bag-of-words model of text, which ignores word ordering and natural-language semantic information. All analysis is based on patterns of associations between words. In addition, our method only captures the most prevalent associations between words; if a single word has multiple meanings in different contexts, the less prevalent patterns may be overlooked. Finally, we employed a model, which assumes that each message contains a single topic. Other approaches can identify a mixture of topics within a single message, but are computationally expensive ,25. GiveWhile preliminarily our findings imply that there is an interest in cancer screening discussions among the ever-growing population using online social media. Our work argues for further transdisciplinary study into cancer screening promotion via social media, whether in a peer-to-peer or in an expert-recommendation fashion.


FESEM and fluorescence microscopic analysis imitates the smooth morphology and even distribution of FA within the polymeric nanofibers at optimum 2 wt% concentration of FA. The average diameters were recorded to be 150 ± 47.4 and 200 ± 79 nm for PLGA/PEO and FA-encapsulated PLGA/PEO nanofibers, respectively. The encapsulation, compatibility, and physical state of FA within the nanofibers were further confirmed by FTIR, TGA and XRD analysis. In vitro drug delivery studies demonstrated initial burst liberation of FA within 24 h followed by a sustained release for the subsequent time. MTT assay revealed the effectiveness of FA-encapsulated nanofibers against human breast carcinoma cells (MCF-7) cells as compared to control. FESEM and fluorescence microscopic analysis further confirmed the apoptotic effect of FA-encapsulated PLGA/PEO nanofibers against MCF-7. These fabricated nanofibers hold enormous potential to be used as a therapeutic agent for various biomedical applications.Ferulic acid (FA) is a polyphenolic phytonutrient which possesses strong antiproliferative effect; however, it has limited therapeutic applications due to its physiochemical instability and low bioavailability at the tumor site. In present study, these shortcomings associated with FA were overcome by fabricating FA-encapsulated poly(The online version of this article (doi:10.1007/s13205-014-0229-6) contains supplementary material, which is available to authorized users. Phenolic nucleus and unsaturated side chain of FA (4-hydroxy-3-methoxycinnamic acid) readily form a resonance stabilized phenoxy radicals which is responsible for its potent antioxidant capacity. This ubiquitous polyphenolic pharmaceutically active constituent arises from the metabolism of phenylalanine and tyrosine, occurs mainly in seeds, leaves and cell walls of plants such as wheat, rice and oats , 3--2,5-diphenyltetrazolium bromide (MTT), cell culture-grade dimethyl sulfoxide (DMSO), phosphate buffer saline (PBS), acridine orange (AO), ethidium bromide (EtBr), Dulbecco’s modified Eagle medium (DMEM) and all analytical grade chemicals were from Himedia (India).PLGA  and PEO  were purchased from Sigma-Aldrich . Michigan Cancer Foundation-7 (MCF-7) and Human Embryonic Kidney (HEK-293) cell lines were procured from National Center for Cell Science (NCCS), Pune, India. FA, dichloromethane (DCM), −1) by means of a single syringe pump . A piece of aluminum foil was used to collect the nanofibers with the horizontal distance of 12 cm from the needle tip. All electrospinning processes were carried out under ambient conditions . The resultant fibers were further dried for 24 h in desiccators to remove the residual organic solvent and moisture.PLGA and PEO in a blend ratio of 1:1 were prepared in a solvent mixture of DCM/DMF  to prepare a blank electrospinning solution at a final concentration of 2 wt%. FA-encapsulated PLGA/PEO solutions were obtained by dissolving FA at different concentrations  w.r.t the total polymer concentration in the PLGA/PEO polymeric solution. The FA-free and FA-encapsulated PLGA/PEO solutions were then carefully placed into a 5 mL syringe, attached with a metallic needle (21 G). Electrospinning was carried out under a fixed electric field (15 kV), feeding rate  of the native FA and nanofibrous samples was performed by using a TGA instrument . Each sample (8–10 mg) was kept under vacuum for 24 h prior to testing and then the precisely weighed samples were heated from 23 to 500 °C at a scanning rate of 10 °C min−1 under a nitrogen atmosphere. Infrared absorptions of the native FA, PLGA/PEO and FA-encapsulated PLGA/PEO nanofibers were recorded by Fourier transform infrared spectrometer . Scans (16) were recorded in the scanning range of 4,000–500 cm−1 with the resolution of 4 cm−1.Surface morphology of the nanofibers was observed using field emission scanning electron microscopy  at an accelerated voltage of 15 kV. For sample preparation the nanofibers were cut into rectangular pieces (1 × 1 cm) and sputter coated  with gold for 1 min. The average diameters of the nanofibers were measured at over 50 different points of FESEM images . The morphology of FA-encapsulated PLGA/PEO nanofibers was also confirmed by fluorescent microscopy . The physical state of FA in the electrospun nanofibers was further examined by X-ray diffraction (XRD) analysis. The XRD patterns were recorded with Cu Kα radiation over the 2The encapsulation efficiency (EE) of FA in the nanofibers was quantified by thoroughly dissolving the FA-encapsulated nanofibers (containing 2 wt% FA) in a mixture of DCM/DMF (4:1) and the amount of released FA was measured by using UV–Vis spectrophotometer (Lasany double beam LI-2800) at 319 nm. The amount of FA in the fibers was calculated from the obtained data against a predetermined calibration curve for the drug. The encapsulation efficiency of the FA was determined as follows:EE (%) = Actual FA content in nanofibers (mg)/Theoretical FA content in nanofibers (mg) × 100.In vitro release studies of FA from the 2 wt% FA-encapsulated PLGA/PEO nanofibers were performed by incubating 10 mg nanofibrous mat in 30 mL PBS (pH 7.4). The incubated samples were maintained in a thermostat (37 °C) at 50 rpm. At predetermined time intervals, 3 ml of samples was withdrawn from dissolution medium and the OD was measured at the 319 nm using UV/Vis spectrophotometry.5 cells mL−1) along with DMEM medium supplemented with 10 % fetal bovine serum (FBS). The plates were then incubated in humidified atmosphere containing 5 % CO2 at 37 °C for 24 h. MCF-7 cells grown in presence of culture medium only were used as negative control, whereas the MCF-7 cells cultured in presence of native FA only were used as a positive control. Subsequently, the MTT solution (60 μL of 5 mg mL−1 stock) was added to each well containing sample, and incubated at 37 °C for 4 h. Then, the solution in the wells was removed carefully and 400 μL of DMSO was added to dissolve the MTT formazan crystals. After that, 100 μL of the dissolved formazan solution of each test sample was transferred to individual wells of 96-well plate to determine the absorbance at 570 nm (A570nm) using micro-plate reader . The cell viability was calculated as follows:In vitro cytotoxicity of the PLGA/PEO and 2 wt% FA-encapsulated PLGA/PEO nanofibers toward MCF-7 cells was evaluated using MTT assay Mosmann . BrieflyAnalysis of change in morphology of MCF-7 tumor cells in presence of native FA and FA-encapsulated nanofibers.5 cells mL−1) and incubated at 37 °C for 24 h in a CO2 incubator. The samples were then washed thrice with PBS (pH 7.4) immediately after the incubation period to remove any unattached MCF-7 cells. The cell morphologies for each sample were first observed using an inverted phase contrast microscope  at 100× magnification. For FESEM examination, the MCF-7 tumor cells adhered to nanofibers were fixed by immersing the samples into 2.5 % glutaraldehyde PBS solution at 4 °C for 4 h. Subsequently, the samples were dehydrated stepwise with graded concentrations of ethanol .The UV-sterilized nanofibrous samples were placed in 24-well plates, seeded with MCF-7 cells (5 × 105 cells mL−1) cultured on the different formulations , as described above were also visualized by double staining with acridine orange (AO) and ethidium bromide (EtBr). Briefly, after 24 h of incubation, the MCF-7 tumor cells seeded samples were rinsed thrice with PBS and instantly stained with equal volumes of AO and EtBr (100 µg mL−1 stock solution) for 10 min. The samples were then visualized under the fluorescence microscope .MCF-7 cells (5 × 104 cells mL−1), seeded in DMEM medium supplemented with 10 % FBS, 100 U mL−1 penicillin, and 100 µg mL−1 streptomycin. The plates were then incubated in a humidified atmosphere with 5 % CO2 at 37 °C for 24 h. Coverslips without nanofibers were used as controls. After 24 h of cell seeding, MTT assay (A570nm) and FESEM studies were performed as described above.The cytocompatibility of different formulation  was evaluated using HEK-293 cells. Briefly, the sterilized formulations were first placed individually in 24-well microtiter plates followed by addition of HEK-293 cells (5 × 10n = 3) and the data are expressed as the mean values with ±standard deviation (SD). Statistical analysis of the differences between mean values obtained from experimental groups was performed using one-way ANOVA test. Values of p < 0.05 or less were considered as denoting statistical significance.Each measurement was performed at least in three independent experiments  the surface morphology of nanofibers changed from smooth, cylindrical, defect free to rough and damaged surfaces Fig. a. Some dθ = 9.02, 10.4, 12.5, 15.6, 17.2, 24.4, 29.4, 35.7, 42.8, 45.8, and 50.7  in the electrospun nanofibers. As shown in Fig. 2O bonding. The second stage of decomposition started at around 260 °C and completed at around 400 °C. This zone was found to be the highest thermal degradation zone which corresponds to a complex process including depolymerization as well as decomposition of bonds and units of polymers  and FA as well as reflected the early onset of FA decomposition in nanofibers than that of native FA. This may be due to the even distributions and presence of amorphous state of FA in PLGA/PEO nanofibers which make FA molecules respond to the heat more sensitively than native FA crystalline particles.The derivative TGA curves of weight loss for native FA, PLGA/PEO and FA-encapsulated PLAG/PEO nanofibers are shown in Fig. −1 (–OH group), 3,015, 2,922, 2,838, and 2,594 cm −1 (C–H bond stretching), 1,690, 1,665, and 1,619 cm −1 (–C=O stretching), 1,272–1,034 cm −1 (–C–O stretching) as also reported earlier by Yang et al. (−1 (–OH stretching), 2,922 and 2,856 cm −1 (–CH stretching), 2,360 cm−1 (C≡C stretching), 1,760 and 1,630 cm−1 (–C=O stretching), 1,271 and 1,100 cm −1 (C–O stretching). All the entire sharp peaks for FA can be observed in the IR spectrum of FA-encapsulated PLGA/PEO-FA nanofibers, indicating that FA in the composite nanofibers more likely tends to form FA dimers that are necessary for constructing a crystal lattice. The shift of 1,640–1,820 cm −1 absorption bands to lower wave numbers in the spectrum of FA-encapsulated PLGA/PEO nanofibers illustrated the presence of weak interactions between the carbonyl groups of PLGA/PEO blend and the hydroxyl group of FA  was achieved, respectively. Moreover, it could be interpret from the results that FA retains its activity even after encapsulation into nanofibrous matrix and is not affected by the electrospinning process. The polymeric nanofibers performed as preservative agent for FA which improved its physiochemical stability as well as protected it from the premature degradation . Therefore, the encapsulation of FA in PLGA/PEO nanofibers resulted into enhanced activity of encapsulated FA in comparison to native FA in solution form. Similar studies have also been reported by Ouimet et al.  and FA-encapsulated nanofibers . The cells cultured on native FA  was executed using equivalent mixture of AO and EtBr (1:1 w/w). The stained cells on different formulations were then analyzed by fluorescent microscopy. AO and EtBr stain DNA, which allows visualization and differentiation of dead and viable cells. Cells with intact membranes stained green due to passage of AO whereas EtBr stained the cells with damaged membranes, as a result, the DNA intercalation of both gives orange fluorescence Fig. . No cell FA Fig. g–i and t FA Fig. j–l displ FA Fig. .Fig. 10Fp < 0.01). Data obtained, imply that the incorporation of FA within the nanofibers did not compromise the cytocompatibility of PLGA/PEO nanofibers. The cytocompatibility evaluation of FA-encapsulated PLGA/PEO nanofibers as compared to the native FA, was also performed via cell surface morphology observations  cells. The findings suggested that the incorporation of FA in nanofibers may reduce the chemotherapeutic side effects and can be useful in providing a high local drug concentration to destroy the tumor cells. Therefore, these fabricated electrospun nanofibers are viewed as future candidates for antitumor drug delivery as well as potential local chemotherapeutic agent for breast tumor formations.NMR analysis data for native FA, PLGA/PEO nanofibers and FA-encapsulated PLGA/PEO nanofibers confirming the stability of FA after encapsulation has been provided as supplementary information.Supplementary material 1 (TIFF 348 kb)Below is the link to the electronic supplementary material.


Phenylketonuria (PKU) is due to a defective hepatic enzyme, phenylalanine (Phe) hydroxylase. Transport of the precursor amino acids from blood into the brain for serotonin and dopamine synthesis is reported to be inhibited by high blood Phe concentrations. Deficiencies of serotonin and dopamine are involved in neurocognitive dysfunction in PKU.(1) To evaluate the effects of sapropterin (BH4) and concurrent use of large neutral amino acids (LNAA) on the peripheral biomarkers, melatonin and dopamine with the hypothesis they reflect brain serotonin and dopamine metabolism. (2) To evaluate synergistic effects with BH4 and LNAA. (3) To determine the effects of blood Phe concentrations on the peripheral biomarkers concentrations.Nine adults with PKU completed our study consisting of four 4-week phases: (1) LNAA supplementation, (2) Washout, (3) BH4 therapy, and (4) LNAA with BH4 therapy. An overnight protocol measured plasma amino acids, serum melatonin, and 6-sulfatoxymelatonin and dopamine in first void urine after each phase.(1) Three out of nine subjects responded to BH4. A significant increase of serum melatonin levels was observed in BH4 responders with decreased blood Phe concentration. No significant change in melatonin, dopamine or Phe levels was observed with BH4 in the subjects as a whole. (2) Synergistic effects with BH4 and LNAA were observed in serum melatonin in BH4 responders. (3) The relationship between serum melatonin and Phe showed a significant negative slope (p = 0.0005) with a trend toward differing slopes among individual subjects (p = 0.066). There was also a negative association overall between blood Phe and urine 6-sulfatoxymelatonin and dopamine (P = 0.040 and 0.047).Blood Phe concentrations affected peripheral monoamine neurotransmitter biomarker concentrations differently in each individual with PKU. Melatonin levels increased with BH4 therapy only when blood Phe decreased. Monitoring peripheral neurotransmitter metabolites may assist in optimizing individualized treatment in PKU. Since newborn screening has been introduced, early diagnosis and dietary management based on blood phenylalanine (Phe) concentrations successfully prevented individuals with Phenylketonuria  from developing significant intellectual disability. Recent studies, however, report that even individuals who are early diagnosed and well controlled have a high prevalence of neurocognitive and neuropsychological symptoms which include decreased executive functioning and internalizing disorders . The dieSome individuals with PKU with excellent control in blood Phe may still be deficient in serotonin and dopamine in the CNS, particularly with restriction of natural protein and/or insufficient use of medical food products containing LNAA including Trp and Tyr ,11. ConvEvaluation of monoamine neurotransmitter metabolism in the CNS could provide essential information to optimize dietary management for individuals with PKU. However, it is impractical to directly monitor the cerebrospinal fluid (CSF) serotonin and dopamine metabolites, i.e., 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA), respectively, due to the invasive nature of the procedure. Therefore, we conducted studies evaluating peripheral melatonin and dopamine in individuals with PKU based on the hypothesis that these potential biomarkers reflect serotonin and dopamine metabolism in the CNS. Our studies to date are consistent with findings by Nielsen and Lykkelund demonstrating increased concentrations of monoamine neurotransmitter metabolites, i.e., 5HIAA and HVA in the CSF in individuals with PKU after implementation of the Phe restricted diet or after supplementation of the precursor amino acids ,12,13,14Since melatonin is synthesized from serotonin in the pinealocytes, we measured this metabolite as a biomarker to reflect serotonin synthesis in the brain in our previous studies ,13. ThesSince tetrahydrobiopterin (BH4) is a cofactor for Phe hydroxylase (PAH) as well as for TPH and TH which are the rate determining enzymes to synthesize serotonin and dopamine, respectively, we conducted the present study in adult individuals with PKU with three objectives: (1) to evaluate the direct effects of sapropterin dihydrochloride (the synthetic form of BH4) on brain TPH and TH by measuring the peripheral neurotransmitter biomarkers, melatonin and dopamine, (2) to determine if any synergistic effects are observed in melatonin and dopamine concentrations with concurrent use of LNAA and sapropterin (BH4), (3) to evaluate the relationship between blood Phe concentration and the peripheral neurotransmitter biomarkers.The study protocol was approved by the University of Southern California (USC) Health Science Institutional Review Board. Study subjects were recruited from our outpatient population based on the past history of compliance with medical instruction. Ten adult subjects with PKU were enrolled after obtaining written informed consent . Table 1post hoc contrasts between the BH4 and Washout phases (Phase 3 vs. Phase 2) and between the BH4+LNAA and LNAA phases (Phase 4 vs. Phase 1), and also between Phase 1 and Phase 2 and between Phase 3 and Phase 4. Within-subject associations between plasma Phe and serum melatonin AUC as well as urine 6-sulfatoxymelatonin and dopamine were examined with equality of slopes mixed models. Statistical tests were 2-sided at a significance level of p<0.05, performed using SAS/STAT® version 9.2 software. P-values between 0.05 and 0.10 were considered suggestive of a trend.Serial concentrations of serum melatonin taken during the overnight stay were summarized as area under the curve of melatonin (AUC) vs. time, calculated by the trapezoidal formula. Additional outcome measures included concentrations of plasma Phe, and urinary concentrations of dopamine and 6-sulfatoxymelatonin. Prior to analysis study variables were log- transformed, as needed to normalize distributions. Comparisons among the 4 phases were made with repeated measures mixed models, with One subject (S10) was removed from the analysis since he received a medication that affects the CNS monoamine metabolism. The other nine completed all 4 phases, except for one subject (S3) who failed to collect a urine specimen in Phase 4. Three subjects  showed serum melatonin increase ≥30% in Phase 3 over Phase 2. In these 3 BH4 responders, plasma Phe concentrations were lower and melatonin AUC was higher in Phase 3 compared to Phase 2  and in Phase 4 vs. Phase 1 , but no statistically significant differences were observed in urine 6-sulfatoxymelatonin or urine dopamine with supplementation of BH4. The remaining six subjects (BH4 non-responder) did not show any significant differences in these 4 variables with BH4 supplementation.1) Serum Melatonin , Fig 2Serum melatonin levels (AUC) at all phases from all subjects are shown in 2) Plasma Phe levels Plasma Phe concentrations from all phases in each study subject are shown in 3) Urine Melatonin Two out of 3 BH4 responders showed increase in urine melatonin in Phase 3 over Phase 2. The data point at Phase 4 in S3 is missing due to sample collection failure. Six BH4 non-responders showed no increase in urine melatonin with BH4 supplementation.4) Urine Dopamine Profiles of urine dopamine concentrations were similar to those of urine 6-sulfatoxymelatonin (C1 and C2) except for S9 whose dopamine concentration was higher in Phase 2 than in Phase 1.5) Plasma Phe vs. Serum Melatonin AUC and Plasma Phe vs. Urine 6-sulfatoxymelatonin Figs  and 56) Plasma Phe vs. Urine Dopamine Urine dopamine concentrations are again quite variable at a particular plasma Phe concentration among the subjects. There was a negative association overall between plasma Phe and urine dopamine (p = 0.047).PKU is caused by a deficiency of PAH which is expressed in the liver but not in the CNS. The neurocognitive and neuropsychological symptoms associated with PKU are likely due to the unique environment in the CNS with high blood Phe and relatively low concentrations of the other amino acids. This may contribute to fetal microcephaly in maternal PKU syndrome through decreased protein synthesis in the rapidly growing stage and to neurocognitive and neuropsychological symptoms after CNS development and growth are completed, possibly due to abnormal neurotransmitter metabolism , 19. ButBurton et al. recently reported improvement in symptoms of attention deficit-hyperactivity disorder and impairments in executive functioning in individuals with PKU who responded to sapropterin therapy with reduction of blood Phe concentrations, suggesting involvement of the monoamine neurotransmitters, serotonin and dopamine, in neurocognitive and neuropsychological symptoms in PKU . Our stuA wide range of serum melatonin AUC as well as urine 6-sulfatoxymelatonin concentrations, which likely reflect brain serotonin synthesis, was observed at a particular blood Phe concentration within this small number of study subjects. One individual (S9) had the highest melatonin AUC (within the range reported for non-PKU adults ) at Phe Urine dopamine concentrations at a given blood Phe concentration also varied widely among study subjects along with a negative association between blood Phe and urine dopamine . EstabliManti el al. recently reported the occurrence of psychiatric disorders in which anxiety and withdrawal are the most frequent self-reported symptoms, is not associated with either the lifelong or concurrent quality of metabolic control, and that even individuals with good metabolic control (Phe<500uM) in the first 11 years of life showed a higher frequency of psychiatric diagnoses compared to healthy controls . This inNardecchia et al. reported that developmental trajectories in some cases of early treated PKU were independent from metabolic control, suggesting individuals vary in their vulnerability to Phe . Our stuAlthough concentrations of neurotransmitters, i.e., serotonin, dopamine or their derivatives 5HIAA and HVA in CSF were not directly measured and compared to concentrations of the peripheral biomarkers, this study supports the hypothesis that peripheral concentrations of melatonin and dopamine, which may reflect serotonin and dopamine metabolism in the CNS, respectively, might be used to evaluate deficiencies of these monoamine neurotransmitters in the CNS. The wide variation of the peripheral biomarker levels at a particular blood Phe concentration, and also the differing responses among individuals in the peripheral biomarkers to Phe concentrations indicates that blood Phe concentrations alone cannot be used to evaluate brain serotonin and dopamine metabolism. Evaluation of urine melatonin and dopamine along with blood Phe concentrations may identify neurotransmitter deficiencies and allow clinicians to determine the most effective individualized management, either by providing more Trp and Tyr through medical food products and/or LNAA, or by reducing natural protein intake to lower blood Phe levels and thus improve serotonin and dopamine metabolism. This individualized, targeted management of PKU may reduce long-term neurocognitive and neuropsychological problems. Establishing reference ranges for these peripheral biomarkers is essential. Larger, long-term follow up studies are needed to evaluate this hypothesis.S1 FileAll of the data obtained from this study including plasma tyrosine, phenylalanine, tryptophan, urine 6-sulfatoxymelatonin, urine dopamine, and serum melatonin AUC are shown.(XLSX)Click here for additional data file.