The positive results of the performance of zirconia for orthopedics devices have led the dental community to explore possible esthetical and mechanical outcomes using this material. However, questions regarding long-term results have opened strong and controversial discussions regarding the utilization of zirconia as a substitute for alloys for restorations and implants. This narrative review presents the current knowledge on zirconia utilized for dental restorations, oral implant components, and zirconia oral implants, and also addresses laboratory tests and developments, clinical performance, and possible future trends of this material for dental healthcare. Some of data provided in this narrative review reflect 7–10 years of extensive studies developed on zirconia for dental applications by three recognized dental institutions that include University of Freiburg - Germany, University of Zurich - Switzerland and New York University - USA.Considering all ceramic materials available for dental healthcare, zirconia offers so far the best mechanical properties. The good results obtained from orthopedic procedures brought significant confidence to dentistry for the utilization of zirconia as a support material  for esthetic restorations as well as for oral implants. However, controversies regarding to the proper interaction between the zirconia substrate and esthetic veneering porcelain arose, and in particular questions regarding veneered zirconia long-term performance for crowns and bridges. It is known however that significant improvements and developments of zirconia-ceramic systems combined with more controlled processing steps have broaden the future perspectives of this material. This narrative review addresses the current knowledge and future perspectives of the utilization of zirconia for dental healthcare emphasizing recent Yttria-tetragonal zirconia polycrystals (Y-TZP) has been utilized for dental applications because of the extensively reported long-term success in the orthopedics field. However, in 2001 [The flexural strength of zirconia rages from 800 to 1,000 MPa , which iThere are four zirconia-containing ceramic systems available for dental healthcare applications, which includes: Magnesium cation-doped partially stabilized zirconia (Mg-PSZ), zirconia-toughened alumina (ZTA), alumina-toughened zirconia (ATZ) and the most widely used yttrium cation-doped (3%) tetragonal zirconia polycrystal (3Y-TZP) . The ATZ2 are difficult to obtain, or else they create magnesium silicates that can reduce the stabilizing Mg content and consequently lower the energy for the t-m transformation [Mg-PSZ has not been successfully used in dentistry because of the presence of porosities and large grain size 30–60 μm) that may lead to surface wear . The Mg-0 μm thatormation . A dentaormation .TM, Brea, CA, USA), contains in one third (12 mol %) ceria-stabilized zirconia (12 Ce-TZP) added to In-Ceram® Alumina® [ZTA utilizes the stress-induced transformation capacity of zirconia in an alumina matrix ,11. One ne third  mol % cee third 1 mol % ceAlumina® . As a drAlumina® .203 as stabilizer) [3Y-TZP (3 mol % Ybilizer)  has beenbilizer) . In dentbilizer) .The 3Y-TZP mechanical properties depend primarily on the grain size. Smaller grain sizes (<1 μm) are associated with lower t-m transformation rates . HoweverThe direct ceramic machining of pre-sintered 3Y-TZP became popular for the dental community in 2001  and seveIf any subtractive procedure is performed after final sintering of the zirconia ceramic, for example sandblasting or grinding of the intaglio surface to increase the roughness for cementation purposes or adjustment of the same surface for better fit, a monoclinic phase will most likely be observed on the treated surface. This monoclinic transformation will in the first instance increase the strength of the restoration ,23. HoweMachining fully sintered 3Y-TZP blocks for dental restorations becomes a concern as this procedure has shown to create monoclinic zirconia  and surfAll-ceramic materials are increasingly being used for the fabrication of crowns and fixed dental prostheses (FDPs). Due to their inferior load-bearing capacity compared to metals, however, ceramics have traditionally been applied in indications with lower loading forces.Systematic reviews of the literature of all-ceramic and metal-ceramic reconstructions revealed that crowns made of ceramics exhibited survival rates similar to those of traditional metal-ceramic crowns . Those cIn contrast to other ceramics, zirconia has the potential to be applied as an alternative material to metal for reconstructions in indications with high loading forces, e.g., the posterior regions ,30. CurrThe six clinical studies published to date on zirconia-based FDPs showed promising results of the FDPs after observation periods of 3 to 5 years ,34,35,36More recently, a randomized controlled clinical trial (RCT) comparing posterior FDPs with zirconia and metal frameworks has been performed . At 3 yeYet, apart from excellent framework survival zirconia-based reconstructions were frequently subject to technical or biological problems ,35,36,37Overall, zirconia veneering ceramics are one major concern for the clinical long-term outcome today. The reasons for the problems with the zirconia veneering ceramics still remain to be clarified. Several factors have been investigated in recent laboratory studies, which possibly affect the rate of veneering fractures. Among the factors analyzed are the thermal compatibility of the veneering ceramics and the zirconia frameworks ,43, diffThe main clinical factor to be considered at zirconia-based FDPs with respect to risk for framework fracture or chipping of the veneering ceramic is the design of the framework. On the one hand the design of the connectors of zirconia-based FDPs have to be adequate, on the other hand space for an even thickness of the veneering ceramic needs to be provided. In several studies computerized fabrication techniques by means of either CAM or CAD/CAM systems were used for the fabrication of the ceramic frameworks ,35,36,37Another observation made at the zirconia-based reconstructions was the occurrence of marginal discrepancies and marginal gaps, indicating problems with the fit of the computer-manufactured frameworks ,31,32,33Finally, the biological outcome of zirconia-based reconstructions generally was favorable in all studies ,35,36,37in vivo fractures for ceramic systems with the main focus on zirconia-based restorations.The understanding of the fracture and chipping mechanisms of zirconia-supported dental restorations became one of the most important steps in developing more predictable all-ceramic restorations. For that, fatigue testing performed on simplified and complex geometries specimens are necessary to firstly mimic oral mastication movements and secondly to follow the crack growth in 2D and 3D layered models. This section addresses the sample preparation and fatigue method employed at New York University College of Dentistry in an attempt to mimic θ = 30° . Fully sintered CAD/CAM zirconia plates  were veneered with an overlay porcelain  by the manufacturer. The cementation surface of the zirconia core was roughened with 600-grit SiC abrasive paper and cemented  to a composite block , simulating veneered zirconia cemented to tooth-dentin structure. The porcelain surface was polished to 1 µm finish. The composite blocks were incubated in water for 4 weeks before cementation to allow for hydroscopic expansion. After cementation, the bonded structures were aged in water for 10 days before fatigue testing. The specimen was mounted on an inclined block  observed clinically [The current fatigue tests were designed to determine the type of damages and the number of cycles to failure inically . Followiinically . Failurein vivo [For the range of loads tested here, a set of partial cones formed at the first contact−load−slide− liftoff cycle in porcelain veneers. Failure was predominately from the deep penetrating occlusal surface partial cone fracture. For example, for a maximum fatigue load of 350 N, a partial cone crack has already propagated ~0.3 mm deep from the occlusal surface after the first loading cycle a; it tooin vivo .In a veneered zirconia system, the stiff zirconia core provides stress shielding of the veneer layer and the underlying tooth structure. The exceptionally high strength of the zirconia core prevents flexure induced cementation surface radial fracture. The high modulus of the zirconia core (compared to porcelain) minimizes flexure of the porcelain veneer and propagation of partial cone cracks. Therefore, with the support of a stiff and strong zirconia core, flexure of the veneer layer is suppressed, resulting in a steady pace propagation of partial cones throughout the entire veneer layer. However, joining porcelain veneer with zirconia core at elevated temperatures poses another problem. Due to the difference in coefficient of thermal expansion and thermal conductivity between the porcelain veneer and zirconia core, residual stresses forms inevitable in the system upon cooling. These residual stresses can superimpose on the mechanical stresses induced from fatigue loading, resulting in premature fracture or chipping of the porcelain veneers. Giving the complex geometry nature of dental restorations, there is currently no effective method to tailor the residual stresses, and chipping or fracture of the porcelain veneer remains a major problem in veneered zirconia restorations.Various testing methods have been used to investigate the mechanical properties of dental materials. Many concerns have been raised lately regarding the clinical significance of simple traditional mechanical testing methodologies (single cycle loading/“crunch the crown test”) . These tAs described above, clinically, all-ceramic restorations commonly fail through stress corrosion and slow crack growth resulting from fatigue caused by repetitive occlusal contact .This complex mechanical scenario of damage initiation and accumulation was reproduced in a recently developed in-vitro test design ,60 by usThe complex and patient unique geometry of posterior all-ceramic dental crowns represents a particularly interesting set of challenges to understanding stress concentration and damage evolution to fracture in response to loading.Similar to flat model topic addressed previously, the sliding contact mouth-motion fatigue testing in water for anatomically correct crowns employed translation of a tungsten carbide indenter 0.5–0.7 mm along the crown surface from the disto-buccal cusp tip down toward the central fossa. Indenter translation simulates approximating tooth surfaces during mastication ,60. FailDuring mouth-motion fatigue, various hand-layer veneered zirconia based crown systems resulted in a limited reliability - approximately 90% of specimens failed from veneer chip-off fracture by 100 K cycles at 200 N ,63 refleGiven the known highly accelerated failure from surface cracks with mouth-motion sliding contacts in water previously described, high failure rates within the veneers can be anticipated . FatigueAs the veneering ceramic material  is weak compared to the high-strength core material (900 MPa) the veneering ceramic is prone to fail at low loads during the evolution of complex tensile fields in function. Thus all tested veneered Y-TZP crowns failed from cohesive fractures within the veneering ceramic, where a thin layer of veneering ceramic still remained on the zirconia coping . As prevThe effect of the coefficient of thermal expansion (CTE) and the highly deleterious impact on core and veneering ceramics caused by residual stresses has been frequently discussed in the dental literature ,68. The Catastrophic failure of the Y-TZP ceramic in terms of core cementation surface radial cracking was not evident in any of the veneered Y-TZP crowns and is consistent with most clinical observations ,70. The in vivo study it was stated that the fracture resistance increases as the core thickness/veneer thickness ratio increases [Framework design and crown geometry plays an important and underappreciated role in fracture failure of all-ceramic crowns . Design ncreases .With the beginning of CAD/CAM technologies in dentistry excessive veneer layer thickness were created due to uniform layer thickness of the copings for crowns as well as bar shaped connectors for FDPs. Ceramic copings are often milled to arbitrary thickness of 0.4 or 0.6 mm. Depending on tooth anatomy this may not provide uniform support or appropriate thickness for the veneering porcelain . This prModern CAD/CAM systems are now able to provide a considerably better anatomically cut back framework design thus future clinical long-term results may be more favorable . The amoet al., unpublished data; Silva, N.R.F.A. et al., submitted data). Until more is known about clinical failure modes and clinical long-term performance parameters, precise recommendations cannot be made with confidence.In the above described standardized crown test model including mouth-motion-step-stress fatigue anatomical support of the veneering ceramic resulted in reduced chip size and significantly increased reliability with press and hand-layer veneering ceramics and appears to be a promising tool to optimize long-term performance of veneered Y-TZP crowns in clinical application  using the above mentioned mouth motion fatigue test set up revealed no core or veneer failure at a load level of 900 N and 170 K cycles and confirmed the high reliability of this all-ceramic system.Besides the high susceptibility to fracture, also from an economical point of view veneering of crown and FDP frameworks involving traditional methods such as the powder layering technique appears to be inefficient. An innovative veneering procedure for all-ceramic crown restorations using a CAD/CAM fabricated high strength lithium disilicate glass ceramic sintered onto a corresponding CAD/CAM fabricated zirconia coping has been described to be very promising with regard to initial mechanical strength testing . UnpubliDue to industrial prefabrication of the blocks and subsequent CAD/CAM processing a high quality material with a minimum of flaws compared to the manual procedures of veneering or heat pressing is provided. Given that appropriate software has been developed to ease the CAD phase for reconstruction the application for FPD frameworks would be of major interest as enhanced clinical performance can be expected. However CAD/CAM manufacturers have to provide the prerequisites regarding software and milling process accuracy before this technique can find general application. Long-term (5–10) year clinical evaluations on zirconia based crowns are however necessary to confirm the presented topics.After the observations regarding the zirconia-based crowns and bridges we shift the focus to the behavior of zirconia as abutment material. The clinical applications of zirconia as abutment can be divided into two areas: 1) Pre- and custom-fabricated zirconia posts for endodontic treated roots. Although this procedure has been advocated by some dental clinicians, the use of single-visit fiber posts have proven to be the choice when an endodontic treated tooth needs to receive an endodontic post and built-up. 2) Zirconia abutments for oral implant reconstructions. Zirconia as implant abutment material  was firsIn order to be qualified for clinical use, ceramic abutments should perform like the “gold-standard“ metal abutments after being at least five years in function .So far, only one randomized controlled clinical trial comparing zirconia and titanium abutments supported by 40 single implants was published . After bin vivo studies demonstrated a decrease of 50% of the fracture toughness, when zirconia was exposed to a simulated 10-year aging process in a humid environment [One specialty of zirconia is its crack resistance, also called “transformation toughening“ , previouironment . As therin vivo study, zirconia abutments showed resistance to high loads of up to 738 N [Based on the results of an to 738 N . These lto 738 N .i.e., mostly for the replacement of premolars [The promising mechanical properties encouraged to apply zirconia abutments also in posterior regions, remolars ,80,81. Tremolars ,81. Yet,in vivo study defined the abutment screw to be the weakest component at titanium abutments, whereas it was not possible to identify the weakest component at zirconia abutments [Implant abutments are attached to oral implants via an abutment screw. Controversy regarding the clinical response of metal or zirconia screw retained implant abutments still remains. The incidence of abutment screw loosening does not differ significantly between metal and ceramic abutments . An in vbutments .In a clinical study on zirconia abutments, 3.7% loosenings of the abutment screws were observed at four years . This isThe fit between implant and abutment is recommended to permit less than 5° of rotational movement in order to create a stable screw joint ,89. Tighin vivo study on customized zirconia abutments fabricated by CAD/CAM procedures, exhibited an excellent fit with a rotational freedom of less than 3° [Furthermore, an  than 3° .No screw loosening occurred at externally connected zirconia abutments  and at iin vivo study analyzed the influence of the type of connection on the fracture load of zirconia abutments and found superior strength when there was an internal connection via a secondary metallic component [When comparing the screw loosening rates at abutments with external and internal implant-abutment connections, a trend towards less problems was found at internally connected abutments in a systematic review . A previomponent . Two-pieomponent .Fracture of veneering ceramic was addressed in a previous section but mainly on teeth-supported restorations. It becomes then imperative to discuss how all-ceramic restorations perform when the under layer support are zirconia abutments attached to titanium implants.According to a systematic review on implant-supported single crowns, fracture of the veneering material was the third most common technical complication and appeared in 4.5% after five years .No chipping occurred in one study at crowns supported by zirconia abutments, whereas chippings were found in 20% of crowns supported by titanium abutments . Other sBased on the promising experiences on zirconia abutments so far, a broadened indication area (for example a more frequent application in molar regions) can be expected in the future. More clinical studies with longer observation periods and increased numbers of observed zirconia abutments are definitively needed to draw final conclusions on the clinical long-term performance of zirconia implant abutments.in vivo and in vivo studies showed less bacterial adhesion on zirconia compared to titanium surfaces [After the discussion on the mechanical aspects related to zirconia abutments, one could question the biological response to such material. In an animal study, it was shown that the collagen fiber orientation was similar around zirconia and titanium implant necks . For botsurfaces ,99,100.In a clinical study, a similar degree of plaque accumulation was found at zirconia and titanium abutments at three years . There wRegarding bone response when zirconia abutments are used as restoration support, there were no significant differences in bone levels between zirconia and titanium abutments after 3-year follow-up . In addiin vivo performance and clinical expectations.In dental healthcare, the use of zirconia implants, as treatment option, is a new topic compared to the other dental applications described in this chapter. This implant system has been a subject of significant controversy among researchers and clinicians. Because of that, this section will review the current knowledge about such implants stating its Oral implants are a clinically and scientifically proven method for the rehabilitation of edentulous areas in the oral cavity ,103,104 Titanium has been the material of choice for the fabrication of oral implants, and many investigations have shown its long-term efficacy ,112. HowThe advantages of zirconia ceramic implants in comparison to titanium implants can be summarized in three points. Firstly, esthetics might be improved because zirconia implants are white and therefore present a better esthetic appeal than titanium . SecondlCeramic oral implants were introduced some decades ago. The material of choice at that time was aluminum oxide ,130,131.versus a single-piece Y-TZP implant in foam blocks was not different. However, the authors did not give any information on the fracture stability of the implants [A PubMed search yielded to laboratory investigations evaluating zirconia oral implants ,134,135.et al. [In a second investigation of the same group  data weret al. , this stet al.  obviousl®). Their results showed that the tested zirconia implant supporting all-ceramic Procera® crowns may be able to withstand in-vitro physiological oral masticatory forces over a simulated 5-year-period [et al. [Two investigations reported on the fracture strength outcomes of prototype two-piece implants fabricated out of zirconia ,132,136. [et al. ,136 eval [et al. ,136. It et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [Eleven animal studies reporting on osseointegration utilizing zirconia oral implants could be located . Investiet al.  investig [et al.  reported [et al. . One par [et al.  compared [et al.  inserted [et al.  evaluate [et al. , the aut [et al.  in rabbi [et al.  and the  [et al.  and furt [et al.  in that  [et al. ,147,148 et al. [et al. [Clinical investigations on zirconia oral implants are rare and of questionable scientific quality ,149,150.et al.  100 zircThe one year results of an ongoing prospective clinical investigation were presented recently in an abstract . The objIt seems that one-piece zirconia implants are stable enough to withstand normal occlusal forces. The ATZ implant systems  have beeThe use of two-piece ceramic implants for intraoral application is of concern. The fact that there is no data available on the mechanical performance of current commercially available implants in the scientific literature is astonishing. Therefore, caution with their usage is highly recommended.This section also discussed animal investigations using zirconia oral implants. All studies that compared zirconia with titanium implants show that the bone-to-implant contact was similar for both materials ,148,151.® one-piece zirconia implant system from Ceraroot®; Sigma implants from Incermed SA; zit-z implants from Ziterion GmbH; Z-Look3 from Z-systems®) no valid clinical data is available in scientific journals. The methodology of the above-mentioned clinical studies is questionable. The only prospective clinical investigation [It is noteworthy to mention that although several zirconia implant systems are available on the market  structure to overcome deficiencies of the veneered zirconia system . This coAs it was shown in the present article, zirconia is being widely applied in dentistry starting from oral implant fabrication to the manufacturing of dental crown and bridgework. Some areas are however better investigated than others. Zirconia and zirconia-supported ceramics are worthy of being further evaluated particularly with improved production methods.


ClinicalTrials.gov is valuable for aggregate-level analysis of trials. The recently published final rule aims to improve reporting of trial results. We aimed to assess variability in ClinicalTirals.gov records reporting participants’ baseline measures.The September 2015 edition of the database for Aggregate Analysis of ClinicalTrials.gov (AACT), was used in this study. To date, AACT contains 186,941 trials of which 16,660 trials reporting baseline (participant) measures were analyzed. We also analyzed a subset of 13,818 Highly Likely Applicable Clinical Trials (HLACT), for which reporting of results is likely mandatory and compared a random sample of 30 trial records to their journal articles. We report counts for each mandatory baseline measure and variability reporting in their formats. The AACT dataset contains 8,161 baseline measures with 1206 unique measurement units. However, of these 6,940 (85%) variables appear only once in the dataset. Age and Gender are reported using many different formats (178 and 49 respectively). “Age” as the variable name is reported in 60 different formats. HLACT subset reports measures using 3,931 variables. The most frequent Age format (i.e. mean (years) ± sd) is found in only 45% of trials. Overall only 4 baseline measures  are reported by > 10% of trials. Discrepancies are found in both the types and formats of ClinicalTrials.gov records and their corresponding journal articles. On average, journal articles include twice the number of baseline measures (13.6±7.1 (sd) vs. 6.6±7.6) when compared to the ClinicalTrials.gov records that report any results.We found marked variability in baseline measures reporting. This is not addressed by the final rule. To support secondary use of ClinicalTrials.gov, a uniform format for baseline measures reporting is warranted. ClinicalTrials.gov is an invaluable resource for researchers, clinicians and patients. Across-trial secondary analysis is made possible using the Database for Aggregate Analysis of ClinicalTrials.gov (AACT).  In theorThe ClinicalTrials.gov registry holds over 224,000 studies, with 23,000 of them reporting summary results. [Originally, mandatory registration of Interventional studies of drugs, biologics, or devices was covered under the FDAAA. Section 801of the FDAAA mandates that all covered studies report results within 1 year of completion.  At the mThe final rule of 2016  expands In this work, we evaluate the degree to which baseline measures reported for trials on ClinicalTrials.gov can be pooled for secondary analysis in their original format—first by analyzing counts and formats of mandatory baseline participant measures and second by comparing these to those reported in published journal articles (using a random sample) in literature.Mandatory baseline measures: According to the final rule, mandatory baseline measures to report are age, sex/gender, ethnicity and region of enrollment. In addition, any number of study-specific baseline measures can be reported . Age repFor each baseline measure, the following three elements are to be included: “(a) Name and description of the measure, including any categories used; (b) Measure Type  and Measure of Dispersion ; and (c) Unit of Measure. Furthermore, data should be reported by study arm or comparison group and overall study population.The September 2015 version of AACT  for whicwww.pubmed.com) search using the ClinicalTrials.gov identifier (NCT number) was used to identify the corresponding journal articles. We manually compared: (a) Age and Gender reporting formats, and (b) the total number of baseline measures reported in each trial. The latter was chosen as a gross measure for discrepancy in reporting of results between the two datasets.We used HLACT studies to assess agreement between ClinicalTrials.gov and published journal articles. A random sample of completed HLACTs was generated to assess the degree of agreement . The first 30 trials in the sample associated with an open access journal article were used for comparison. A PubMed  report baseline participant measures and 13,818 are HLACTs . There aHighly Likely Applicable Clinical Trials use 3,931 variables in baseline measures reports. The top baseline measures reported are listed in Manual comparison of ClinicalTrials.gov records and corresponding journal articles  reveals In this work, we analyzed studies registered on ClinicalTrials.gov to assess reporting patterns of participant’s baseline measures. Overall, our findings suggest inconsistent reporting of these measures. As in other work–10 we foFurthermore, our results show that ClinicalTrials.gov records and their corresponding journal articles differ substantially in the selection of reported measures and their format. On average, there are about twice as many measures included in journal articles than in the corresponding ClinicalTrials.gov record. Thus, we assert that ClinicalTrials.gov reports include only some of the parameters considered by researchers important enough to be published.ClinicaTrials.gov is an invaluable resource for patients, their families and healthcare providers to navigate the complex and dynamic world of clinical trials. However, ClinicalTrials.gov targets researchers as well, providing a platform for conducting across-study analyses including meta-analyses, general  and domaYet, a pre-requisite for any across-trial analysis is that trial reports are comparable. As long as many different formats are used to report baseline measures, any attempt to pool this data from multiple trials would require manual alignment of variables. Thus, aligning the 178 different Age formats found in our work is labor intensive and error-prone. Furthermore, our results suggest that the discrepancies between format of baseline measures in published articles and ClinicalTrials.gov may hinder the use of ClinicalTirals.gov as an authoritative resource for secondary analyses of journal articles.In this sense, the final rule is incomplete. While expanding mandatory reporting requirements, it does not mandate the use of uniformly coded, machine interpretable format in result reporting. Following the Notice of Proposed Rulemaking (NPRM) for Clinical Trials Registration and Results Submission, nearly 900 comments were received. The NPRM invited comments on the adequacy of the lists of proposed choices for Measure Type and Measure of Dispersion, however there were no specific comments on this topic. There is an inherent tension central to this matter. On one hand, secondary use of ClinicalTrials.gov requires standard formatting of trial reports. On the other, those conducting and sponsoring clinical trials want to have the freedom to collect, analyze and report their data as they see fit. These conflicting approaches are reflected in the comments made on the NPRM.  Authors Formulating the format of a structured results report is far from being straight forward. Considering the diversity of trials, aligning all components of their reports can also pose a real challenge. For example, when comparing trial-unique outcome definitions, such alignment may not even be possible. Yet when participant baseline measures are concerned, there can potentially be a core set defined for the most common and useful measures for machine-understandable, uniform reporting format. Experience from past shows, that when the National Library of Medicine (NLM) and the scientific community commit to the task, a standard nomenclature is generally definable. Successful terminology standardization including LOINC for lab results  and the In the digital age, unlike in printed press, space is unlimited. The redundancy-tolerant digital medium can support a parallel, flexible reporting scheme. This means that researchers reporting trials results can use any format they deem right, as long as this is done in addition to-, and not instead of- the core uniform format.Adherence to posting results of studies listed on ClinicalTrials.gov is poor, yet even among studies reporting results, inconsistent formats make automated across-trial analysis of these data impractical. The final rule aims to improve adherence to results reporting but does not solve the problem of multiple reporting formats. Our findings call for a discussion around implementing standards for unambiguous reporting of at least a core set of measures to support clinically and scientifically important secondary use of the ClinicalTrials.gov repository. Defining the set of measures to be reported and the format to be used should involve the entire research community, including funders and researchers.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


B-cell epitopes on the envelope (E) and premembrane (prM) proteins of dengue virus (DENV) were predicted using bioinformatics tools, BepiPred, Ellipro, and SVMTriP. Predicted epitopes, 32 and 17 for E and prM proteins, respectively, were then characterized for their level of conservations. The epitopes, EP4/E (48–55), epitope number 4 of E protein at amino acids 48–55, EP9/E (165–182), EP11/E (218–233), EP20/E (322–349), EP21/E (326–353), EP23/E (356–365), and EP25/E (380–386), showed a high intraserotype conservancy with very low pan-serotype conservancy, demonstrating a potential target as serotype specific diagnostic markers. EP3 (30–41) located in domain-I and EP26/E (393–409), EP27/E (416–435), EP28/E (417–430) located in the stem region of E protein, and EP8/prM (93–112) from the prM protein have a pan-serotype conservancy higher than 70%. These epitopes indicate a potential use as universal vaccine candidates, subjected to verification of their potential in viral neutralization. EP2/E (16–21), EP5/E (62–123), EP6/E (63–89), EP19/E (310–329), and EP24/E (371–402), which have more than 50% pan-serotype conservancies, were found on E protein regions that are important in host cell attachment. Previous studies further show evidence for some of these epitopes to generate cross-reactive neutralizing antibodies, indicating their importance in antiviral strategies for DENV. This study suggests that bioinformatic approaches are attractive first line of screening for identification of linear B-cell epitopes. Dengue is a mosquito-borne systemic viral infection caused by any of the four antigenically related dengue viruses (DENV). An estimated 400 million people worldwide are infected with dengue annually, leading to approximately 100 million cases of dengue and 21,000 deaths .DENV is a positive-sense, single-stranded RNA virus containing a genome of approximately 10.6 kb. The single open reading frame encodes a polyprotein precursor, which is cleaved by cellular and viral proteases into three structural proteins, capsid (C), precursor membrane (prM), and envelope (E), and seven nonstructural proteins . The E pAs shown in previous studies, B-cell responses are known to be directed against the viral structural proteins E and prM of DENV –19, whicTraditional epitope selection methods are usually cumbersome and require large resources. However, the advent of technologies related to immune epitope prediction and databases could aid the prediction of B-cell epitopes. Sophisticated bioinformatic tools enable the systematic scanning for candidate epitopes from large sets of protein antigens. This approach saves considerable time and cost, especially for researchers in countries with limited resources . In thisAs predicted and analyzed in this study, seven epitopes on the E protein demonstrated the potentiality to be used as serotype specific diagnostic markers. Several epitopes on the E protein and prM proteins were having high dengue group conservancies and located in positions with previous evidence for generating NAbs and therefore indicate a potential use of them in antiviral strategies or in developing as dengue group diagnostic markers.http://www.ncbi.nlm.nih.gov/). Each serotype consisted of fifty sequences each for both E and prM protein. The retrieved data set is representative of a wide geographical coverage  and a time span of approximately 50 years (isolates from 1963 to 2014). Isolates with partial sequences in NCBI were excluded. The variable and conserved regions were compared among the downloaded isolates after aligning the isolates using Clustal W on MEGA6 (http://www.megasoftware.net/).The E and prM protein sequences from 200 variants belonging to all 4 serotypes of DENV  were retrieved from National Center for Biotechnology Information (NCBI)  is a combination method, produced by combining the predictions of a Hidden Markova model and the propensity scale by Parker et al. [http://tools.iedb.org/ellipro/), is a web-tool that implements a modified version of Thornton's method  [http://sysbio.unl.edu/SVMTriP/), utilizes Support Vector Machine in combination with tripeptide similarity and propensity scores (SVMTriP) [BepiPred   , and togpitopes) , and theSVMTriP) . The lenThe selected sequences of the E and prM protein from one serotype were uploaded to each of the 03 computational tools . Resultshttp://www.iedb.org/). Epitopes conservancy was measured at two levels: first within each serotype (intraserotype conservancy) and then among all four serotypes (pan-serotype conservancy)  .http://weblogo.berkeley.edu/logo.cgi)  .The neighbor-joining method on MEGA6 was used to construct phylogenetic tree for each predicted epitope using the multiple sequence alignments (MSA) generated for each epitope .The epitopes were predicted independently using three selected prediction tools and the results were compared. The predicted epitopes were characterized in terms of their predictability, conservation, phylogenetics, and so forth. Several observations on the significance and the potential use of the predicted epitopes made in the present study are described below.Total of forty-five epitopes of E and prM proteins were predicted by the three tools used. The same protein regions were predicted as epitopes with respect to each serotype with one to two amino acid differences to the length of the epitopes between the serotypes. Thirty-two out of forty-five predicted epitopes are on E protein: seventeen were predicted by BepiPred, eleven by Ellipro, and four by SVMTriP . The remWith most protein locations, although the epitopes predicted are not identical, similar locations have been predicted by the three different tools, as epitopes, such that they are overlapping. The sequences of overlapping epitopes as predicted by the three tools are indicated in The percentage conservancy analysis was carried out on the entire sequence of proteins studied and for the predicted epitopes by IEDB tools. The pan-serotype conservancy ranged from 63% to 100% for both E and prM proteins. But intrastereotypic conservancies of the four serotypes for both proteins resulted in percentages more than 85%. These results indicate the similarity of isolates within a serotype and also give evidences for genetic variability among serotypes.All the E epitopes (13 in number) showed a pan-serotype conservancy ranging from 10 to 83%, nineteen with pan-serotype conservancy above 50% . For prMSeveral of the predicted epitopes were found located on the surface of the respective protein, with significant functional roles in either host attachment or infection. Some of them were also found highly conserved across the serotypes. Those epitopes with high pan-serotype conservancy and also located on the protein where it is crucial for viral infection would be very interesting. The rationale behind this approach is that conserved epitopes constitute the regions in DENV proteins with minimal or no amino acid differences among different DENV serotypes/variants and therefore are expected to cause the least if not no variability in immune response against different DENV viral serotypes/strains. Thus, such epitopes with neutralizing immunogenicity will be excellent candidates for broadly reactive vaccine development. The neutralization ability of those epitopes, however, needs verification through biochemical investigations, as any significant variability in the immune responses across serotypes, which could be caused by even a single amino acid difference, may lead to the development of ADE rather than protection. On the other hand some of the predicted epitopes were noted to have a low pan-serotype conservancy level, at the same time having high intraserotype conservancies. Such epitopes could be potential candidates for serotype specific diagnostic markers. The characteristics of the epitopes which we have identified as potentially significant in dengue diagnosis and therapeutics are discussed in the following in detail.Seven of the thirteen E epitopes with less than 50% pan-serotype conservation level, the epitopes EP4/E, EP9/E, EP11/E, EP20/E, EP21/E, EP23/E, and EP30/E, are with very low pan-serotype conservancy (less than 40%) but high intraserotype conservancies (more than 80%). In particular, the epitopes EP4/E and EP23/E are suggestive to be promising candidates to be serotypic diagnostic marker, owing to their pan-serotype conservancy below 15%. To add on to the evidence, EP4/E and EP9/E of the above are located on DI of E protein. This is complementary with the findings of Roehrig et al.  that DI Unlike the above mentioned E protein epitopes, except for EP13/prM (which has a pan-serotype conservancy of 33% and intraserotype conservancy more than 80%), none of the other predicted prM protein epitopes had striking characteristics of a potential candidate for a serotypic diagnostic marker, as determined through conservation analysis in this study. The epitope EP7/prM (Ellipro), corresponding to the peptide region 55–65 a.a. on the prM protein, showed a pan-serotype conservancy of 18%. However it only showed higher intraserotype conservancies for DENV2 (81%), DENV3 (81%), and DENV4 (90%), whereas it was only 36% for DENV1. Therefore these epitopes could be useful in the differentiation of the former serotypes. A previous study on DENV infected mice and human using prM protein of DENV2 has established the production of specific antibodies against the region 57-71 a.a., indicating the potential use of EP7/prM in specific identification of certain serotypes . This evEP5/E (62–123 a.a.) predicted by Ellipro includes highly conserved fusion loop (FL) (97–111 a.a.) and bc loop (73–79 a.a.). EP6/E (63–89 a.a.) which is predicted by BepiPred contains bc loop within the peptide stretch. Further EP7/E (97–108) predicted by the same tool is more or less within the fusion loop. The above epitopes, which are located on the DII of E protein, show pan-serotype conservancies of 64%, 67%, and 83%, respectively. According to Rey et al. , DII conα helices (EH1 and EH2) in the stem region (396–452 a.a.) and two transmembrane domains (ET1 and ET2) in the anchor region (452–495 a.a.) [Seven epitopes have been identified in the C-terminus of E protein, where there are two 95 a.a.) . EpitopeAnalysis of WebLogo revealed a partially conserved region on prM protein spanning from 65 to 117 a.a., which includes EP8/prM and EP9/prM. It is worth noting that EP9/prM showed a pan-serotype conservancy of 75% and an intraserotype conservancy higher than 90% within each of the four serotypes. This result suggests that this epitope could be a potential universal vaccine candidate, if it also proves to be neutralizing upon verification with laboratory experiments.Several other epitopes are located on the protein at locations suggestive to be important in viral infection, host cell binding, and so forth. As such, DIII of E protein is the putative receptor binding domain based on several factors: DII has an immunoglobulin-like fold characteristic of many cell receptors, DIII has loops that project further from the virion surface than either DI or DII, and various soluble forms of DIII have been shown to block infection of cells by DENV . PeptideSome of the predicted epitopes are located partially on two different domain regions: EP17/E (287–313 a.a.) is located partially on DI and partially on DIII and EP24/E 371–402 a.a.) is partially on DIII and stem region. EP26/E (393–409 a.a.) represents the DENV complex conserved peptide 393-KKGSSIGQ/KM-401 . The seq2 a.a. isFinally, we have visualized all the epitope sequences of E and prM proteins on WebLogo, as shown in Tables This study concludes that the bioinformatic approach is an effective initial step to screen potential linear epitopes of DENV E and prM proteins. These predicted epitopes, however, need verification through experimental approaches in order to confirm their immunogenicity and neutralization abilities, before confirming their potential use in diagnostic or therapeutic applications. According to the analysis of the current study, the epitopes, predicted bioinformatically, prove promising being carried to the next step of experimental verification as future work.


Identifying hierarchies and rankings of nodes in directed graphs is fundamental in many applications such as social network analysis, biology, economics, and finance. A recently proposed method identifies the hierarchy by finding the ordered partition of nodes which minimises a score function, termed agony. This function penalises the links violating the hierarchy in a way depending on the strength of the violation. To investigate the resolution of ranking hierarchies we introduce an ensemble of random graphs, the Ranked Stochastic Block Model. We find that agony may fail to identify hierarchies when the structure is not strong enough and the size of the classes is small with respect to the whole network. We analytically characterise the resolution threshold and we show that an iterated version of agony can partly overcome this resolution limit. Identifying ranking hierarchies in complex networks is of paramount importance in many disciplines and applications. An exact hierarchical organisation in a directed network means that the set of nodes can be divided in an ordered collection of classes such that links exist only from a node of a low rank class to a node of a higher rank class. Clearly it is equivalent to define exact hierarchical structures when links exist only from upper to lower classes.Recently the relevance of measuring ranking hierarchy was pointed out in the context of ecosystems , in whicoptimal ordered partition of nodes into classes such that the structure has a maximal level of hierarchy.Since real networks are not necessarily exactly hierarchical, the problem considered here is to find an Framed in this way, the procedure is to choose a suitable hierarchy metric and to devise feasible algorithms which find the ordered partition of nodes in such a way that the hierarchy metric is maximised. In , this maranking hierarchy we employ in this paper, introduced in .Define ∀dlinks}].Define ∀Remarks 2A possible interpretation of the RSBM is thatp, qgive the backbone of the hierarchical structure, whilesrepresents the noise.As mentioned in Remarks 1.2, the definition of the cost function implies that links between nodes of the same rank have a positive cost. This means that those links are classified as backward, and for this reason they are assigned a probabilitysas the other backward links in the model.Since RSBMs are random graphs, different realisations of the model give different values of agony and hierarchy. We will compute below the expected value of these quantities.hd, the hierarchy of the planted ranking of RSBM graphs. Note that we make a little abuse of notation indicating with hd the value 1 − Ad/m, i.e. we do not consider the minimisation of agony. For this reason h is not necessarily bounded between 0 and 1 as We estimate the expected value of hd, we obtaineij. We consider a second order Taylor expansion:Indicating with ni = O(N)∀i, then the last two terms in N → ∞, henceIf we assume that Ad on realisations of such graphs, it is not a priori obvious that the optimal ranking is the planted one. We therefore ask the following question:RSBMs are constructed with a specific ranking, the planted one, which is determined by the choice of the classes and the model parameters. When minimising a generalised agony Given a RSBM, find the rankingrwhich minimises the generalised agonyAd. In particular check when the planted rankingrp) are not considered and we expect to play a minor role when the number of planted classes is large. In any case in the subsection Simulation we use numerical simulations to test our intuition.This is in general a complicated problem and we do not have a complete answer to this question, despite the fact that it is possible, at least for the number of classes changes either by merging adjacent classes or by splitting each class; due to homogeneity, merged or split classes have all the same size;j = 1, ‥, R, i.e. nodes in highest ranks of the planted ranking are given lowest ranks in the alternative. Moreover we consider also the case when the number of classes is arbitrary, but again their size is assumed to be uniform.the rank is inverted, For this reason we shall compute the generalised agony of the following alternative rankings:direct, in contrast with inverted for the latter. For each of these alternative rankings we compute the value of optimal the ranking with highest value of global optimum. We will see for example that numerical simulations of some RSBM indicate that the globally optimal ranking is a partial inversion of the planted hierarchy. However this analysis serves to show that planted ranking might not be globally optimal for some generalised agony and to provide an upper bound for the resolution threshold as well as getting intuition on the characteristics of the optimal ranking in a RSBM.To distinguish between the two families of ranking, we will denote the former as p ≥ q > s, termed a twitter-like hierarchy;q = 0, p ≠ 0 termed a military-like hierarchy.In the following we will focus on two regions of the parameter space of RSBMs:s. As we will see the global optimal ranking of the two hierarchies is quite different.In the former hierarchy forward links can connect low rank nodes with nodes of any higher rank, while in the latter the forward links can connect a node only with nodes in a direct superior class. In both cases backward links can exists with a probability b < 0) or merging (b > 0). The parameters a > 1 and b < a are such that a−b classes as rb), the first order estimate of p, q, s, a are the parameters of the RSBM, while b refers to the modified ranking rb)(. Moreover it is clearly As for merging (p ≥ q > s) it is In the twitter-like hierarchy . The parameters p = q = 0.5, R = 32 are fixed, while different curves refer to different values of s. We plot the variable s is small the maximum value of sm of the parameter describing the probability of a backward link, the planted ranking is no longer optimal and merging classes gives a ranking with higher hierarchy. Notice that for h* ∈ . The condition To show this, in the left panel of s. As explained, when s < sm it is s = smax it is sm sets a resolution threshold, since twitter-like graphs with a probability of backward links larger than sm will not be correctly identified by agony with d = 1. More precisely sm is an upper bound of the resolution threshold, since other rankings, not considered here, could have higher hierarchy than the planted and the merged ones when s < sm.The right panel of R, as we prove in the following Proposition 1, the resolution threshold scales as sm ∼ (6p − 3q)/R2, i.e. the more communities are present the more it is difficult to detect them. The same happens for large networks (N → +∞). Taking the number of classes constant and letting p and q scale as 1/N to keep the connectivity fixed, one immediately sees that sm = O(N−1), i.e. for large networks and fixed number of classes the detectable structures are those with very strong hierarchical structure. Thus agony with d = 1 has strong resolution limits for large graphs, similarly to what happens with modularity and community detection.Interestingly for large number of classes q = 0) because for large s inverted rankings become better than direct ones. To show this, we refer to the left panel of s, and circles to identify p = 0.5 and R = 32. For small values of s (solid blue lines), s reaches the critical value si (dashed red lines), the optimal choices for both the direct and inverted rankings give the same value of hierarchy. For higher s (dotted green lines) the only direct ranking with non negative hierarchy is the trivial one, i.e b. Therefore in this regime inverted rankings outperfom the planted one.The situation is more complex in the military-like hierarchy .The right panel of d = 1 in the following proposition.We summarise the results for Proposition 1Whend = 1 andp ≥ q > s, (Twitter hierarchy) the first order estimate for the optimal value ofhwhereFurthermore, whenq = 0, (Military hierarchy)whered = 1) than the planted one. The merging of the classes for the twitter hierarchy is due to fact that for a large number of classes it might be more convenient to aggregate classes paying a penalty equal to one than to leave them separate but paying a higher penalty for the distant backward links. Similarly, for the military hierarchy, when the number of backward links is relatively large, it is more convenient (in terms of agony) to invert the ranking because forward links do not enter the cost minimisation. Thus even if p is much larger than s and the number of forward links is much larger than the number of the backward links, it is more convenient to invert the ranking to avoid to pay large penalties of backward links between very distant classes.In conclusion, we explicitly showed that for RSBMs there exist alternative rankings with a smaller agony  the optimal value for the first order estimate ofhis given by (for both Twitter and Military hierarchy)N, so in specific realisations of the RSBM the optimal ranking can have a number of classes smaller than N.Finally we note that the value of d = 2. Similarly to the case d = 1, splitting is never optimal, both for the direct and inverted rankings, while merging can give rankings with higher value of b > 0:Finally, we consider the case of p ≥ q > s), the behaviour is similar to the d = 1 case. Since b, inverted rankings are never optimal. The planted ranking is optimal up to the critical value sm2, for the probability of backward links. After that, merged rankings outperform the planted one, and the number of classes decreases with s. When s2,1 < s ≤ smax the optimal choice is the trivial ranking, i.e. d = 1 case, the resolution threshold is now higher, since it can be shown that sm2, ≤ sm. Moreover, while, as noted before, in the d = 1 case sm = O(R−2), in the d = 2 case the resolution threshold is not only stricter but also it decreases faster as the number of classes increases, since it scales as d = 2 the large s case has the trivial ranking as the optimal one, whereas in the d = 1 case the optimal ranking has two classes.As before we describe the behaviour for the two considered hierarchies and then we state the proposition summarising our results. For the twitter-like hierarchy (q = 0), the planted ranking is proven to be optimal with respect to the direct rankings up to the critical value s, starting from d = 1, in this case it holds p/R3, while inverted rankings are to be preferred for any For the military-like hierarchy (d = 2 in the following proposition.We summarise the results for Proposition 3Whend = 2 andp ≥ q > s (Twitter hierarchy), the first order estimate for the optimal value ofhwhereandis givenFurthermore, whenq = 0 (Military hierarchy),whered, for which the sums in the estimates of agony have a closed formula. Intuitively as s. In other words agonies with d > 1 are strongly suboptimal and are able to identify very strong structures.With this last proposition we showed that hierarchy detection with quadratic cost function has a behaviour very similar to the linear case. However the resolution limits we highlighted before escalates in this case, and, as a result, only very strong hierarchies are detected correctly when the number of class is large. The same computations can be done also for greater integers d < 1. For at least some of those d one can expect to soften the resolution limits associated to integer d. However the approach to study the regime cannot rely on analytical formulae.Following this remark, better candidates as penalty functions are likely those with 0 < In this Subsection we show the results of numerical simulations to test the propositions we presented before. This is important for two reasons. First, to show that the guessed rankings, obtained by merging, splitting, or inverting the planted one, are indeed the optimal ones or have a hierarchy close to the optimal one. Second, to prove that the first order approximation and other simplifying assumptions give analytic expressions close to numerical simulations.igraph [agony  for brevity, and which gives the exact solution for the optimisation problem when d = 1. Finally, we use the MCMC algorithm in the GraphTool [We use igraph  to sampligraph , which wraphTool  package p, q, s, R, N and the results are consistent, thus in the following we present only representative cases. We use the adjusted Rand Index (RI) [We perform the same analysis with different choices for the parameters dex (RI)  to measuN = 212 = 4096, and we vary the value of s. agony for different values of s. The heat-maps are constructed as follow: a square in position  refers to the number of nodes that belong to class i in the planted rank and are placed in class j by agony: the darker the colour, the higher the number. For small s  the algorithm recovers faithfully the planted ranking and the RI is high. When the hierarchical structure becomes weaker, the ranking obtained by agony is the merging of contiguous classes in the hierarchy, as postulated in the theoretical part above. For this choice of p, q, R the resolution threshold for s is sm = 0.00151 consistently with our simulations. As we predicted, classes merge more and more when s increases. The inferred rankings are close to uniform, and the main exception is the first and last class which are smaller than the other ones.We generate RSBM with parameters agony on the simulated graphs against the hierarchy of the planted rank GraphTool. We estimate p = q and s as the average elements of the inferred affinity matrix on the corresponding classes and we leave free the number of classes. For s < sm (red symbols) the two methods agree and give a value of hierarchy consistent with the real one. When s > sm  and not necessarily to the model itself. In fact it is well known that SBM have a resolution threshold both when inference is done using Maximum Likelihood methods [GraphTool) and we compute the RI of the planted ranking versus the one obtained with agony and the SBM fit. The result is shown in s. We see that the SBM fit outperforms agony. It is clear that, since we are using SBM for generating the graphs, its fitting will be better. However what we want to stress is that there is remarkably wide interval of values of s for which agony is not able to detect a hierarchical structure even if it is strong enough to be detected by another method. Hence the limit in resolution is not embedded in the RSBM but in the objective function associated to agony.Finally in  methods  and spec methods . To this methods  for the p = 0.5 and nine values of s. With these parameters our formulas give si = 0.00280 and s1 = 0.00284. We see that for strong hierarchical structures  agony recovers well the classes. However when s increases a partial inversion of the hierarchy is observed and only for large s we recover the fully inverted ranking we studied in the previous Section. Thus simulations show that the latter is not always the optimal ranking but rather there are partially inverted rankings with a larger hierarchy. The purpose of the above analysis on the military-like hierarchy is to show that there exist values of the parameters for which the planted ranking is not optimal and to demonstrate that partial inversion can outperform the planted one. Moreover the partial inversion is observed for s = 0.002 < s1, hence our computations provide a upper bound of the true resolution threshold.For the military-like hierarchy things are more complicated. agony on the simulated graphs against the hierarchy of the planted rank GraphTool. The main message of the Fig is that, despite the fact the symmetrically inverted ranking is not the optimal one according to numerical simulations, its value of hierarchy is very close to the one of the optimal ranking, while the planted one strongly mis-estimates the value of h. Thus our computation in the previous Section can be used to reliably estimate the hierarchy of a military-like ranking. This is obviously a partial answer and analytical calculations of the hierarchy of partially inverted rankings are left for a future study.In the previous Sections we have shown theoretically and numerically that inference of ranking hierarchies based on agony suffers from significant resolution limit. In twitter-like hierarchies, the identified classes are merging of adjacent classes and thus small classes are not identified. In military-like hierarchies inversions start to play a significant role.An heuristic method to overcome this problem is to iterate the application of agony. As done with modularity, one can apply agony to each class found in the first iteration of the algorithm, in order to find subclasses. In principle one could continue to iterate, even if the fact that agony finds two classes in an Erdös-Renyi graph suggests a careful design of the stopping criterion. The purpose of this Section is not to propose a full criterion for the improvement of agony via iteration, but to show that indeed improvement is possible, both considering model graphs and real networks.agony with d = 1. For small values of s the second iteration does not improve the inference because one iteration already recovers the planted structure. For larger values of s, i.e. weaker structures, the second iteration dramatically outperforms the result of the first one, indicating that iterated applications of agony can significantly improve the hierarchies detection. h after the second run is actually smaller than the one from the first run, despite the fact that the RI follows the opposite pattern. This is expected since agony finds the optimal value of h, while the RI looks at the similarity with the planted ranking. A closer look to the results of the two iterations  but they are all quite sparse.agony algorithm. Specifically, for each network the table contains: the number of nodes N, the density (m is the number of edges), the percentage of nodes in the largest strongly connected component (SCC), the value of R) and the total number of classes after the second run (R′) of agony.It is clear that the second application the algorithm to the classes detected in the first iteration increases significantly the number of classes, suggesting that the classes identified in the first iteration could be aggregation of smaller classes. In agony penalises links among nodes in the same class, the subgraphs in some cases have no links  agonies have a resolution limit, being unable to detect small classes in large networks. This is somewhat similar to the well known resolution limit of modularity in community detection. In some cases we have strong numerical indications that the proposed alternative rankings, are actually close to the global optimal one. Finally we have shown that in these cases the iterated application of agony can lead to significant improvement of the hierarchy detection.d (or other agony functions). We have shown some results indicating that the resolution threshold depends on d, however numerical simulations cannot be performed because of the lack of heuristic methods for optimisation of agony with d ≠ 0, 1. Finally, other methods to identify ranking hierarchies could be investigated, for example suitably modifying the agony function or by considering optimisations for a set of functions.There are several directions along which our work can be extended. First, we have investigated in detail the case of uniform cardinality of the classes, even if our formulae can be used to study more complex structures, such as a pyramidal hierarchy with a small top class and larger bottom classes. With a careful choice of the sizes one might be able to maintain analytical tractability, however the study of these structures are left for future investigation. The second direction is to consider, at least theoretically, other values of We leave these extensions for future work and we are confident that the results will be of interest in the general problem of hierarchy detection in networks.S1 Appendix(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Information is missing from the Funding section. Yashpal S. Kanwar was also a recipient of National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (US)(NIHNIDDK)RO1DK60635. The correcting Funding Disclosure Statement should read as follows: This work was supported by the following sources of funding: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases(US) (NIHNIDDK)RO1DK097240, Recipient: Janardan K. Reddy; National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (US) (NIHNIDDK)RO1 DK083163, Recipient: Janardan K. Reddy; National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (US)(NIHNIDDK)RO1DK60635, Recipient: Yashpal S Kanwar; and National Institutes of Health (US), National Institute of Allergy and Infectious Diseases(NIH) (AI)R21AI1094296, Recipient: Bayar Thimmapaya.


In case of keratoconus, rigid gas-permeable contact lenses as the correction method of first choice allow for a good visual acuity for quite some time. In a severe stage of the disease with major cone-shaped protrusion of the cornea, even specially designed keratoconus contact lenses are no more tolerated. In case of existing contraindications for intrastromal ring segments, corneal transplantation typically has a very good prognosis.In case of advanced keratoconus – especially after corneal hydrops due to rupture of Descemet’s membrane – penetrating keratoplasty (PKP) still is the surgical method of first choice. Noncontact excimer laser trephination seems to be especially beneficial for eyes with iatrogenic keratectasia after LASIK and those with repeat grafts in case of “keratoconus recurrences” due to small grafts with thin host cornea. For donor trephination from the epithelial side, an artificial chamber is used. Wound closure is achieved with a double running cross-stitch suture according to Hoffmann. Graft size is adapted individually depending on corneal size . Limbal centration will be preferred intraoperatively due to optical displacement of the pupil. During the last 10 years femtosecond laser trephination has been introduced from the USA as a potentially advantageous approach.Prospective clinical studies have shown that the technique of non-contact excimer laser PKP improves donor and recipient centration, reduces “vertical tilt” and “horizontal torsion” of the graft in the recipient bed, thus resulting in significantly less “all-sutures-out” keratometric astigmatism (2.8 vs. 5.7 D), higher regularity of the topography (SRI 0.80 vs. 0.98) and better visual acuity (0.80 vs. 0.63) in contrast to the motor trephine. The stage of the disease does not influence functional outcome after excimer laser PKP. Refractive outcomes of femtosecond laser keratoplasty, however, resemble that of the motor trephine.In contrast to the undisputed clinical advantages of excimer laser keratoplasty with orientation teeth/notches in keratoconus, the major disadvantage of femtosecond laser application is still the necessity of suction and applanation of the cone during trephination with intraoperative pitfalls and high postoperative astigmatism. However, even special keratoconus lenses can no longer be tolerated after a certain level of cone-shaped deformation of the cornea . If ther2). Still, a lack of donated tissues with long waiting lists for keratoplasty presents an unsolved problem, above all for those young patients who are in the midst of their professional years between the ages of 20 and 40, who deal with psychological and economic problems of keratoconus. The indication for keratoplasty with keratoconus has fortunately become more rare after the introduction of Riboflavin UV A Crosslinking . The Hom1.1Today, a clear graft after normal risk keratoplasty in keratoconus with high and/or irregular astigmatism can no longer be considered “successful” -14. TherDecentration, especially with host trephination [hination -18.“Horizontal torsion”, i.e. the second cardinal suture is not placed exactly 180° opposite to the first ccardinal suture [l suture .Vertical tilt” i.e. non-congruent cut edges are compensated by suture tension in order to achieve a water-tight wound closure [“ closure , 19.1.23), consideration should be given as to whether cyclosporin A orally can be administered at a dosage of 150 mg twice a day for 4 weeks before keratoplasty [As a matter of principle, systemic underlying diseases such as rosacea or neurodermitis - in which problems with the surface of the eye are very common - must be identified and consistently treated before keratoplasty. In cases of very severe neurodermatitis Fig.  and as small as necessary . Mostly, transplants ranging from 8.0 to 8.5 mm are ideal for keratoconus , 14, 20.1.44) should be avoided, because suture anchoring in gelatinous tissue is insufficient, and one can regularly count on post-operative thread loosening and corresponding adverse consequences, such as infectious infiltration, neovascularisation and immune responses. The prevalent fear of the doctor and patient of penetration is largely unjustified in corneal hydrops! Nourishing and decongestant drugs are topically applied, and keratoplasty is carried out after 3 to 6 months with good success, after the previously oedematous areas of the cornea have become sufficiently scarred and solid [Immediate penetrating keratoplasty in the acute stage of keratoconus resulting from rupture of Descemet’s membrane   ). In chiTypically, the pupil is constricted with pilocarpine in order to protect the lens of the phakic eye.Horizontal positioning of the head and limbal plane is an indispensable precondition for the avoidance of decentration, “vertical tilt” and “horizontal torsion” .Paracentesis at the limbus is recommended before trephination.In cases of doubt, limbal centration should be preferred over pupil centration, because the optical displacement of the pupil must be taken into consideration . In no cDonor and recipient trephination should be performed from the epithelial side with the same system. This is the prerequisite for congruent cut surfaces and angles in the donor and recipient. An artificial anterior chamber is typically used today for donor trephination .Orientation structures in donor and recipient facilitate the correct placement of the first four or eight cardinal sutures and thereby contribute towards preventing “horizontal torsion”. The second cardinal suture is absolutely crucial for a correct graft fit .It is clearly not advised to under-size the graft in comparison to the recipient bed to compensate myopia, because with that, no tension-free adjustment of the transplant is possible , resulti5) serves as prophylaxis against so-called Urrets-Zavalía syndrome in young people with keratoconus [6A-C). We assume that Urrets-Zavalia syndrome results from peri-operative angle closure with a maximum pressure gradient and iris sphincter atrophy resulting from wound leakage.A peripheral iridotomy at 12 clock  is preferred, since it results in higher topographic regularity, earlier visual rehabilitation and a lower rate of suture loosening [8), posterior steps are allowed, and often unavoidable with keratoconus.As long as Bowman's layer is intact, a double running cross-stitch suture according to Hoffmann  with tension adaptation of the continuous sutures or re-placement of the simple interrupted sutures should be used after the lid speculum and cardinal sutures have been removed.1.6i.e. the graft does not clear up at any time after PKP), the aim should be to replace it at an early stage, i.e. after no more than 6 weeks. Here, if the donor tissue has been documented as good at the cornea bank and the surgical technique was uncomplicated, it is always important to consider latent herpes simplex virus infection of the graft as the cause of primary graft failure [In cases of primary graft insufficiency . This is not the only reason why trephination today is done preferredly non-mechanically with a laser, especially for keratoconus. In clinical practise, non-mechanical trephination may today be performed with the 193-nm excimer laser (since 1989) or a femtosecond laser (since 2006). In Homburg/Saar and Erlangen, we have, since 1989, successfully trephined more than 1,500 keratoconus eyes with the Zeiss-Meditec MEL60®, MEL70® and the Schwind Amaris Excimer laser using metal masks with eight “orientation teeth/notches” [Each mechanical trephination in thinned keratoconus corneas leads to non-round openings in the host cornea due to compression and distortion. Prof. Herbert Kaufman has recommended for more than 20 years not to use an obturator in trephination of the keratoconus with a manual trephine Fig.  is fixed in an artificial anterior chamber and toned to a physiological intraocular pressure. Subsequently, a 0.5 mm thin metal mask is centred and placed in position and a laser beam (Ø approx. 1 mm) is guided by a helium-neon aiming beam moving along the outer edge of the metal mask to the perforation. The central cornea is protected from stray radiation by viscoelastics. In recipient trephination, a 0.5 mm thin recipient mask is placed directly on the patient's cornea. Here the laser beam is moved along the inner edge of the metal mask . Further acceleration and standardisation of excimer laser trephination has been achieved since 11/2014 in Homburg/Saar using the pseudo ring profile of the Schwind-Amaris Excimer laser (750 Hz).10A) after having assured the horizontal direction of the limbal plane of the eye. The recipient mask rests on the cone like a “ruff” without exerting distorting forces during trepanation . Due to the use of the mask technique, an identical configuration of the donor and recipient is assured for both the vertical and horizontal dimensions.A major benefit of excimer laser trephination is the ability to lay the mask freely around the cone-shaped protruding cornea  is made considerably easier during the subsequent course of the operation because the first 8 cardinal sutures provide the microsurgeon with a waterproof wound early on.Symmetrical adjustment of the graft in the recipient bed is ensured through so-called orientation teeth/notches , 34. The2After suture removal, the astigmatism in the control group (Geuder Motor Trephine) - at 6.1 ± 2.9 dpt - was twice as high as in the excimer laser group at 3.0 ± 2.1 dpt. The decisive factor is that in the excimer laser group - at 2.8 ± 2.0 dpt - almost the entire keratometric astigmatism had been subjectively tolerated in the spectacles as a cylinder, whereas this was true only for two-thirds in the control group at 4.2 ± 2.4 dpt. This may be dependent upon the regularity of the graft surface. After suture removal, the SRI in the excimer laser group - at 0.91 ± 0.45 - was significantly lower (p< 0.01) compared to the control group at 1.0 ± 0.46. In the excimer laser group, the astigmatism diminished after suture removal by 0.2 ± 3.1 dpt. In the control group it increased by 2.3 ± 3.4 dpt. Overall, there was a decrease in astigmatism in 52% of the eyes of the excimer laser group after suture removal, whereas in the control group there was an increase in astigmatism in 76% of the eyes after suture removal.Concerning mechanical trephination, the Guided Trephine System GTS with donor and recipient trephination from the epithelial side appears to be the highly beneficial, especially for keratoconus. Objective astigmatism results appear to be only slightly less favourable with GTS than excimer laser trephination .However, the only crucial aspect for the patient is the visual acuity with spectacle correction: After suture removal, the best spectacle-corrected visual acuity in the excimer laser group increased to 0.8 ± 0.2 and was therefore significantly higher than in the control group (0.6 ± 0.2) (P<0.001). These results are of special significance above all for patients with keratoconus, who are usually young, have a career and benefit in particular from early and complete visual rehabilitation . We were2.111).Last but not least, the 193-nm excimer laser enables non-contact (!) trephination with a primary unstable cornea. This includes, among others, the situation of iatrogenic keratectasia as a consequence of LASIK. With a so-called ‘recurent keratoconus’ where the graft diameter was too small initially, a well-centred repeat keratoplasty with a larger diameter  allows less suture tension without creating a so-called ‘barrel-top configuration’ at the graft-host-junction with good vision even in the presence of sutures  , analogue). The eight lines which are applied, for example with the Intralase femtosecond laser, in the donor and recipient can often not be brought into complete alignment intra-operatively in the keratoconus eye [12). There is not much data about the potential advantages of femtosecond laser keratoplasty after suture removal. Only the Freiburg working group has published results pertaining to the situation after complete suture removal. After a mean follow-up of 14±5 months, topographic astigmatism without sutures in the mushroom profile was 6.4±3.0 dpt, and in the top hat profile 5.8±4.6 dpt [13).Femtosecond laser keratoplasty has caused a good deal of excitement for the past 10 years. The advantages of femtosecond laser keratoplasty are the arbitrary horizontal and vertical shapes, including the “top hat”, “mushroom”, “zigzag”, “Christmas tree”, “octagon”, “decagon”, “dovetail” etc . The prionus eye  . In add±4.6 dpt  . The special characteristics of the mushroom technique researched here are shown in the following details: (1) thickness of the anterior peripheral rim is 2/3 of the corneal thickness, (2) donor oversize by 0.1 mm, (3) intended pre-Descemet’s suture placement with a double-continuous cross-stitch suture.Recruiting for a prospective, randomised study which compares the results between excimer laser- and femtosecond laser-assisted trephination for keratoconus, has been concluded in the Homburg Keratoconus Centre (HKC) ,topographic astigmatism (not only refractive cylinder!),topographic regularity60 patients aged 20 to 81 years  were randomised into four groups in this single-centre study. There were 15 keratoconus and 15 Fuchs-dystrophy patients, each operated with an excimer laser (groups I and II) and with femtosecond laser (groups III and IV). Exclusion criteria included repeat PKP and/or simultaneous lens surgery. A ‘mushroom’ (keratoconus) or ‘top hat’  profile was created using FSL with an internal diameter of 7.5 mm and an external diameter of 8.5 mm as well as a so-called ‘side cut’ in 2/3 of the corneal thickness. The diameter of the cylindrical profile for the excimer laser PKP was 8.0 mm. The overdimension of the graft was 0.1 mm in all groups. Fixation was done in a standardised way with a double,-running cross-stitch suture. With FSL PKP, the stitch depth was intended pre-Descemet’s and not to the plane of the side cut. The first suture were removed after 1 year, the second suture was removed after 1.5 years under topical anaesthesia .The following results were obtained: In particular for keratoconus, the FSL group showed more decentration, more Vis à tergo, and more often individual sutures were required in order to ensure donor-recipient-apposition without steps and gaps . At leas2.5Deep anterior lamellar keratoplasty (DALK) can be considered as an alternative to PKP in keratoconus. This procedure is surgically demanding and time-consuming. Furthermore, the course of the operation is not anywhere near as possible to standardise as with PKP , 43. It  This was described originally by Anwar  in 2002,In Homburg/Saar we prepare the donor and recipient trephination with the excimer laser along the metal mask in the typical way , 46, but2.6We recommend prednisolone acetate AT initially 5 times a day, then taper every 6 weeks by 1 drop to prevent immune reactions. In addition, we recommend nourishing gel 5 times per day (such as Hylogel or Corneregel) because corneal sensitivity is dramatically reduced in the first months after PKP. The first suture is removed after 1 year, the second suture after 1.5 years under outpatient conditions. Additional microsurgical procedures  or final eyeglass adjustments are carried out at least 6 weeks after complete suture removal.1). A few published results in relation to astigmatism and topographic regularity after suture removal demonstrate that the high technical and financial cost of FSL use for PKP - especially in keratoconus - is not justified.In order to avoid high and/or irregular astigmatism after suture removal, a trephination system should be used for penetrating keratoplasty which ensures symmetrical, tension-free fitting of a circular donor disc in a circular recipient bed with congruent and easily waterproof-adapting incision edges. These demands for optimal trephination are best fulfilled at this time with a non-mechanical excimer laser assisted trephination procedure, which has been conducted for more than 25 years on more than 4,000 patients, and shows proven benefits in regards to topographic astigmatism, regularity of the topography and vision after suture removal. The effort is particularly valuable in (1) young patients with keratoconus – especially after corneal hydrops, (2) repeat keratoplasty (due to high astigmatism), (3) paediatric keratoplasty, (4) unstable cornea . Femtosecond laser assisted keratoplasty has significantly more expense, and the need for suction and applanation of the conus are combined with intraoperative disadvantages such as decentration and step/gap formation at the graft-host-junction – often requiring additional single sutures at the end of surgery. Superiority of femtosecond laser assisted PKP could not be demonstrated by us and also in the literature over the past 10 years (Table


Fluorescent nanostructures (NSs) derived from polysaccharides have drawn great attention as novel fluorescent probes for potential bio-imaging applications. Herein, we reported a facile alkali-assisted hydrothermal method to fabricate polysaccharide NSs using starch and chitosan as raw materials. Transmission electron microscopy (TEM) demonstrated that the average particle sizes are 14 nm and 75 nm for starch and chitosan NSs, respectively. Fourier transform infrared (FT-IR) spectroscopy analysis showed that there are a large number of hydroxyl or amino groups on the surface of these polysaccharide-based NSs. Strong fluorescence with an excitation-dependent emission behaviour was observed under ultraviolet excitation. Interestingly, the photostability of the NSs was found to be superior to fluorescein and rhodamine B. The quantum yield of starch NSs could reach 11.12% under the excitation of 360 nm. The oxidative metal ions including Cu(II), Hg(II)and Fe(III) exhibited a quench effect on the fluorescence intensity of the prepared NSs. Both of the two kinds of the multicoloured NSs showed a maximum fluorescence intensity at pH 7, while the fluorescence intensity decreased dramatically when they were put in an either acidic or basic environment (at pH 3 or 11). The cytotoxicity study of starch NSs showed that low cell cytotoxicity and 80% viability was found after 24 h incubation, when their concentration was less than 10 mg/mL. The study also showed the possibility of using the multicoloured starch NSs for mouse melanoma cells and guppy fish imaging. Novel nanostructures (NSs) with multicoloured fluorescence have attracted much attention because of their potential biological applications including molecular labelling, contrast medium, sensors and image-guided drug release ,2,3. TraUp to now, many of kinds of methods, such as arc-discharge , electroIn this work, the synthesis and characterization of multicoloured NSs were reported using starch and chitosan as raw materials. As linear polysaccharides, the starch and chitosan contain a large number of glucose or glucosamine units joined by glycosidic bonds. We compared the properties for these two kinds of polysaccharide-based NSs. The metal ion effects on the fluorescence intensity were investigated. The multicoloured NSs derived from starch were successfully used as multicoloured probes for the imaging of mouse melanoma cells. In addition, we further demonstrated that the polysaccharide NSs may also be used as unique nanostructural probes for the imaging of small aquatic craniate animals.The polysaccharide-based NSs were synthesised through a facile one-pot hydrothermal route using starch or chitosan as raw materials, respectively. As shown in −1 corresponding to the stretching vibration of O–H or amino groups. Therefore, the presence of a large number of functionalised groups, such as hydroxyl or amino groups, not only leads to the good water solubility of NSs, but also makes the surface modification easier and more convenient. The peaks at 2924–2932 cm−1 correspond to the C–H vibrations of methylene. Moreover, there is an absorption peak at about 1635–1643 cm−1, which might be due to the vibrations of carboxyl groups and aromatic C=C structures. The absorption peaks at 1375 to 1380 cm−1 demonstrate the presence of the respective –CH3. Unlike the spectrum of chitosan, a strong absorption peak in the range of 1011–1167 cm−1 is observed, thus indicating the presence of a large number of hydroxyl groups for the starch NSs, while only weak absorption peaks at 1045 cm−1 corresponding to the C–O stretching vibration are found for the chitosan NSs. As for starch NSs, the peaks of the FT-IR remained unchanged with different degrees of carbonization by varying the reaction time from 0 to 4 h, while the peak at 1660 cm−1 for chitosan NSs fades away with the increased reaction time from 0 to 2 h, indicating the formation of amide groups. The zeta-potential measurement indicated that the starch NSs had a negative value less than −18 mV at pH 6.5, and a positive value of 17.5 mV for chitosan NSs (Fourier transform infrared (FT-IR) spectra in osan NSs .Moreover, it is essential for the polysaccharide-based NSs to possess good photostability against bleaching when they are used as multicoloured agents in bio-imaging. As shown in The influence of metal ions on the FL intensity of the polysaccharide-based NSs is shown in The pH effect on the FL intensity of the polysaccharide-based NSs was also investigated . Both ofSince the starch NSs possess relatively higher quantum yields, they would be more suitable for fluorescence cell imaging to gain a higher signal-to-noise ratio. In vitro cytocompatibility of the starch NSs with mouse melanoma cells was investigated by a MMT -2,5-diphenyl-2-H-tetrazolium bromide)) assay. As shown in ex = 405 and 488 nm, as compared to the control cells without the addition of NSs. This result demonstrated that the starch NSs entered into the cells resulting in multicoloured fluorescence, and multicoloured imaging due to the excitation-dependent emission property. Moreover, the starch NSs were also evaluated for organisms imaging by treating small guppy fish with these polysaccharide-based NSs. As shown in The potential use of these starch NSs was explored with in vitro uptaking by mouse melanoma (B16-F10) cells. The B16-F10 cells were incubated with RPMI1640 medium containing the starch NSs for 12 h at 37 °C. 3PO4, >85%) was purchased from Hengxing Chemical Reagent Co. Ltd. . Boric acid  was purchased from Bodi Chemical Reagent Co. Ltd. . All other chemicals and reagents were purchased from local reagent store. The reagents were used without further purification unless otherwise stated.Chitosan (molecular weight 30 KDa) was prepared through the degradation of raw material . Starch with a molecular weight 49k Da was purchased from Yufeng Starch products Co. Ltd. . Fluorescein and rhodamine B were purchased from Sinopharm Chemical Reagent Co. Ltd. . Quinine sulfate was purchased from Kermel Chemical Reagent Co. Ltd. . Mouse melanoma cell line B16-F10 was bought from KeyGen Biotechnology Co. Ltd. . Size-exclusion Sephadex gel G-25 was purchased from GE Healthcare . Quinine sulphate, fluorescein and methyl thiazolyl tetrazolium (MTT) were ordered from Aladdin Reagent Co. Ltd . Dialysis bags with a molecular cut-off 3000 were purchased from Genestar Biotechnology Co. Ltd. . Muffle furnace was produced by Zhonghuan Experimental Electric Furnace Co. Ltd. . A JEOL model JEM-2000EX transmission electron microscope  was used to measure the shape and size of the NSs. Fourier transform infrared (FT-IR) spectra were measured using a VECTOR 22 FT-IR spectrometer  with a KBr pellets. A Perkin Elmer LS 55 spectrofluorometer was used for fluorescence spectra measurements. UV-Visible absorption spectra were measured on a Shimadzu UV2550 UV-Vis spectrophotometer . FV 1000 confocal microscope  was used for cell imaging experiment. Ex vivo imaging was carried out with a Cri Meastro Ex in vivo imaging system . Dimethylsulfoxide (DMSO) was bought from Damao Chemical Reagent Co. Ltd. . Sodium hydroxide  was purchased from Dalu Chemical Reagent Co. Ltd. . Phosphate (HThe carbon sources of starch (1 g) or chitosan (1 g) and 0.1 g sodium hydroxide were added to 15 mL of deionised water with vigorous stirring. The reaction mixture was then put into a Teflon-lined stainless-steel autoclave and sealed heating at 160 °C for 4 h. Subsequently, the pH of the solution was adjusted to 7 using 1 M hydrochloric acid. The resulting NSs were thoroughly dialyzed for 48 h against distilled water through dialysis bags with the molecular weight cutoff of 3000. Further, the solution was centrifuged at a speed of 4000 rpm to remove impurities with larger molecules. Relatively small impurity was removed by microporous membrane made from mixed cellulose with a pore size of 0.22 μm. The obtained solution of polysaccharide-based NSs was then heated with a microwave oven (800 W) for 5 min. Later, the solution was cooled and purified with sephadex G-25 column. The fluorescent components were collected, freeze dried and stored at 4 °C for further characterization and use.wt/wt) to analyse the surface groups of NSs. Fluorescence quantum yield (Φ) of the NSs was calculated according to the following equation  = (ODtreated/ODcontrol) × 100% .In vitro cytocompatibility of the NSs with mouse melanoma (B16-F10) cells was investigated by seeding the cells (5 × 104 cells/well at 37 °C in a humidified 5% CO2 atmosphere. 100 µL multicoloured starch NSs (3.8 mg/mL) and 400 µL medium were added to each well. The glass slide with cells was washed thoroughly with PBS buffer after 5 h incubation, and the cells were fixed with formalin. In vitro tumour cell imaging was conducted with a FV 1000 laser confocal microscope and the fluorescence images were collected in blue and green region with exposure time 100 ms.B16-F10 mouse melanoma cells were seeded in 24-well tissue culture plates with a glass slide at the bottom at a density of 3 × 10Guppy fish were put in the water mixed with the starch NSs (~5 mg/mL) and the control fish were put in fresh water. After 10 min, the fish were thoroughly washed with distilled water and taken image on a CRi Meastro Ex in vivo imaging system. Spectral fluorescence images could be produced by selecting appropriate filters for NSs with excitation 455 nm, emission 515 nm long-pass filter, acquisition settings 500–750 nm in 10 nm steps). In this study, the exposure times were automatically calculated and1500 ms was selected.In summary, we demonstrated a facile one-pot hydrothermal method for the preparation of polysaccharide-based NSs using starch or chitosan as raw materials. The obtained NSs are nano-sized and highly water-soluble due to the presence of hydroxyl or amino groups on the surface for starch and chitosan NSs, respectively. Under ultraviolet excitation, the NSs exhibited strong fluorescence with an excitation-dependent emission behaviour. The polysaccharide-based NSs are very stable against photo-bleaching as compared with the traditional fluorescent compounds. Some oxidative metal ions, Hg(II), Cu(II), and Fe(III), displayed a quenching effect on the FL intensity of the NSs. The NSs showed a maximum FL intensity at physiological pH. The cytotoxicity study demonstrated that the starch NSs are safe for B16-F10 cells at a concentration less than 10 mg/mL. The multicoloured starch NSs showed success as fluorescent probes for B16-F10 melanoma cells and guppy fish imaging. The results of this work represent a step toward the design of polysaccharide-based NSs for bio-imaging applications.


International Journal of Environmental Research and Public Health (IJERPH) [The authors wish to update the Introduction in their paper published in the (IJERPH) . In the middle of the third paragraph in the Introduction, they would like add a citation to the following sentence:For example, the ability to mentally conceptualize a problem, store information temporarily in the visual-spatial sketchpad, develop a plan, evaluate and adapt complex goal-directed behavior has been identified as a product of working memory and executive function .The changes do not affect the results. The manuscript will be updated and the original will remain online on the article webpage, with a reference to this addendum.


The Brazilian Journal of Cardiovascular Surgery (BJCVS) is grateful for the reviewers,listed below, which collaborated in this edition. Without their work would be impossibleto keep the high scientific standard of our journal.Carlos Manuel de Almeida BrandãoClaudia Bernardi CesarinoEduardo Augusto Victor RochaEktor Correa VrandecicElaine Soraya Barbosa de OliveiraFábio Antônio GaiottoFábio Binhara NavarroHenrique MuradJarbas Jakson DinkhuysenJosé Carlos Dorsa Vieira PontesLuciano AlbuquerqueOmar Asdrúbal Vilca MejíaPaulo Roberto Barbosa ÉvoraTomas SalernoValéria PapaVinicius José da Silva Nina