The nature of police work often necessitates use of Individual Light Armour Vests (ILAVs) for officer protection. Previous research has demonstrated various biomechanical and physical performance impacts of ILAVs, however, little knowledge exists on the individual officer’s perceptions of ILAV. The aim of this study was to investigate officers’ perceptions of the impacts of three different ILAVs and normal station wear whilst performing police occupational tasks.A prospective, within subjects, repeated measures design was employed in which 11 serving police officers wore each of three different types of body armour  and normal station wear for a full day while performing tasks including a simulated victim drag, a patrol vehicle exit and a marksmanship shoot. Ratings of Perceived Exertion (RPE) and a Visual Analogue Scale  were used to examine officer perceptions of each ILAV. Finally, officers were asked to indicate areas of both discomfort and comfort of each ILAV on a mannequin chart.Officers perceived less effort was required for the victim drag whilst wearing ILAV B (RPE = 3.6/10) when compared to ILAV A, ILAV C and even station wear . A positive impact on performance was perceived for ILAV B (VAS = + 0.26) when performing a patrol vehicle exit and sprint task but not for the other two ILAVs . Officers perceived a positive impact of ILAV B (VAS = + 2.7) and station wear (VAS = + 1.4) and a negative impact of ILAVs A and C  on marksmanship. Despite all armour types being criticized for discomfort, ILAV B received lower ratings of discomfort overall, and some positive comments regarding both comfort and performance.Officers perceived ILAV B to have positive effects on task performance. It was also rated more comfortable than the other two, possibly due to a longer torso design which shifted load from the shoulders to the hips and pelvis. Officer perceptions of comfort and effects on occupational performance should be considered when designing and procuring armour systems. Although ILAVs may be similar, perceived impacts may vary between officers. Policing duties may intermittently involve periods of running, jumping, crawling and engaging in combat without warning , 2. NumeMore recently, lighter Individual Light Armour Vests (ILAV) weighing between 2.7–3.8 kg have been implemented to reduce officer fatalities from stabbing, blunt trauma and small calibre bullets .The importance of acceptance of additional loads by the tactical personnel who must carry them was evident in a study by Ramstrand et al. , who exaGiven the importance of acceptance by the wearer before implementation of ILAVs, the opinions of individuals required to wear the equipment should be sought during the ILAV design or procurement phases, if user engagement with the proposed ILAVs is to be achieved. Therefore, the aim of this study was to investigate officers’ perceptions of the comparative impacts of three different types of ILAV and normal station wear when they were worn while performing occupational tasks associated with policing duties.A prospective, within-subjects, repeated measures design was employed, using a counterbalanced randomization procedure to determine the order of types of ILAV and normal station wear worn by each participating officer. Each officer served as their own control and wore each of three types of ILAV for one full day, and normal station wear for one full day. On each day, all measures were taken at the same time of day while the officers wore the ILAV or standard station wear assigned for that particular day. The counterbalanced randomization procedure controlled for any potential activity learning effects and other factors (eg ambient conditions) that might have varied across the 4 days of data collection.Data collection for the study was conducted at an Australian State police college in 2016. Eleven research volunteer officers who were all qualified and serving members of the Australian State Police Force, served as participants for this study. The officer’s characteristics can be seen in Table To improve the translation of this research across the general State police force population, equal numbers of female and male officers were initially recruited, with two participants per gender sized as each of small, medium and large, with respect to the standard sizes used by the ILAV suppliers. This process and diversification allowed for the comparison of the perceived impacts of three ILAV and normal station wear in relation to both gender and body size. All of the 12 initially-recruited officers were provided an initial briefing regarding the program and, if they expressed willingness to participate , they were invited to provide written informed consent for participation. One recruited female officer was unable to commence the research due to medical concerns which were identified at this stage and so the final sample was reduced to 11 officer participants. All participants formally consented to participation and the study was approved by the Bond University Human Research Ethics Committee (protocol number 15803).The three different types of ILAV  were all weighed  prior to provision. Each type of ILAV was then fitted with standard equipment worn by each officer as part of their normal duties. This equipment was individualised and varied slightly due to each officer’s daily taskings and preferences. This equipment carried as part of normal duties also made up the fourth condition; normal station wear (N). On the days of testing, officers wore their allocated load configuration for the duration of the day. Officers were randomly allocated to one of four initial conditions  using a lot draw on the first day of data capture which employed a counterbalanced approach. Following the first day of data collection, each participant progressed to the next ILAV to be trialled, in the specified sequence, which progressed from ILAV A to ILAV B to ILAV C to normal station wear to ILAV A.To minimise any diurnal variations a standardised program was followed on each day. Noting that this program of data capture was part of a larger project, with other measures also captured, the aspects of the daily program relevant to this study are shown in Table The Rating of Perceived Exertion  is a measure of a person’s perception of the rate of effort required for a given task. While the original scale was rated between a score of 6 (no exertion) and 20  , the modThe 10 m victim drag scenario utilized a mannequin fitted with a ballistic vest (80 kg). Officers were required to lift the shoulders of the mannequin off the ground and, moving backward, drag the mannequin 6 m, complete a 90-degree right hand turn through a doorway, and continue to drag the mannequin another 4 m to the end of the track. This configuration was designed to mimic retrieving a victim from the centre of a road and then dragging them back and behind cover. The distances officers covered for the victim drag task were measured using a digital mini-measuring wheel . Officers were allowed an initial practice run at their estimated 80% of maximum capacity, to familiarize themselves with the scenario and as a warm up.The Visual Analogue Scale (VAS) is a commonly used tool to quantify perceptions of a particular type of experience . On comprapid patrol vehicle exit task, a police patrol vehicle  was parked so that the driver’s side of the vehicle opened onto a track. Officers were seated in the driver’s seat of the vehicle without their seatbelt on and with both hands on the steering wheel. A researcher gave a verbal command ‘go’ to start the scenario whilst simultaneously breaking the beam of the electronic timing system light gate . The officer exited the driver’s side of the vehicle and ran to the rear of the vehicle through two opposing timing gates placed 5 m away, as measured using a digital mini-measuring wheel   with a Glock model 22 pistol, firing 26 rounds in total, with marksmanship scored over three separate sequences. These three sequences were: Point/proximity shooting (9 rounds); Immediate distance / kneeling (5 rounds); and Transition drills / reloading (12 rounds). Each sequence assessed a single or related multiple skill set which was deemed a mandatory and necessary skill set for the operational policing environment. As soon as officers completed their marksmanship scenarios they were asked to rate on the VAS how the ILAV or normal station wear they were wearing impacted on their shooting ability. For the duration of the marksmanship task, the volunteer officers were under the direction and authority of the State police force Range Safety Officers.The participating officers were instructed not to remove their assigned ILAV, for the duration of each day (e.g. during lunch or short breaks), and following completion of activities on each day, the officers were requested to indicate on a manikin figure Fig.  any areaAll recorded data except for data relating to the participants’ subjective evaluations of ILAV were entered into a spreadsheet in SPSS version 23 (IBM 2015) and were then cleaned for analysis. Initial descriptive analyses were conducted to provide counts, means, standard deviations and ranges for the included variables, as relevant depending on levels of measurement. These descriptive statistics were derived for each ILAV type and for the normal station wear.Following these descriptive analyses, a multivariate repeated measures analysis of variance (ANOVA) was conducted to examine the relative impacts of the different ILAV types and normal station wear, with post hoc pairwise comparisons using a Bonferroni adjustment. Finally, subjective evaluations documented by the participants regarding comfort of the ILAV and normal station wear were aggregated onto a single body chart for each type of ILAV assessed, in order to provide an overview for each ILAV type of the evaluations from all participants. Comments on each body chart were numbered according to the identification number assigned to the participant who made the comment, so that multiple comments by the same participant could be identified and so that where comments were more frequent from particular participants, commonalities in those participants’ characteristics  that might have affected their evaluations could be considered.p < .04 in all instances).An overview of the measured weights of each ILAV type is provided in Table p = .048). Average RPE scores attributed to the victim drag task by officers when wearing each type of ILAV are seen in Table Subjectively, ILAV type significantly affected required effort in the victim drag, as assessed by participants using the RPE scale .p = .026). The average impact officers perceived each ILAV condition to have on their performance in the range shoot, as measured by the VAS, was positive in both ILAV B and normal station wear and negative in ILAV A and ILAV C (Table The repeated measures ANOVA indicated that ILAV condition significantly affected officer self-assessments using the VAS of ILAV impacts on task performance (marksmanship) during the range shoot  and normal station wear on subjective measures of performance and comfort whilst police officers performed occupational tasks associated with their normal policing duties. Despite numerous previous investigations reporting the effects of wearing armour on performance and mobility, there has been minimal focus on the opinion of the individual who wears the armour and no known studies comparing multiple types of ILAV.There were significant differences in the weights of the three types of body armour used in this study, with ILAV A being the heaviest and ILAV C being the lightest. Also of note, body armour weight increased as the body weight, height and chest circumference of the officer increased, since the size of the body armour required to fit them increased and so also its weight.The results from this study suggest that overall, ILAV B appeared to be the most preferred by officers when compared to other ILAV configurations, across all three tasks. For both the 10 m victim drag and marksmanship, ILAV B received significantly better ratings that the other ILAV configurations in relation to its impact on task performance. For the 10 m victim drag task the least amount of effort required to complete the task across all ILAV conditions was reported by participants to be associated with ILAV B. Likewise, ILAV B received positive  ratings regarding impacts on marksmanship performance. Interestingly, the quickest times of the Victim drag and the best score on the marksmanship task were whilst wearing ILAV B, so officer perceptions matched objective measures of performance on those tasks.In contrast to previous research , 16, 17 When data from each configuration’s subjective evaluation sheet was collated, it was apparent that the major areas of discomfort for all ILAV were the chest, shoulders and hips. This is in agreement with previously published research which found that, apart from the foot, the low back and hips were the areas of least comfort for carriers of load , 19, 20.The outcomes of this study demonstrate that the subjective feedback of officers regarding both comfort and performance are important to consider for the issuing of ILAV and that their perceptions regarding performance are accurate. For example, there was a notable preference for one of the three types of ILAV and whilst wearing this preferred ILAV, the subjective perceptions of performance were in most cases reflected in the objective measures of performance in the tasks.The future development and large-scale implementation of ILAVs should ensure that the user’s feedback and opinion is sought to ensure that first and foremost they are going to be worn, and that perceived comfort and performance impacts are considered. Seeking the opinion and acceptance of the officers who will be required to wear the equipment is vital to ensure a balance between user acceptance and equipment requirements are obtained, and given the costs associated with the procurement of new equipment for law enforcement, studies on subjective acceptance should be performed before any procurement decisions are made.There are some limitations to this study which need to be acknowledged. The results from this study are only applicable to the 3 variants which were utilised in this study and therefore may not be representative of all options available to law enforcement officers. The opinions expressed by the sample of officers in this study may also not be representative of the entire police force and a larger scale study may therefore be warranted.The results from this study suggest that participants perceived that wearing ILAV B assisted them in performing several key occupational tasks . The ILAV B also had fewer negative comments with respect to comfort and fit throughout the testing period and these findings may at least in part have been due to the longer torso design of ILAV B, shifting load of the ILAV from shoulders and chest to the hips and pelvis. Conversely, the other ILAVs were found to be more uncomfortable on the neck and shoulders, and detrimental to occupational task performance, when this was assessed both subjectively and also objectively. Nevertheless, ILAV B did cause more pressure on hips and pelvis and obstructed deployment of belt-mounted tactical equipment to some degree, according to participants, and this may be of operational concern. Overall, the results from this study suggest that feedback from wearers is important when considering the implementation of ILAV and involving personnel in this way may form an important part of organisational acceptance of new ILAV.


An unsupervised specific clustering approach was designed to search for recurrent new turns in the type IV category. The classical rules of β-turn type assignment were central to the approach. The four most frequently occurring clusters defined the new β-turn types. Unexpectedly, these types, designated IV1, IV2, IV3 and IV4, represent half of the type IV β-turns and occur more frequently than many of the previously established types. These types show convincing particularities, in terms of both structures and sequences that allow for the classical β-turn classification to be extended for the first time in 25 years.The functional properties of a protein primarily depend on its three-dimensional (3D) structure. These properties have classically been assigned, visualized and analysed on the basis of protein secondary structures. The β-turn is the third most important secondary structure after helices and β-strands. β-turns have been classified according to the values of the dihedral angles φ and ψ of the central residue. Conventionally, eight different types of β-turns have been defined, whereas those that cannot be defined are classified as type IV β-turns. This classification remains the most widely used. Nonetheless, the miscellaneous type IV β-turns represent 1/3 These properties have classically been assigned, visualized and analysed on the basis of protein secondary structures, which are composed of repetitive parts , in which the distance between Cα(s) of residues i and i+n must be smaller than 7 Å 29n = 4), α-turns (n = 5)31n = 6)33Turns comprise i.e., i+1 and i+2, should not be helical. Similarly, β-turn residues must not consist solely of β-strand residues. β-turns have been classified according to the values of their central residue dihedral angles, φ and ψ. A deviation of ± 30° from these canonical values is allowed on 3 of these angles, whereas the fourth can deviate by ± 45°β-turns have been the most analysed among the turn conformations. Apart from the distance between Cαs, a second rule applies to the characterization of their secondary structure; because helices can easily be confused with a succession of turns, the central residues of β-turns, i and i+310-helix and types I and I’, whereas turns V, V’ and VII are rare, and their definitions are inaccuratea and VIb. Hutchinson and Thorntona into the 2 sub-types VIa1 and VIa2. Wilmot and Thornton have precisely defined type VIIIb and was proposed after the removal of type VII3927The β-turns, as defined by C.M. Venkatachalam, are characterized by a hydrogen bond between the N-H and C = O of residues E and βP), γ, δ, α, ε and αL (αL and γL). This rough clustering allows various classes to be defined, with some being associated with amino acid specific behaviours. The turns are also divided into full turns  and half turns (with a polypeptide chain angle of 90°). The first category represents 7 major clusters, and the second one represents 8 major clusters44464849E, βP, αR, ε, αL and γLE → γL (8%) and γL → αR (4%). The 6 other clusters represented only 1% eachThe difficulty with using such an approach is the ‘strict’ rule(s) used to define the β-turn types. Efimov has used a Ramachandran plot simplified to 6 and 8 regions: β . The selected chains had X-ray crystallographic resolutions less than 1.6 Å or 2.5 Å and R-factors less than 0.25 or 1.0. They comprised between 2,542 and 23,943 protein chains. Each chain was automatically examined with geometric criteria to avoid bias from zones with missing density. The main purpose of such diversity was to examine (i) the poorly populated turns and (ii) the stability of the clustering approach (see below).To remove representative bias regarding protein resolution or sequence identity, non-redundant datasets were used. These datasets were generated using the PISCES database1210 and π-helices; the β-strand, containing only the β-sheet; and the coil, comprising everything else . Turn assignment was performed as described previously2728i and i+3 should be less than 7 Å; the central residues of the turns must be non-helical; and in the case of strands, at least one residue must be associated with a coil. The types of turns  were assigned according to the classical definition by using the φ and ψ dihedral angles of the central residues . The PBs were obtained with an unsupervised classifier similar to Kohonen Mapsm and d are prototypes for the central regions of α-helix and β-strands respectively. PBs a through c primarily represent the N-cap of a β-strand, whereas e and f correspond to the C-caps; PBs g through j are specific to coils, PBs k and l correspond to the N cap of an α-helix, and PBs n through p correspond to C-caps. PBs were assigned by using in-house Python software, although similar assignment can be performed through the PBE web serverhttps://github.com/pierrepo/PBxplorelocks PBs63 corresD. Thus, each dataset was associated with T type IV β-turns.A specific clustering approach was designed to cluster type IV β-turns by using the classical rule, allowing +/− 30° for all angles, with the exception of one at +/− 45° for the defined values. The clustering derived from Self-Organizing Maps . k type IV β-turns were taken randomly to initialize the clusters.1. T type IV β-turns was randomly selected from the dataset D (denoted V2) and compared with each of the k clusters.2. One of the V1 (representing the clusters) and V2 of dihedral angles was defined as the Euclidean distance among the M links, the RMSDA , Ψi(V1)}(resp. Ψi(V2), Ψi(V2)) denotes the series of the (2M) dihedral angles for V1 (resp. V2). The angle differences were computed modulo 360°. Thus, in the training, this distance was used for assessing the dissimilarity of any fragment in the database with the different clusters.where {ΦRMSDA value was used to define the winning cluster W, i.e., the closest to the observation. W values were modified according to the learning coefficient α:3. The minimal j(Vw)} and Ψj(Vw) are the values of the winner at time t, with j ranging from 1 to 2, similar to the values of the real data .where {ΦT represents the total amount of data to learn (here the number of type IV β-turns). t represents the number of β-turns already used. The process goes back to step 2. One cycle of training corresponds to the learning of the whole dataset α0, which is then equal to α0/2; after 5 cycles, it is equal to α0/5, etc. Initially, α0 = 0.35, as in68The decrease of α was performed similarly to that for SOM53i.e., 20 times T; these steps were important to diminish the potential effect of the initialization.The process was iterated for 20 cycles, k clusters were used as initial values. α0 was still equal to 0.35.The final values of the T type IV β-turns was randomly selected from the dataset D (denoted V2) and compared with each of the k clusters. Instead of using only RMSDA, the β-turn rule was used: 3 angles can be at +/− 30° and 1 angle at +/− 45°.The winner positively applied this rule; otherwise no training was performed.One of the Modification of the winner weights was performed as in step one −3.The process was iterated for 20 cycles.Step two:k. k was first set at 50 and then reduced. The obtained clusters were compared in the order of largest to smallest k values.An important point is the choice of The amino acid occurrences for each local structure conformation were normalized into a Z-score:i in position j for a given secondary structure, and j with the frequency of amino acid i in the entire databank equals −5 and 5.10−2, respectively). The same computation was also performed for the protein blocks.whereMost of the quantitative analysis was performed using in-house Python scripts, and statistics and visualization were performed with R software (version 3.2.2)ori to differentiate them from the new types in the current analyses. 3671The different amino acid datasets showed the expected amino acid and protein block occurrences, with no peculiarities in the rate of redundancy and the resolution quality see . As note2727As a first step, before searching for new types, the previously discarded types were analysed.10 helices and to type I (and I’) β-turns. The type V β-turn has been considered to be a rather unusual departure from the type II β-turn .From the above section, it is apparent that nearly 1/3clusterability of the data was manageable.A major difficulty in every classification approach is the choice of the clusters. Here, it was slightly different; the idea was not to have an optimal number of clusters but to assess the most frequently occurring and recurrent clusters to define the new pertinent types. In related research, Micheletti and collaborators have decided to take the largest cluster each time and iteratively repeat the clustering, each time removing the largest clusterThe training was performed with different datasets beginning with a large number of clusters (50 at first), which was progressively reduced (to 10). A notable feature of the learning was that four clusters appeared at the beginning and remained the most frequently occurring cluster for each of the different datasets. The deviation in the dihedral angle values between the different simulations (and different datasets) was never higher than 0.3°, thus indicating that the clustering was reasonably stable . These numbers are reasonable because they were highly consistent across all of the datasets. misc.The four new type IV β-turn sub-types were named IVurns see , composi1 and type II β-turns , which comprise 70% and 49% hydrogen bond turns. The evolution was strikingly lower for the type IV1 β-turn, with less than 30% of residues associated with hydrogen bond turns. Although all the new type IV β-turns were linked to neither α-helices nor β-sheets, type IV1 β-turns were often observed at the ends of β-sheets (in nearly 2/3 of the cases).To describe the type IV β-turns more precisely, we examined their former DSSP assignments (hydrogen bond estimation) as turns or bends. Interestingly, more than 2/3 of the residues of IVAs mentioned in the introduction, two major efforts were made in the 1980 s and 1990 s to define β-turns. Both were based on a Ramachandran plot divided into 6 to 8 large regions. The size and shape of these regions were largely different from the strict rule of +/−30° (and 45°). Notably, these previous classifications were performed with all turns, whereas in the current analyses the classification was performed on only a subset of type IV β-turns.1 might seem as if it could be characterized as βEαL because it looks like the proposed βαL-half-turn; however, the type IV1 β-turn is not a half-turn but a complete turn. The type IV3 β-turn is the only local conformation that can be described as a half-turn, but instead of being a αγ-half-turn, it is mainly α/γ- > α. Type IV4 β-turns can be described as γγ; a similar type has been described in2 β-turns were the only ones that seemed to be directly related to Efimov’s analyses, because they could be characterized by a γδ connection between α-helices, as described in44R represents the γ, δ and α regions; very diverse conformations were found in type IV3 and IV4 β-turns as well as type I β-turns . Type IV2 β-turns had the same description as type VIII . Interestingly, only two non-classical turns, βE → γL (8%) and γL → αR (4%)1 β-turn is not βE → γL, but βE → αL.Wilmot and Thornton have also used a simplification of the Ramachandran plot in 6 major regions, with 12 combinations1 β-turn. Additionally, it showed the specificity of the type IV3 and IV4 β-turns in regards to their fine description. The type IV2 β-turn was the only one to have been clearly characterized previously by both studies45Hence, these comparisons illustrate that the specific clustering performed in the current analyses highlighted one new main cluster that was not observed previously: the type IVnon-turn-like structures) and 18 reverse KK β–turns presented in 3 and IV4 β-turns were associated with the KK type I β-turn (18% of the true turns). Type VIII β-turns were associated with the KK type VIII3 β-turn (6.5% of the true turns). Interestingly, type IV2 β-turns were not associated with any KK β-turn types.Koch and Klebe (KK) used a sophisticated approach to unify the assignment of turns of different lengths3 and IV4 β-turn), whereas type VIII β-turns were easily found by using this approach. Similarly to previous results, the type IV2 β-turn remained specific to our clustering. However, differences between the studies should be taken into account, such as the different learning method used by Koch and Klebe, considered more angles than ours and their training was conducted on the complete set of turns and not just the type IV β-turns.Hence, this comparison between studies indicated some similarities because the major turn (type I β-turn) could not distinguish between the two new less frequent turns . Similarly, in type IV2 β-turns, the PB motif was [fjkl] [bklno] [bglp] [cg] and was comparable to the type IVori β-turns but also had some differences compared with the type VIII β-turns. Hence, only half of the over-represented PBs in type VIII β-turn were found in type IV2 β-turns and 5 under-represented PBs were over-represented .For type IV3 and IV4 β-turns were mainly associated with the most frequent β-turn, the type I β-turn, because their dihedral angles were in the same restricted area.PB motifs of type IVi+2.β-turns have been widely analysed in terms of sequence – structure relationships, which have been incorporated in various prediction approaches2774ori and IVmisc β-turns remained strongly linked, because erasing half of the occurrences did not change the general trend of the unassigned turns.Type IV3 and IV4 were clearly distinct in terms of dihedral angle distributions but had very similar amino acid compositions. Indeed, they shared the same over- or underrepresented amino acid trends in 80% of the cases; only one inversion of amino acid preference was observed for the type IV3 β-turns at position i+2 ,IV2 β-turns were structurally close, with high sequence similarity. We found only one inversion between these types at position i+2 for the valine residue.The type VIII and IV1 and II β-turns were close structurally but had strongly divergent sequences. At position i, no common amino acid over- or under-representation was observed. In the Ramachandran plot’s αL region, glycine represented 88% of the residues, whereas in γL, it was only 38% . Interestingly, the type IV1 encompassed mainly the non-glycine residues at i+2 . For each simulation, the clusters were always found at similar frequencies and with similar dihedral values. However, the other clusters were substantially more variable. A simple analysis was also performed to evaluate the possibility of the presence of sub-clusters inside the different clusters by diminishing the authorized dihedral angle deviation allowed during the training. Similarly, the centre of the four main clusters always appeared, thus supporting their stability.A classical question raised by any clustering methodology is the relevance of the results. Here, our results can be considered reliable, owing to their reproducibility and stability. The use of 10 different datasets ranging in quality and sequence identity highlighted the high stability of the four main clusters (1 β-turn) was not highlighted, although it is the 5th most occurring turn (including type IVmisc β-turns). Only the type IV2 β-turns were previously included.Comparisons with the previous alternative classification proposed by Efimov45i.e., IVori) during the simulations is very different when the turn is in fact a type IV2 or IVmisc. The former case (type IV2 β-turn) is a simple extension of this conformation, whereas the latter (type IVmisc β-turn) is really a different independent conformationmisc); thus, this classification provides a better description of local protein conformations and resolves the spectrum of IVmisc turns to a greater extent.This extended classification is relevant because it does not modify the currently accepted β-turn types, is highly stable , and proposes new ways to analyse the architecture and dynamics of the protein or peptide structure of β-turns. Hence, we envision two potential applications of this classification system. The first one addresses molecular dynamics simulations in which researchers follow the dynamic evolution of type VIII β-turnsAn interesting point is that turns are often observed as tandem repeats, sometimes leading to long series of γβ, βγ, ββ or γγ turns83How to cite this article: de Brevern, A.G. Extension of the classical classification of β-turns. Sci. Rep.6, 33191; doi: 10.1038/srep33191 (2016).


To obtain such a heterostructure in high quality via controllable synthetic approaches is still challenging. In this work, in‐plane epitaxy of graphene/h‐BN heterostructure is demonstrated on Cu–Ni substrates. The introduction of nickel to copper substrate not only enhances the capability of decomposing polyaminoborane residues but also promotes graphene growth via isothermal segregation. On the alloy surface partially covered by h‐BN, graphene is found to nucleate at the corners of the as‐formed h‐BN grains, and the high growth rate for graphene minimizes the damage of graphene‐growth process on h‐BN lattice. As a result, high‐quality graphene/h‐BN in‐plane heterostructure with epitaxial relationship can be formed, which is supported by extensive characterizations. Photodetector device applications are demonstrated based on the in‐plane heterostructure. The success will have important impact on future research and applications based on this unique material platform.Graphene/hexagonal boron nitride ( It has been both theoretically predicted and experimentally verified that the band gap can be opened and tuned by embedding h‐BN domains in graphene layer.h‐BN in‐plane heterostructures.Monolayer graphene and hexagonal boron nitride  has been extensively studied to form in‐plane heterostructure.h‐BN starting from CVD graphene edges on copper has been realized with lattice coherency of the two crystals.h‐BN growth. Methods in the reversed order were also reported by growing h‐BN first, followed by graphene growth, either on patterned h‐BN or isolated h‐BN grains. However, imperfections still could be observed in graphene.h‐BN in‐plane heterostructure have also been achieved by etching regrowth or chemical conversion methods after growing a continuous h‐BN film.h‐BN, while the long growth process also creates many defects in h‐BN lattices.Sequential growth of graphene/h‐BN in‐plane heterostructure by sequentially growing h‐BN single‐crystals and graphene grains on Cu–Ni alloy. The alloy's enhanced ability in decomposing polyaminoborane residues improves the cleanliness of the alloy surface during h‐BN growth.h‐BN grains and follows the lattice orientation of h‐BN, indicating the epitaxial relationship between the two crystals. As the growth of graphene on Cu–Ni alloy occurs in isothermal segregation,h‐BN lattices. As a result, extensive characterizations confirm that both graphene and h‐BN are in high qualities. Photodetector devices were demonstrated by integrating monolayer 2D semiconductor materials to the in‐plane graphene/h‐BN heterostructure. The method will facilitate both future 2D electronics and physical studies based on large‐area high‐quality graphene/h‐BN in‐plane heterostructure.Here we report the successful synthesis of large‐area graphene/85Ni15) were used as the catalytic substrates, and ammonia borane (H3BNH3) and methane (CH4) were chosen as the precursors for h‐BN and graphene growth, respectively. The time dependence of experimental parameters is given in Figure S1 (Supporting Information), while the experimental details for h‐BN growth were described in our earlier report.In this work, a two‐step low pressure chemical vapor deposition (LPCVD) method was employed. Cu–Ni alloy foils containing 15 atom% Ni . Dendrite‐shaped graphene nucleus was observed at each corner of h‐BN grain but nowhere else on the substrate. With extension of growth time, hundred micrometer sized graphene grains grew, coalesced and eventually resulted in a continuous film with embedded single‐crystal h‐BN grains, as shown in Figure Experiments were carried out to investigate the influence of nickel introduction on the formation of the high‐quality in‐plane heterostructure. As shown in Figure h‐BN. Another one‐third of graphene grains grow along the edges of h‐BN. And the remaining one‐third of graphene grains appear on the copper surface away from h‐BN grains. Such randomness of graphene nucleation on copper is in qualitative agreement with literature studies.For comparison, the experiments were repeated on copper foils with similar processing condition, and the results are presented in Figure h‐BN in‐plane heterostructures grown on Cu and Cu–Ni substrates. Figureh‐BN in‐plane heterostructure grown on Cu–Ni and copper surfaces and transferred onto Si substrates with a 90 nm SiO2 capping layer, respectively. Figure h‐BN (cyan dot) requires an integral time 20 times longer than that for graphene . The peak observed at 1370 cm−1 corresponding to E2g phonon mode indicates that the h‐BN grain (cyan dot) is well crystallized after graphene growth.−1) to G band (≈1585 cm−1) is 2–3, and the full width at half maximum of the 2D band is about 32 cm−1, confirming that the graphene grains are monolayered and with high quality.−1) intensity of graphene grown on Cu–Ni is more than one order of magnitude lower than that of graphene grown on copper foil . On the other hand, for Cu–Ni alloy substrate, the polymeric residues were completely decomposed.h‐BN corners as the unique sites for graphene's heterogeneous nucleation.We would attribute the much decreased nucleation density as well as the highly improved quality of graphene to the higher catalytic power of Cu–Ni in decomposing polyaminoborane, a polymeric residue precipitated during h‐BN grains, and eventually helps to make h‐BN and graphene both in high qualities.Moreover, Cu–Ni substrates were also reported for a higher capacity of dissolving the introduced carbon atoms,h‐BN grains. Figureh‐BN heterostructure transferred onto TEM grid, respectively. Figure h‐BN and graphene grains are well crystallized. Furthermore, although the three h‐BN grains (red labels) show different orientations with two parallel aligned and one rotated by 30°, graphene grains (blue labels) show exactly the same orientation as the h‐BN grains they stitch to. Such alignment was further confirmed by low energy electron microscopy (LEEM) and corresponding low energy electron diffraction (LEED) measurements . The precise alignment of graphene to its matching h‐BN grains, its preferential nucleation uniquely at the corners of the h‐BN, together with literature studiesh‐BN crystals.Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) measurements were used to study the lattice coherence between the graphene and the h‐BN in‐plane heterostructure. Figureh‐BN and graphene for 40 min and 75 s, respectively. Figure h‐BN (red dot) and graphene (blue dot), respectively, with the corresponding B (KLL), C (KLL), and N (KLL) spectra given in Figure h‐BN grains respectively, together with a weak C (KLL) peak at 275 eV because of surface absorption. In addition, the graphene shows an intense carbon peak with no traces of boron or nitrogen detectable. Figure h‐BN grains together with sharp contrast at the interfaces, implying the existence of high‐quality in‐plane heterostructure. Similar characterizations were taken on graphene/h‐BN heterostructure synthesized on copper substrate . It is found that both boron and nitrogen elements were detectable on exposed copper surface and even on graphene grains. More data about the characterizations on the graphene/h‐BN in‐plane heterostructure, including Raman maps, X‐ray photoemission spectroscopy (XPS), and optical absorption spectrum are presented in Figures S6 and S7 (Supporting Information), respectively.Auger electron spectroscopy (AES) was utilized to investigate the spatial distribution of specific elements in the graphene/h‐BN both with high qualities brings in hope for various electrical and physical applications. For example, with an h‐BN grain serving as the dielectric substrate while the surrounding graphene serving as the bias electrodes, the transferred film provides an easy and effective platform to support phototransistors based on 2D semiconductor materials such as MoS2 . Moreover, other types of interesting optoelectronic structures such as p–n junction devices could also utilize this platform. As shown in Figure2 (p‐type) and MoS2 (n‐type) crystals were stacked on an h‐BN grain area while touching isolated parts of graphene. The structure is confirmed by Raman and photoluminescence (PL) spectra given in Figure I−V curve without illumination, while the corresponding current, especially the reverse current value, increases dramatically by increasing the illumination intensity, within a reasonably fast response time of ≈1 ms, as shown in Figure h‐BN in the in‐plane heterostructure are thus well characterized and utilized, paving a way for further applications in logic and photoelectric circuits .The success in synthesizing graphene and h‐BN in‐plane heterostructure on Cu–Ni alloy by a two‐step LPCVD method. Graphene was found to preferentially nucleate at the corners of triangular h‐BN grains deposited in advance and follow the atomic orientation of h‐BN. Extensive characterizations confirm that the synthesized heterostructure is of high quality in crystallinity. Cu85Ni15, an alloy catalyst with higher catalytic power than copper, results in cleaner substrate surface, lower nucleation density, fast growth rate of graphene, and eventually large‐scale graphene/h‐BN in‐plane heterostructure. Based on the success, photodetector devices built on the unique platform were successfully demonstrated. This study may have significant impact on scalable synthesis of large area 2D heterostructures for future research and applications.In summary, we have successfully synthesized graphene/Growth: Cu–Ni alloy foils containing 15 atom% Ni (Cu85Ni15) were used as substrates, and time dependence of experimental parameters is given in Figure S1 (Supporting Information). Details of the substrate preparation and h‐BN growth were described in our earlier report.h‐BN growth ended, the substrates were first maintained at 1050 °C for 10 min in an H2 flow at a low pressure to make sure that the borazane precursor was completely cooled down. Then an H2/CH4 (100/50 sccm) mixture were introduced for graphene growth. After that, the substrates cooled down to room temperature in a mixed Ar/H2 flow.Characterizations: The graphene/h‐BN in‐plane heterostructures were characterized by SEM , Raman spectroscopy , TEM , and AES .MoS2 (WSe2) Transfer: Graphene/h‐BN in‐plane heterostructure film grown on Cu–Ni was transferred onto SiO2/Si substrate first. CVD‐grown MoS2 crystals covered by a thin Poly (methyl methacrylate) (PMMA) film were lifted off in etching solution, washed in deionized water, and then picked up by a fresh SiO2/Si wafer. A fine tungsten probe was employed to cut a selected piece of PMMA/MoS2 out of the film and gently load it onto specific location shown in Figure 2 crystal was transferred using the same method.Device Fabrication and Measurement: Device fabrication was done by e‐beam lithography, electron beam evaporation (Ti/Au), and lift‐off sequentially to contact graphene. Continuous graphene film was split by a tungsten probe. Electrical measurement was conducted in a home‐built probe station connected with Keithley 2634B source meter. Photocurrent was measured with a 543 nm laser .The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Graphene plasmonics has become a highlighted research area due to the outstanding properties of deep-subwavelength plasmon excitation, long relaxation time, and electro-optical tunability. Although the giant conductivity of a graphene layer enables the low-dimensional confinement of light, the atomic scale of the layer thickness is severely mismatched with optical mode sizes, which impedes the efficient tuning of graphene plasmon modes from the degraded light-graphene overlap. Inspired by gap plasmon modes in noble metals, here we propose low-dimensional hybrid graphene gap plasmon waves for large light-graphene overlap factor. We show that gap plasmon waves exhibit improved in-plane and out-of-plane field concentrations on graphene compared to those of edge or wire-like graphene plasmons. By adjusting the chemical property of the graphene layer, efficient and linear modulation of hybrid graphene gap plasmon modes is also achieved. Our results provide potential opportunities to low-dimensional graphene plasmonic devices with strong tunability. In the context of light-matter interactions, the concentration of electromagnetic fields on materials is a critical issue for the performance of tunable optical devices, such as photodetectors3678Due to its two-dimensional (2D) structure with extremely large conductivity from the massless Dirac point111314161719212223242126272931Here, we focus on low-dimensional waveguide systems for the improved light-graphene overlap factor. We firstly reveal the existence of hybrid graphene gap plasmon (H-GGP) modes the field profile of which is strongly confined inside the graphene gap between metallic and dielectric graphene layers. We demonstrate that the H-GGP mode has larger field concentration on graphene layers than those of edge33w (the sheet conductivity σ(G) where Im{σ(G)} < 0) is inserted in-between semi-infinite metallic domain M (Im{σ(M)} > 0) and dielectric domain D (Im{σ(D)} < 0). The gap region G satisfies the condition of Im{σ(D)} < Im{σ(G)} < 0, as the analogy of plasmonic gap modes in noble metals8212627μ(x). μ-dependency of the graphene conductivity calculated by Kubo formula1121f = ω/2π = 20 THz, charged particle scattering rate8Ω(M))−1 > 0.6 and 0.5 < (Ω(D))−1 < (Ω(G))−1 < 0.6, where Ω−1 = μ/(ℏω) is the normalized chemical potential.We consider the 2D metal-gap-dielectric waveguide system composed of three distinct graphene domains ; a dielew = 5 nm, (Ω(M))−1 = 4, (Ω(G))−1 = 0.54, and (Ω(D))−1 = 0.5002). In the numerical analysis, the graphene is considered as the filmδ = 0.2 nm and the relative bulk permittivity of εg(ω) = 1 + jσg(ω)/(ωε0δ), where σg is sheet conductivity of graphene obtained from Kubo formula33σ~ − σ for all z) have improved confinement compared to that of the GEP mode with much stronger structural asymmetry | ≪ |σ| and |σ| = |σ| for z ≠ 0), H-GGP exhibits more confined transverse (Ex) field on the gap region than that of the 1D-SPP mode, as similar to the difference between gap plasmons and surface plasmons in noble metals8Im{σ(G)}·Ex(G)~Im{σ(D)}·Ex(D), deriving the enhancement of Ex(G) from the condition of Im{σ(D)} < Im{σ(G)} < 0. For the practical realization, we also note that H-GGP modes can be obtained at any finite temperatures when the conductivity of each domain satisfies the condition of Im{σ(D)} < Im{σ(G)} < 0 < Im{σ(M)}, as shown in f = 100 THz.w = 0) corresponds to the σ(M) − σ(D) 1D-SPP system with the effective mode indexneff = Re{q}/k0 ≈ 2·(3/2)1/2·εε0·c/(Im{σ(M)} + Im{σ(D)}), while the H-GGP system with infinite w is converged to the σ(M) − σ(G) 1D-SPP system. The effective mode index (de index  and the de index  of the Hw case is converged to that of the σ(M) − σ(G) 1D-SPP system, smaller w cases exhibit the intensity profiles focused of the graphene gap. Such a distinct in-plane intensity distribution imposes the unique property on out-of-plane confinement, in terms of the light-graphene overlap factor ρ = ∫∫graphene|E|2·dS/∫∫|E|2·dS: the concentration of electromagnetic fields on graphene. ρ as a function of the gap width w, which demonstrates the improved light-graphene overlap for the structures with apparent field concentration on the gap (0 < w < 40 nm). We note that the H-GGP mode acquires much higher field concentration on the graphene layer (ρ = 2.07 × 10−3 at w = 5 nm), when compared to those of 1D-SPP modes (σ(M) − σ(D) system of ρ = 1.81 × 10−3 and σ(M) − σ(G) system of ρ = 1.70 × 10−3) and the GEP mode (ρ = 0.728 × 10−3).Most importantly, there exist differentiated features of the H-GGP mode when compared to 1D-SPP modes, as shown in Ω(M))−1 = 4 + ΔΩ−1, (Ω(G))−1 = 0.54 + ΔΩ−1, and (Ω(D))−1 = 0.5002 + ΔΩ−1, with the spatially global (wmax modulation width) modulation range. As seen, the effective mode index of the H-GGP mode can be controlled with an order of smaller modulation of ΔΩ−1 when compared to the GEP mode. The H-GGP mode also provides more efficient regime of ΔΩ−1 for controlling effective index compared to 1D-SPP modes (ΔΩ−1 ≤ 0.015). Such improved efficiency is more apparent for the case of the finite modulation region for ΔΩ−1 (dotted lines in wmax modulation width around the graphene gap), due to the improved transverse localization of the H-GGP mode  will be required for increasing excitation efficiency, for example, by applying adiabatic procedure or matched layers.We demonstrated the existence of low-dimensional gap plasmon modes on graphene, which supports large light-graphene overlap factor. The system with spatially-varying chemical potential (or doping level) for H-GGP modes can be realized by several existing schemes such as electric field bias332137How to cite this article: Kim, Y. et al. Low-dimensional gap plasmons for enhanced light-graphene interactions. Sci. Rep.7, 43333; doi: 10.1038/srep43333 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Small cell lung cancer (SCLC) is an aggressive malignancy with a distinct natural history and dismal prognosis. SCLC is characterized as a recalcitrant neoplasm with limited therapeutic options and platinum-based chemotherapy is the treatment of choice. Programmed cell death-ligand 1(PD-L1)-mediated immune escape may be a suitable target for specific therapy, but its role in SCLC is unclear.In total, 186 SCLC cases were investigated. Paraffin-embedded tumor sections were stained with a PD-L1 antibody. PD-L1 overexpression was denoted by moderate-to-strong PD-L1 membrane staining in ≥ 5% of tumor cells. Tumor cells and infiltrating lymphocytes were scored separately.The overall frequency of PD-L1 overexpression, in tumor cells and tumor infiltrating lymphocytes (TILs) was 78.0% and 54.3%, respectively. High tumor PD-L1 expression was significantly correlated with high TIL PD-L1 expression (P=0.001) and stage IV disease (P=0.048). Multivariate analysis revealed that high tumor PD-L1 expression and stage IV disease were two independent risk factors for poor overall survival.High PD-L1 expression was observed in SCLCs compared with their expression in conventional NSCLCs. The aggressive behavior of SCLC could be partially related to PD-L1-mediated immune escape. High PD-L1 expression correlated with poor prognosis and may provide a rationale for immunotherapy for high-grade SCLC. Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer that secretes and responds to a wide variety of mitogenic peptide growth factors, SCLC accounts for 10-20% of all lung cancers . The devProgrammed cell death ligand 1 (PD-L1) couples with programmed cell death 1 (PD-1), a coinhibitory receptor on T-cells, to play an important role in the ability of tumor cells to evade the host immune system . In theoThe goal of this study was to conduct a comprehensive investigation of PD-L1 expression in a large series of patients with SCLC and to correlate the expression with clinicopathologic parameters and clinical outcomes.One-hundred-sixty-seven (89.8%) of the patients were men, and 19 (10.2%) patients were women , followed by the right upper lobe (23.7%). The distribution of tumors in the left lower lobe, right middle lobe and right lower lobe was the same: 13.4% each.Given the very limited role of surgery in SCLC, most materials were collected via biopsies, including: bronchoscopic biopsy , sonography-guided biopsy  or CT-guided biopsy . Only a few patients underwent surgery: lobectomy  or wedge resection .In the histological analysis, the tumors were characterized by small cells with scant cytoplasm, poorly defined cell borders, finely dispersed granular nuclear chromatin, and absent or inconspicuous nucleoli. The cells were round, oval, or spindle-shaped Figure . In the Immunostaining for PD-L1 was observed in the membrane of the tumor cells and stromal lymphocytes. Tumor PD-L1 membranous reactivity Figure  was deteHigh PD-L1 expression was associated with advanced age (≥ 60 years) (P<0.001) and PD-L1 expression in TILs (P=0.001) , stage IV diaease (P<0.001) and positive tumor PD-L1 expression (P<0.001) Table . In the SCLC is a heterogeneous and genetically complex disease with a very high mortality rate. The current standard of care includes concurrent chemoradiation with cisplatin and etoposide for stage I-III disease SCLC and a combination of platinum and etoposide or irinotecan for stage IV disease SCLC . DespiteFor the past few decades, the choices for the systemic disease control of SCLC have been limited. Recently, many clinical trials have reported that PD-L1 blockade provides durable tumor control with minimal-related adverse events , 8. The Contradictory to the results reported by Schulthesis et al., 145 of 186 cases (78.0%) of SCLC in this series showed positive membranous PD-L1 staining in tumor cells. Our results of frequent PD-L1 expression in SCLC are supported by two recent studies by Ishii et al . and KomThe reasons for the discrepancies among studies are unclear but may include the following: sample collection, methodology for tissue fixation, different antibodies, cut-off points and scoring methods. The cut-off value for PD-L1 positivity is a concern when interpreting the study results. IHC testing has yet to be standardized and optimal IHC assays have not been validated. The IHC antibody we used was also utilized in several studies on various neoplasms, including lymphoma,  lung canOur study revealed that tumor PD-L1 expression and a stage IV disease were significantly associated with poorer prognosis independent of the other factors examined. This result corresponds with previous studies showing that PD-L1 is associated with poorer prognosis in patients with NSCLC, , 30 esopPD-L1 staining on immune cells in lung cancer has been reported in a few studies , 20, 21 SCLC has long been associated with PNS. Endocrine paraneoplastic disorders are characterized by ectopic production of peptide hormones and the neurological complications are related to antibody-mediated damage to the central nervous system. Hyponatremia is the most common PNS (up to 15%)  but the In conclusion, this large cohort study demonstrates that tumor PD-L1 expression in a membranous pattern is a common feature in SCLC, supporting the hypothesis that PD-L1 expression might lead to enhanced immune evasion by the tumor. High tumor PD-L1 expression was significantly correlated with TIL PD-L1 expression and stage IV disease. In addition, patients with positive PD-L1 expression had a poor clinical outcome. The results also indicate that clinical trials targeting PD-1 and/or PD-L1 may benefit patients with this otherwise difficult to treat disease, and the utility of such targeted therapeutics in clinical practice needs to be determined in randomized trials.st January 2010 and 31st December 2015. The Hospital's Research Ethics Committee approved the study, and all the patients provided written informed consent. SCLCs was diagnosed according to the criteria set by the World Health Organisation [The investigations were performed in a cohort of 186 individuals with SCLC, who were treated at the National Taiwan University Hospital between 1Clinical data were tabulated from each patient's medical records and analyzed in conjunction with microscopic findings. The tumor samples used for IHC analysis included surgically resected specimens, bronchoscopy tissue and sonography or computed tomography (CT)-guided lung biopsy tissue from inoperable patients.For IHC staining of PD-L1 expression in tumor tissue, 4-μm-thick sections from each formalin-fixed, paraffin-embedded tissue blocks were de-waxed with xylene and rehydrated in a graded series of ethanol. For PD-L1  , 28, 29 PD-L1 immunostaining results were classified into two groups according to the intensity and extent of staining: (1) negative, no staining or staining detected in < 5% of the cells; and (2) positive, when membranous staining was present in ≥ 5% of the cells and the staining intensity was moderate to strong. The 5% threshold was based on a previous phase 1 trial of anti-PD-1 agents and studies of other malignances , 11, 12.The correlation was correlated with various clinicopathologic parameters using Fisher's exact test. Overall survival (OS) was assessed using the Kaplan-Meier method, and the log-rank test was used for comparisons. Prognostic factors of OS were analyzed by univariate and multivariate analyses. Hazard ratios and 95% confidence intervals were calculated for all variables in the regression model. All tests were two sided, and P < 0.05 was considered significant. PASW Statistics 18.0  was used for all the analyses.


L1 minimization may not produce the sparsest solution. In this paper, we propose a novel reconstruction method by minimization of the difference of L1 and L2 norms. To solve the nonconvex L1-2 minimization problem, an iterative method based on the difference of convex algorithm (DCA) is presented. In each DCA iteration, the update of solution involves an L1 minimization subproblem, which is solved by the alternating direction method of multipliers with an adaptive penalty. We investigated the performance of the proposed method with both simulated data and in vivo experimental data. The results demonstrate that the DCA for L1-2 minimization outperforms the representative algorithms for L1, L2, L1/2, and L0 when the system matrix is highly coherent.Sparse reconstruction inspired by compressed sensing has attracted considerable attention in fluorescence molecular tomography (FMT). However, the columns of system matrix used for FMT reconstruction tend to be highly coherent, which means  Fluorescence molecular tomography (FMT) has become a promising molecular imaging modality since it has the ability to provide localization and quantitative analysis of the fluorescent probe for preclinical research , 2. Howe a priori information, such as anatomical information, optical properties, permissible region, and sparsity of target distribution, has been successfully incorporated in FMT reconstruction [L2 norm regularizer is a common penalty term in spite of its over-smoothness and results with lower resolution [L0 norm, which is nondeterministic polynomial (NP) hard and can be solved by a greedy approach such as the orthogonal matching pursuit (OMP) [L1 norm regularizer as the convex relaxation of L0 has become a widely used sparsity-inducing norm for FMT reconstruction [L1 norm regularizer is not always providing the sparsest solution for the inverse problem of FMT [Lp (0 < p < 1) norm regularizer, which has been applied to optical tomography and was found to have better results than L1 does [Lp (0 < p < 1) norm regularizer with p near 1/2 performs the best result among regularizers of Lp (0 < p < 1) norm [To alleviate the ill-posedness of FMT, sometruction –7. In adsolution . Anotherit (OMP) . Inspiretruction –13. Howem of FMT . This giL1 does . Some co 1) norm .L1-2 has been proposed and produced better solution than Lp (p = 1/2) norm regularizer when the sensing matrix was large and highly coherent [L1-2 norm regularizer outperforms L1/2 and L1 for highly coherent matrix [Recently, a new nonconvex regularizer named coherent , 18. Thet matrix . Meanwhit matrix .L1-2 norm regularization was proposed to improve the FMT imaging. In our method, a difference of convex algorithm (DCA) was presented to solve the nonconvex L1-2 minimization problem. And the alternating direction method of multipliers (ADMM) with an adaptive penalty was used to solve the subproblem with fast convergence for each DCA iteration. The performance of the proposed method was validated with simulated data and in vivo experimental data.In this paper, new L1-2 norm regularization algorithm.  in vivo experimental data. Finally, we conclude the paper and discuss relevant issues in The outline of this paper is as follows. r ∈ Ω is the domain under consideration. Dex = 1/3 and Dem = 1/3 are diffusion coefficients with μa,ex, μa,em as absorption coefficients for excitation and emission wavelengths, g is the anisotropy parameter, and μs,ex′, μs,em′ are the reduced scattering coefficients. Φex and Φem denote the photon density. ημaf is the unknown fluorescent yield to be reconstructed. Using the finite elements method (FEM), , the Diffusion Approximation associated with Robin boundary conditions has been widely used for modeling the light transportation in biological tissues , 21. Forap and aq are different columns of A. The mutual coherence of system matrix A derived by FEM method is always as high as above 90% [Lp (p = 1/2) and L1-2 norm regularizers are expected to yield the sparest solution that L1 regularization always fails to [The CS theory provides sufficient conditions for the exact recovery of the sparse signals from limited number of measurements. One commonly used concept is the mutual coherence , 22 whicbove 90% . In the fails to , 18.L1-2 algorithm was proposed and theoretical properties of L1-2 minimization have been proved in papers [L1-2 minimization, we converted linear matrix equation  = G(x) − H(x), whereTo resolve minimization problem , the difx‖2 is differentiable with gradient x/‖x‖2. An iterative scheme was used to solve F(x) as follows:In , ‖x‖2 isL1-regularized convex subproblem that needs to be solved:In each DCA iteration, there is a We use the augmented Lagrangian method and transform  into theLδ with respect to x, minimizing Lδ with respect to y, and updating u successively. In order to solve  [L1) [L2) [L1-2 algorithm, respectively, as the corresponding method for the above regularizers.In this section, the simulations both on 3D digital mouse model andS-L1/2) , incompl2) [L1) , Tikhono1) [L2) , and OMP1) [L2)  were comThe qualities of reconstruction results are quantitatively evaluated in terms of the absolute location error (LE) , reconst−1. For excitation, we used 18 excitation sources being located on the plane of Z = 16.4 mm as shown in A 33 mm height torso extracted from a 3D mouse atlas was utilized to simulate the heterogeneity of biological tissues .  FigureThe forward FEM mesh was discretized into 24231 nodes and 128300 tetrahedral elements. Meanwhile, the FEM mesh for inverse reconstruction was discretized into 2601 nodes and 12752 tetrahedral elements. The mutual coherence of the system matrix for inverse reconstruction was 99.96%.Figures L1-2 and L1/2 were remarkable. Compared to the other three methods, results of DCA-L1-2 and IRLS-L1/2 have fewer artifacts, lower LE, lower NRMSE, and lower PNZ. Meanwhile, the Recon. FY by DCA-L1-2 and IRLS-L1/2 were closer to 0.05 mm−1. The proposed DCA-L1-2 completely outperforms the other methods, except for a slightly larger time consumption compared to OMP.As shown in Figures L1-2 are generally satisfactory even in the case of 4 excitation sources.Generally speaking, the quality of FMT reconstruction is influenced by the number of excitation sources. To test the stability of the algorithm, different numbers of excitation sources were used for reconstruction. L1-2 algorithm is quite resilient with Gaussian noise.The quality of reconstructed results is sensitive to measurement noise because of the severe ill-condition of system matrix. For stability test, four different levels of Gaussian noise  were added to the synthetic measurements. L1-2 and IRLS-L1/2 was remarkably compared to the other three methods in the simulation experiments. In this section, we further evaluated the performance of the proposed algorithm with in vivo experimental data [The performance of DCA-tal data .−1 μM−1 and quantum efficiency of 0.23 at the peak excitation wavelength of 671 nm) [−1 [In this experiment, an adult BALB/C mouse with a glass tube implanted into its abdomen was used. The experimental data was acquired by a hybrid FMT/Micro-CT system . The gla 671 nm) . The cen nm) [−1 . For rec nm) [−1 .L1-2 and IRLS-L1/2 are shown in  in vivo experiments via DCA-L1-2 are shown in For inverse reconstruction, the segmented mouse torso data was discretized into a mesh with 3049 nodes and 14932 tetrahedral elements. Mutual coherence of the system matrix for inverse reconstruction was 99.87%. Comparison results between DCA-L1-2 norm regularization was proposed to solve the inverse problem of FMT with highly coherent system matrix. To accurately recover the small fluorescent target, an iterative method based on DCA algorithm was presented to solve the nonconvex L1-2 minimization problem. And the ADMM method with an adaptive penalty was used to get fast convergence for the subproblem.In this paper, novel  in vivo experimental data acquired by a hybrid FMT/Micro-CT system were used to demonstrate the feasibility of the DCA-L1-2 algorithm for FMT. The comparative results of single target show that the DCA-L1-2 algorithm has better performance compared to other typical algorithms based on L1/2, L1, L2, and L0 norm regularizer. The robustness tests further illustrate that the DCA-L1-2 algorithm is stable and robust to measurement noise. In addition, decreasing the number of excitation sources from 18 to 4, DCA-L1-2 still yields satisfactory results.Simulated data on a 3D heterogeneous mouse model andL1-2 norm regularizer in other imaging modalities in the near future.However, the reconstructed fluorescent yield of the proposed method was still smaller than the true value. So new strategies that may further improve fluorescent yield will be our future research focuses. Moreover, we will also focus on investigating the multitargets resolution and new application of  in vivo experiments validated the good performance of L1-2 regularizer for FMT. Moreover, comparative experiments indicate that L1-2 outperforms the iterative reweighted strategies for lp with p = 1/2 when system matrix is highly coherent.In conclusion, both numerical experiments and


Expression of PD-L1, the ligand for T-cell inhibitory receptor PD-1, is one key immunosuppressive mechanism by which cancer avoids eradication by the immune system. Therapeutic use of blocking antibodies to PD-L1 or its receptor PD-1 has produced unparalleled, durable clinical responses, with highest likelihood of response seen in patients whose tumour or immune cells express PD-L1 before therapy. The significance of PD-L1 expression in each cell type has emerged as a central and controversial unknown in the clinical development of immunotherapeutics. Using genetic deletion in preclinical mouse models, here we show that PD-L1 from disparate cellular sources, including tumour cells, myeloid or other immune cells can similarly modulate the degree of cytotoxic T-cell function and activity in the tumour microenvironment. PD-L1 expression in both the host and tumour compartment contribute to immune suppression in a non-redundant fashion, suggesting that both sources could be predictive of sensitivity to therapeutic agents targeting the PD-L1/PD-1 axis. PD-L1, the ligand for T-cell inhibitory receptor PD-1, can be expressed by various cell types in the tumour microenvironment. Here, the authors show that, in mouse models, the expression of PD-L1 from both cancerous and normal host immune cells is important for suppressing anti-tumour immune responses. Cancer cells elicit multiple mechanisms of immunosuppression to avoid obliteration by the immune system. Expression of PD-L1, a ligand for the T cell inhibitory receptor PD-1, plays a key role in attenuating anti-tumour responses in both mice and human cancer patients346789678128Here we directly evaluate the relative roles of PD-L1 expression by the tumour and by the host's immune cells in the suppression of anti-tumour immune responses. Using genetic chimeras, we find that both tumour and host play non-redundant roles in regulating the PD-1 pathway, suggesting a key role for infiltrating immune cells in both generating and negatively regulating anti-tumour immunity.in vivo (PD-L1 immunohistochemistry (IHC) analysis of human lung and breast tumours has identified three distinct patterns of positive PD-L1 expression: malignancies with predominant epithelial tumour cell PD-L1 expression, those with infiltrating immune cell expression only, or tumours with PD-L1 on tumour and immune cells . Althoug14in vivo , with inin vivo . Concordin vivo , validatWe next characterized tumour infiltrating immune cells in PD-L1-deficient hosts  and the in vitro . Tumours grown in PD-L1-deficient hosts were characterized by cytokines directly supporting lymphocyte infiltration such as CXCL9/10. By contrast, PD-L1-deficient tumours exhibited a different array of T-cell chemoattractants , and an increase in general inflammatory cytokines, especially those associated with neutrophil/granulocytic MDSC infiltration, for example, CXCL1/3/5. Concordant with these alterations, the neutrophil/granulocytic MDSC population made up a larger proportion of the total immune infiltrate in PD-L1-deficient tumours  into PD-L1-deficient tumour cell clones. Ectopic expression of PD-L1 in deficient clones was sufficient to prevent tumour rejections and re-established ent line . To furt tumours . These din vitro setting. We exposed antigen-specific cytolytic T cells to cognate antigen-positive target cells expressing varying levels of PD-L1 in vitro. While lack of PD-L1 on target cells led to a high degree of target cell lysis, PD-L1 was able to partially suppress T-cell-mediated cell lysis in a dose dependent manner (We further investigated the dose dependency and potency of this direct cell protective mechanism in an t manner . These rt-test P<0.05). Similarly, mice re-challenged after initial complete regression showed rapid tumour rejection when re-challenged with the same PD-L1-deficient tumour cells . Most profound PD-L1 increases in the myeloid compartment were detected in DCs and monocytic MDSCs (t-test P<0.05). PD-L1 expression on antigen presenting myeloid cells has been implicated in delivering an inhibitory signal during the early priming phase and hence may inhibit T-cell activation16As a substantial subset of tumour cells remained MHC I positive, we next investigated if additional immune suppressive mechanisms could contribute to tumour escape in PD-L1-deficient tumours. We observed increases in tumour cells expressing the immune regulatory B7 family member PD-L2, the second inhibitory ligand that mediates T-cell suppression through PD-1 . Increasic MDSCs . GranuloAltogether, these data suggest that total levels of PD-L1 expressed on host immune cells serve as a sensitive read out of heightened immune activation within the tumour microenvironment, and that the overall level of PD-L1 from various cellular compartments act to influence the degree of direct anti-tumour immune activity together.To test whether increased immune cell PD-L1 expression can be a mechanism of tumour escape in PD-L1-deficient tumours, we compared tumour growth in MC38 models with PD-L1 deficiency on the tumour, the host, and both compartments. In line with previous experiments, PD-L1 loss in the tumour or host compartment led to tumour regressions , with a These results support a model in which increased immune cell PD-L1 expression, as a result of heightened immune activation in PD-L1-deficient tumours, is sufficient to allow tumour escape by dampening the cytotoxic activity of T-cells. More importantly, it suggests that PD-L1 from disparate cellular sources, including tumour cells, myeloid or other immune cells can similarly modulate the degree of cytotoxic T-cell function and activity in the tumour microenvironment. Furthermore, PD-L1 expression in both the host and tumour compartment contribute to immune suppression in a non-redundant fashion, and that both could be predictive of sensitivity to therapeutic agents targeting the PD-L1/PD-1 axis.We have shown that expression of PD-L1 by both tumour and host cells play critical roles in mediating immune suppression of anti-tumour T cell responses. Although elimination of the PD-L1 gene from tumour cells significantly impaired their growth in immune competent hosts, some tumours did escape. In these instances, high PD-L1 expression by infiltrating myeloid cells appeared to provide a compensatory source of the inhibitory ligand. Furthermore, PD-L1-positive tumours implanted in PD-L1-deficient mice also exhibited markedly repressed growth. These findings indicate that infiltrating immune cells play a critical role in negatively regulating T cell responses even at steady state, independent of PD-L1 expression by tumour cells. Although mouse models do not fully recapitulate the complexity of human cancer, they offer a reductionist approach to definitively assess the contribution of tumour- and host-derived PD-L1 in the prevention of anti-tumour immunity. Despite the limitation of mouse models in predicting human disease dynamics, our results offer a potential explanation for the clinical observation that expression of PD-L1 by infiltrating cells is highly correlated with response to anti-PD-L1/PD-1 therapy in human cancer patients. Infiltrating myeloid cells may respond more rapidly and effectively to IFNγ released by T effector cells, thus suppressing initial T cell responses at the level of antigen presentation, including the activation of memory T cells or priming of naive T cells, before the surrounding tumour cells have the chance to react and upregulate PD-L1 themselves. Our results clearly demonstrate that in preclinical models, regulation of T cell function in tumours by PD-L1/PD-1 involves more than just a simple adaptive response by the tumour cells themselves, but rather involves an intimate and complex interchange between the tumour and its immune microenvironment. These results have potential broad implications for the clinical setting, where measuring PD-L1 expression in both compartments may more faithfully predict therapeutic benefit from antibodies interfering with PD-L1 activity.S. pyogenes Cas9 was cloned into a pRK vector and expressed via the human cytomegalovirus (CMV) immediate-early promoter. Individual gRNAs  targeting mouse PD-L1 were cloned downstream of and expressed from the human U6 promoter of the pLKO.5 vector . Lentiviral plasmids were constructed into a pINDUCER11 vector containing constitutive EGFP and a tetracycline response element driving TurboRFPHuman codon optimized −1 of mouse interferon-γ (R & D Systems) to induce PD-L1 expression before they were single cell sorted on a BD FACS Aria (BD Biosciences). We confirmed PD-L1-deficient clones by flow cytometry and RT-PCR with TaqMan Assays Mm00452054_m1 (PD-L1 01) and Mm03048247_m1 .Parental  and PD-L1-deficient MC38 and CT26 (ATCC) colon carcinoma cell lines were maintained in RPMI with 10% fetal bovine serum and 2 mM GlutaMAX (Life Technologies) and routinely tested for mycoplasma contamination . Plasmids containing gRNAs and CAS9 were co-transfected into MC38 or CT26 parental cell lines with Lipofectamine LTX with PLUS Reagent (Life Technologies) in Opti-MEM I (Life Technologies). After transient transfection, cells were expanded and stimulated for 48 h with 20 ng ml−1). Infected cells were induced with doxycycline (1 μg ml−1) for 48 h and then bulk sorted for uniformly induced EGFP+, RFP+ or EGFP+, PD-L1+ populations.Lentivirus was produced following established protocolIn vivo tumour studies were performed as follows−/− (Taconic) mice were inoculated subcutaneously in the right unilateral flank with 1 × 105 tumour cells suspended in Hanks's Buffered Saline Solution (HBSS) and phenol red-free Matrigel (BD Bioscience). For studies using doxycycline inducible PD-L1 cell lines, mice and cells were dosed with doxycycline (1 ug/ml) for 48 h before tumour inoculation, and then kept on doxycycline throughout the remainder of study. For anti-PD-L1 treatment studies, mice with tumours starting at ∼150 mm3 were treated via intra-peritoneal injection with anti-PD-L1 (clone 6E11), 10 mg kg−1, three times per week for one week. For CD8 T-cell depletion studies, mice were treated via intra-peritoneal injection with anti-CD8 (clone 2.43), 25 mg kg−1, 1 day before tumour inoculation and then at day 3 and 7 post inoculation. Tumour volumes were measured and calculated twice per week using the modified ellipsoid formula ½ × (length × width2). Tumours <32 mm3 were considered completely regressed, whereas tumours >2,000 mm3 were considered progressed and animals were killed. Similarly, animals whose tumours ulcerated before progression or complete response were killed and removed from study. All animals studies performed were in compliance with protocols approved by the Genentech Institutional Animal Care and Use Committee.5 wild-type, PD-L1-deficient, or -inducible PD-L1 tumour cells. In certain studies, mice were also inoculated with 1 × 105 EMT6 breast carcinoma cells in the fourth mammary fat pad on the opposing right side. Tumour volumes were measured and calculated twice per week as described above. Animals whose single tumour volume exceeded 2,000 mm3 or combined tumour volume exceeded 3,000 mm3 were killed. All animals studies performed were in compliance with protocols approved by the Genentech Institutional Animal Care and Use Committee.After a 2-month tumour-free period, mice with complete regression of PD-L1-deficient tumours, as well as age matched naive control mice, were re-inoculated subcutaneously in the left unilateral flank with either 1 × 10CD274/PD-L1 gene and replace it with a neomycin resistance gene cassette (neo), thereby eliminating the start codon and the IgV domain required for PD-1 binding. Homologous recombination events were screened by PCR and verified by Southern blot analysis. Two targeted embryonic stem (ES) cell lines were injected into C57BL/6 blastocysts. Founder mice showing germ-line transmission were interbred to produce homozygous PD-L1-deficient mice. Mice were screened by PCR to confirm genotype: wild-type (WT), knockout (KO) or heterozygous (HET). Inactivation of the PD-L1 locus was verified by Southern blot analysis and loss of protein expression was confirmed by flow cytometry.We constructed a gene-targeting vector (TNLOX1-3) that by homologous recombination deletes the first two coding exons of the mouse 4Cl, 10 mM KHCO3, 0.1 mM Na2 EDTA, pH 7.2). Between days 14–24 post-inoculation when tumours reached ∼150–300 mm3, tumours were isolated, homogenized and digested in 1 mg ml−1 Collagenase D (Roche) and 0.2 mg ml−1 Dnase I (Roche) in RPMI media with 5% fetal bovine serum to generate single cell suspensions for flow cytometry analysis. Cells were stained using standard protocols in Brilliant Stain Buffer (BD Biosciences) for surface markers CD45 , CD3 , CD4 , CD8 , CD25 , PD-1 , CD44 , CD62L , H-2Kb/H-2Db , H-2Kd/H-2Dd , PD-L1  and LIVE/DEAD Fixable Near-IR Dead Cell Stain (Life Technologies). Cells stained for FoxP3  and/or Ki-67  were fixed/permeabilized with the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) before staining for intracellular proteins. Cells were analysed on the BD LSR Fortessa and sorted on the BD FACS Aria. In analysis, dendritic cells (DC)=CD45+, CD11b+, CD11c+; myeloid derived suppressor cells (MDSC)=CD45+, CD11b+, CD11c−; granulocytic MDSC=CD45+, CD11b+, CD11c−, Ly6C+, Ly6G+; monocytic MDSC=CD45+, CD11b+, CD11c−, Ly6C+, Ly6G−.Tissue collection of spleens and draining lymph nodes were in RPMI with 5% fetal bovine serum on ice, and processed through 40 μM cell strainers to make single cell suspensions. Red blood cells were lysed in ACK lysis buffer , Ki-67 (ThermoFisher), cleaved caspase 3 or phosphor-STAT3 , and counter-stained with hematoxylin on a Ventana Discovery XT instrument. Brightfield whole slide scans were obtained on a Nanozoomer slide scanner (Hamamatsu). Quantification of total number of positive objects per mm2 were completed over one section per animal per group, with n=5–7 per group.Formalin-fixed, paraffin-embedded human tumour samples were obtained through commercial vendor  with each patient individually consented in accordance with the Food and Drug Administration good clinical practice guidelines. Samples were processed into 4um tissue sections. Mouse tumours were collected at ∼250 mm2. Images were acquired every 4 h for one week, with two images per well. Data was analysed using IncuCyte analysis software to detect and quantify live cell confluence (phase-contrast), and dead or dying cells (fluorescence) from the same well. Averages with standard error of the mean at each time point were plotted in Prism (Graphpad).Parental and PD-L1-deficient MC38 and CT26 cell lines were plated in triplicate at 1,500 cells per well in a 384 well plate with CellToxTM Green (Promega) at a 1:5,000 dilution. Cells were imaged at × 10 magnification in an IncuCyte Zoom Live-content imaging system (Essen Bioscience) at 37 °C with 5% CO−1 with 20 units per ml of mouse IL-2 (Roche) in RPMI with 10% fetal bovine serum, 20 uM HEPES, 55 μM 2-mercaptoethanol, and 1 × concentrations of the following supplements from Life Technologies: GlutaMAX, sodium pyruvate, penicillin/streptomycin, and non-essential amino acids. On day 5, primed CD8+ T-cells were purified by negative selection kit (Miltenyi Biotec). MC38 target cells  were pre-treated with or without doxycycline at 1 μg ml−1 for 2 days pre-treatment. Surface PD-L1 levels on MC-38 target cells were confirmed by FACS prior to labelling with CFSE at 1 μM (Life Technologies). Labelled MC-38 target cells were co-cultured with purified CD8+ T cells. Co-cultures were set-up in triplicate wells at an E:T ratio of 10:1 in 96 well flat-bottom plates in the presence or absence of SIINFEKL at 0.001 or 0.0001, ng ml−1 overnight before flow cytometric analysis of CFSE plus propidium iodide (PI) (BD Biosciences). The percent lysis was calculated as follows: % lysis=[%CFSE+PI+]/[total CFSE+] × 100.Single cell suspensions of splenocytes collected from C57BL/6-Tg(TcraTcrb)1,100 Mjb/J (OT-1) mice (JAX) were cultured for 5 days with ovalbumin SIINFEKL peptide at 1 ng ml3  as described above. RNA integrity and concentration of RNA samples were determined respectively by Agilent 2100 Bioanalyzer (Agilent Genomics), Fragment Analyser  and NanoDrop 8000 (Thermo Scientific) before their processing by RNA-seq. 1 μg of total RNA was used as input material for library preparation using TruSeq RNA Sample Preparation Kit v2 (Illumina). Size of the libraries was confirmed using Fragment Analyser  and their concentration was determined by qPCR-based method using Library Quantification Kit (KAPA). The libraries were multiplexed and then sequenced on Illumina HiSeq2500 (Illumina) to generate ∼25 million uniquely mapping reads per sample. Reads were mapped to mm9 using GSNAPTotal RNA was extracted from MC38 derived P values .Differential expression analysis between knockout and wild-type lines was conducted using the voom/limma pipeline26Gene set enrichment analysis (GSEA) between PD-L1 knockout and WT tumours was performed using CAMERA28−1 mPD-L1-Fc in PBS overnight, washed with 0.05% Tween-20 in PBS and blocked with 0.5% BSA. Wild-type and PD-L1-deficient MC38 or CT26 clones were treated with or without IFNγ  for 48 h. Supernatants were collected, serially diluted and incubated to test for binding to biotinylated mouse PD-1-Fc (250 ng ml−1). Plates were washed and visualized by Streptavidin-HRP  and TMB (Kirkegaard and Perry Laboratories) at 450 nm absorbance. Anti-mouse PD-L1 (clone 6E11) and recombinant mouse PD-L1-Fc protein were used (starting at 15 μg ml−1) as a positive control for binding to mouse PD-1.Mouse PD-1-Fc was biotinylated with EZ-Link sulfo-NHS-LC-biotin (Pierce) for 30 min at room temperature as described by the manufacturer. Excess non-reacted biotin was removed with Quick Spin High Capacity Columns, G50-Sephadex (Roche) as described by the manufacturer. Nunc Maxisorp 384 well plate was coated with 250 ng mlThe RNASeq data have been de[posited at the NCBI Sequence Read Archive (SRA) under BioProject ID PRJNA356678. All other data supporting the findings of this study are available within the article and its How to cite this article: Lau, J. et al. Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice. Nat. Commun.8, 14572 doi: 10.1038/ncomms14572 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary TableGene set enrichment analysis of differential expression between wt_host-wt_tumour and wt_host-ko_tumour.Gene set enrichment analysis of differential expression between wt_host-wt_tumour and ko_host-wt_tumour.


MTT showed that 12.5–50 μg/mL MIRB could promote cell proliferation and MIRB over 100 μg/mL exhibited toxic effect on hDPSCs. In vitro MRI showed that 1 × 106 cells labeled with various concentrations of MIRB (12.5–100 μg/mL) could be visualized. In vivo MRI showed that transplanted cells could be clearly visualized up to 60 days after transplantation. These results suggest that 12.5–50 μg/mL MIRB is a safe range for labeling hDPSCs. MIRB labeled hDPSCs cell can be visualized by MRI in vitro and in vivo. These data demonstrate that MIRB is a promising candidate for hDPSCs tracking in hDPSCs based dental pulp regeneration therapy.Tracking transplanted stem cells is necessary to clarify cellular properties and improve transplantation success. In this study, we investigate the effects of fluorescent superparamagnetic iron oxide particles (SPIO)  on biological properties of human dental pulp stem cells (hDPSCs) and monitor hDPSCs in vitro and in vivo using magnetic resonance imaging (MRI). Morphological analysis showed that intracellular MIRB particles were distributed in the cytoplasm surrounding the nuclei of hDPSCs. 12.5–100  Mice were anesthetized with 30 mg/mL pentobarbital sodium and then imaged with a customized mice coil  on a 3.0-Tesla MR scanner . Spin Echo T2-weighted (SE T2WI) images were taken . Initially, the color of original MRI images of all mice was reversed and the following parameters were set for the area of interest: R, 0–255; G, 0–255; B, 0–255. Then the average integrated optical density values (IOD) for the area of interest, which represented the intensity of MIRB, were measured.μm. Then Prussian blue staining was performed to evaluate the change of iron content as a function of time.After MRI analysis, the anesthetized nude mice were sacrificed 0 d, 1 month, and 2 months, separately. The implanted root fragments were harvested and fixed by 4% paraformaldehyde (PFA) at 4°C overnight. All specimens were decalcified in 10% EDTA containing 2.5% PFA for 6–8 weeks, dehydrated, embedded in paraffin, and serially sectioned at 5 P < 0.05 was considered as statistically significant.All data are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed to examine the effect of differing concentrations of MIRB on cell proliferation, cell cycle, apoptosis, and osteogenic differentiation. All statistical analyses were performed using the SPSS software package . A value of In this study, human DPSCs were successfully isolated from the pulp tissue of 6 extracted third molars. The primary cells presented clone-like growth after they were incubated for 72 h . The floμg/mL MIRB  images of hDPSCs labeled with MIRB are shown in μg/mL–50 μg/mL groups increased in accordance with the increasing concentrations of MIRB, which were nearly 2-fold higher than the previous one. However, the intracellular iron content in 100 μg/mL group was only 49.76 pg/cell, which did not reach 2-fold higher than that of 50 μg/mL group (The intracellular iron content in 12.5 mL group .μg/mL to 50 μg/mL significantly enhanced the proliferation of hDPSCs during 1 d to 7 d (P < 0.05), while 100 μg/mL MIRB did not affect the cellular viability of hDPSCs from 5 d to 7 d (P > 0.05). Therefore, MIRB under 100 μg/mL was safe for labeling hDPSCs and 12.5 μg/mL coupled with a 24 h incubation time was a preferable choice for labeling hDPSCs  for 24 h, the S-phase percentages were much higher than that of unlabeled hDPSCs (P < 0.05)  , indicatiability . TherefoAfter induction of 7 days and 14 days, the ALP activity of hDPSCs in response to different concentrations of MIRB is indicated in Figures The expression levels of odonto-/osteogenic genes including ALP, BSP, DSPP, and OCN were determined by RT-PCR . At day 6 cells labeled with various concentrations of MIRB (12.5 μg/mL–100 μg/mL) could be visualized and the signal intensity increased with increasing concentrations of MIRB , so the amount of primarily cultured hDPSCs is limited. But, with several properties, such as noninvasive way to access, high proliferative potential, the capacity of self-renewal, and multilineage differentiation, hDPSCs represent a novel adult stem cell population , not onlμg/mL–100 μg/mL) in the absence of transfection reagents could effectively label hDPSCs with sufficient intensity to be detected by the fluorescence of the MIRB rhodamine conjugation , have been well characterized and widely used for cell labeling and tracking by MRI . Traditi Figures . The iro Figures . A recen/mL MIRB . Here, wntration .μg/mL did not affect the cellular viabilities of SD rat BMSCs [μg/mL MIRB, the cell proliferation decreased at passage 5 and passage 6, but there was no difference with respect to cell surface antigens and differentiation ability between MIRB-treated and untreated BMSCs [μg/mL did not affect cell vitality and proliferation, but MIRB over 30 μg/mL interfere with cell vitality [μg/mL PLL with 15 μg/mL SPIO  could significantly increase relative metabolic activity of hDPSCs [2O2 through intrinsic peroxidase-like activity. Meanwhile, SPIO could accelerate cell cycle progression, which may be mediated by the free iron (Fe) released from lysosomal degradation [3O4-MNPs could result in significant increases in ROS production and eryptosis in the end [μg/mL–50 μg/mL could promote cell proliferation, accelerate cell cycle, but have no effect on cell apoptosis. However, 100 μg/mL MIRB could accelerate cell cycle and induce apoptosis as well. Finally the proliferation in 100 μg/mL group showed no difference with control group. Therefore, we presume that intracellular intact SPIO nanoparticles at low concentration can accelerate cell cycle and promote proliferation. When SPIO concentration is too high, the cell uptake has been saturated and redundant free iron may induce Fenton reaction. In addition, exposed iron oxide core may generate ROS [μg/mL MIRB might counteract each other and finally resulted in no effect on cell proliferation. Additionally, there was no difference regarding stem cell and hematopoietic lineage epitope patterns in unlabeled and labeled hDPSCs, indicating that stem cell characteristics were retained after MIRB labeling.Multiple types of cell have been successfully labeled with MIRB and cell vitality, proliferation, and differentiation ability were investigated , 15, 30.at BMSCs . On the ed BMSCs . Similarf hDPSCs . Huang eradation . On the radation . A recen the end . In addi the end . In our rate ROS , which mrate ROS  and finarate ROS , 37. So,μg/mL–50 μg/mL) had no influence on osteogenic differentiation capacity.Currently, there still existed controversy about whether SPIO would affect the osteogenesis of mesenchymal stem cells. In cynomolgus monkey, untreated and MIRB-treated mesenchymal stem cells show no difference in osteogenic differentiation , 15. Simμg/mL SPIO, which was the optimal concentration of SPIO for labeling hDPSCs. At that moment, intracellular iron content reached 13.4 pg iron/cell, which was the minimum request for MRI detection [μg/mL, which was lower than the minimum request for MRI detection. In vitro MRI result showed that 1 × 105 cells labeled with 12.5 μg/mL could not be clearly detected in 3.0-Tesla MR scanner. However, the labeling efficiency in 12.5 μg/mL group still reached 100%. Therefore, we presume that the optimal labeling concentration is not consistent. It has no relationship with cell type but has close relationship with cell condition and cell number. Additionally, the quality of MR imaging depends on the quality of MRI scanner as well.Struys et al. pointed out that hDPSCs could be detected in 9.4 T MR scanner after being labeled with 15 etection . Here, wMany studies have verified cell migration and homing by tracking transplanted cells with MRI –40. Someμg/mL–50 μg/mL MIRB could efficiently label hDPSCs without significant effects on cell viability, phenotype and differentiation. 100 μg/mL MIRB showed cytotoxicity and promote cell apoptosis. MIRB-labeled hDPSCs were detected in vitro and we found the intracellular iron content of 12.5 μg/mL group was not sufficient to be detected by MRI scanner. Furthermore, we found that MIRB-labeling did not interfere with the generation of cell sheet and cell sheet/root fragment complex could be clearly detected in vivo by MRI. Our study suggests that MIRB is a useful tracer for dental pulp regeneration therapy and further in vivo histological study should be investigated.This study used multiple methods to investigate the biological changes of MIRB-labeled hDPSCs from many aspects. The cytoskeletal structures were not changed obviously after labeling. 12.5