RNA-seq was performed using the NextSeq 500 system, and genes with |fold change| ≥ 2 and p < 0.05 relative to the controls were defined as differentially expressed genes (DEGs) in IgG4-ROD. To validate the results of RNA-seq, real-time polymerase chain reaction (PCR) was performed in 30 IgG4-ROD and 30 orbital MALT lymphoma tissue samples. RNA-seq identified 35 up-regulated genes, including matrix metallopeptidase 12 (MMP12) and secreted phosphoprotein 1 (SPP1), in IgG4-ROD tissues when compared to all the controls. Many pathways related to the immune system were included when compared to all the controls. Expressions of MMP12 and SPP1 in IgG4-ROD tissues were confirmed by real-time PCR and immunohistochemistry. In conclusion, we identified novel DEGs, including those associated with extracellular matrix degradation, fibrosis, and inflammation, in IgG4-ROD biopsy specimens. These data provide new insights into molecular pathogenetic mechanisms and may contribute to the development of new biomarkers for diagnosis and molecular targeted drugs.High-throughput RNA sequencing (RNA-seq) uses massive parallel sequencing technology, allowing the unbiased analysis of genome-wide transcription levels and tumor mutation status. Immunoglobulin G4-related ophthalmic disease (IgG4-ROD) is a fibroinflammatory disease characterized by the enlargement of the ocular adnexal tissues. We analyzed RNA expression levels via RNA-seq in the biopsy specimens of three patients diagnosed with IgG4-ROD. Mucosa-associated lymphoid tissue (MALT) lymphoma, reactive lymphoid hyperplasia (RLH), normal lacrimal gland tissue, and adjacent adipose tissue were used as the controls ( Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a relatively new disease concept that was originally proposed in Japan. This disease causes fibrosis in the organs throughout the body including the eyes, and the organs become swollen . InvolveIgG4-related ophthalmic disease (IgG4-ROD) is an orbital lymphoproliferative disorder that show enlargement of the ocular appendages, including the lacrimal gland. IgG4-ROD is classified as a benign disease, though malignant lymphoproliferative diseases, such as mucosa-associated lymphoid tissue (MALT) lymphoma, can also occur in the orbit. Clinical symptoms, diagnostic imaging, histology, molecular analysis, and flow cytometry using biopsy specimens are the most commonly used diagnostic tools for orbital lymphoproliferative disorders ,6. DiffeDifferential diagnosis between IgG4-ROD and orbital MALT lymphoma, both of B-cell lineage, remains complicated and challenging in routine clinical practice because of the lack of specific diagnostic biomarkers. Elevated serum IgG4 is not sufficiently sensitive or specific enough for this purpose ,14. TherRNA sequencing (RNA-seq) is a type of next-generation sequencing (NGS) technology that allows the comprehensive evaluation and quantification of all subtypes of RNA expressed in tissues . CompareSeveral previous studies used NGS to analyze IgG4-RD for the purposes of elucidating the effects of steroid treatment and discovering for biomarker ,19, but This study included 71 patients with lymphoproliferative disorders who visited Tokyo Medical University Hospital of Ophthalmology between 2016 and 2020.n = 3). As controls, biopsy specimens of adjacent adipose tissue from the same IgG4-ROD patients, orbital MALT lymphoma possibly derived from the lacrimal gland, RLH, and normal lacrimal gland tissues were used (n = 3 each). The normal lacrimal gland tissues were collected to examine them for infiltration at the time of removing other orbital tumor  or for the purpose of diagnosing lacrimal gland enlargement . All biopsy specimens were stored frozen within 30 minutes after collection and each sample was stored at −80 °C until analysis. The demographic and laboratory features of the subjects and controls studied by RNA-seq are summarized in RNA-seq was conducted using biopsy specimens containing typical lesions obtained from IgG4-ROD patients  serum IgG4 level above 135 mg/dL. Definite IgG4-ROD was diagnosed when (1), (2) and (3) were present. Probable IgG4-ROD was diagnosed when (1) and (2) were present. Twenty-eight patients had definitive and five had probable IgG4-ROD. The diagnoses of IgG4-ROD, orbital MALT lymphoma, and RLH were based on clinical, radiographic, histologic and flow cytometric studies, and molecular genetic analyses, such as gene rearrangement, in the biopsies. In particular, the diagnosis of IgG4-ROD was made in accordance with the published criteria . BrieflyWritten informed consent was obtained from all the participants of the study. The study was also approved by the Ethics Committee of the Tokyo Medical University Hospital, Tokyo, Japan (number: SH3281). All investigations were conducted according to the principles of the Helsinki declaration.p < 0.05 and absolute FC (|FC|) ≥ 2 and were calculated using Empirical Analysis with the DEG tool.Total RNA was extracted from each tissue using TRIZOL ), and RNA integrity was measured by a Bioanalyzer RNA 6000 Pico Kit . The depletion of ribosomal RNA (rRNA) and RNA-seq library preparation was performed with a NEBNext rRNA Depletion Kit and a NEBNext Ultra Directional RNA Library Prep Kit, respectively . Sequencing for 2 × 36-bp paired-end reads was performed with NextSeq500 . FASTQ files were imported to the CLC Genomics Workbench . Reads were mapped to the hg19 human reference genome and quantified against 57,773 genes. Differentially expressed genes (DEGs) in IgG4-ROD were defined as p values were analyzed. The Reactome provides many cellular processes as a network of molecular transformations under a single consistent data model [Pathways associated with up-regulated DEGs were analyzed using Reactome . Up to 3ta model .β-actin were detected using TaqMan probes  with a 7900 HT Fast Real-Time PCR system (Thermo Fisher Scientific). The PCR cycling conditions were 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Data were normalized to the expression of the β-actin. Relative quantification was performed using a calibration curve with placental cDNA.The total RNA was extracted using a miRNAeasy Mini Kit  and reverse transcribed using a High Capacity RNA-to-cDNA kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Matrix metallopeptidase 12 (MMP12), secreted phosphoprotein 1 (SPP1), and Immunostaining by mouse anti-human MMP12  and rabbit anti-human SPP1  was performed using paraffin-embedded biopsy tissues. The intensity of immunostaining and the distribution of immunoreactivity were examined.p value less than 0.05 was considered statistically significant. Principal component analysis (PCA) was performed using R (3.6.2.) [p < 0.05 were extracted. The results of the first and second principal components were used for plotting the results in two dimensions. Unsupervised hierarchical clustering analysis using DEGs was performed using an algorithm based on Pearson correlation and the average-linkage method.Real-time PCR data were analyzed with SPSS statistical software version 26. The statistical significance of difference was determined using a Mann–Whitney U test. A (3.6.2.) . PCA wasRNA-seq detected 35 up-regulated DEGs and nine down-regulated DEGs in IgG4-ROD when compared with all the controls . An unsuWhen compared to adjacent adipose tissue, 2698 DEGs were up-regulated and 3057 were down-regulated in IgG4-ROD. Pathway analysis using the up-regulated genes revealed that 59 pathways (including duplications) were related to IgG4-ROD , Table 4Compared with orbital MALT lymphoma, 1718 DEGs were up-regulated and 705 were down-regulated in IgG4-ROD. Pathway analysis using the up-regulated genes revealed that 64 pathways were related to IgG4-ROD , Table 5When compared with RLH, 439 DEGs were up-regulated and 342 were down-regulated in IgG4-ROD. Pathway analysis using the up-regulated genes revealed that 43 pathways (including duplications) were related to IgG4-ROD , Table 6p values in RLH compared with the lacrimal gland; all these genes showed similar dysregulated patterns in IgG4-ROD. However, RNA-seq detected 781 DEGs in IgG4-ROD compared with RLH, which could be used for further analysis. The normalized mRNA expression levels in IgG4-ROD and the other lymphoproliferative disorders obtained from RNA-seq were subject to PCA, and the plot of the first principal components versus that of the second principal components is shown in In total, 2897 up-regulated and 3554 down-regulated DEGs were detected in RLH compared with normal lacrimal gland tissue; among them, 2262 and 2875 DEGs, respectively, were also detected in IgG4-ROD. An unsupervised hierarchical cluster analysis was then performed to investigate the variations in DEGs between IgG4-ROD and each control b. In allPathway analysis identified many pathways related to the immune system , suggestn = 30; orbital MALT lymphoma, n = 30). MMP12 and SPP1, two genes that showed low p values when compared to any of the control tissues in RNA-seq     were selctively) . There wRepresentative micrographs of immunostaining for MMP12 and SPP1 in IgG4-ROD, MALT lymphoma, RLH, and lacrimal gland tissues are shown in Immunostaining indicated expression of MMP12 and SPP1 at the protein level in IgG4-ROD biopsy tissues, suggesting that these molecules play a role in pathogenesis and could thus serve as new biomarkers for IgG4-ROD.In this study, we performed a comprehensive RNA analysis of lacrimal gland-derived IgG4-ROD using adipose tissue adjacent to the IgG4-ROD lesion, orbital MALT lymphoma, RLH, and normal lacrimal gland tissue as controls. Orbital MALT lymphoma and RLH were disease controls, while adipose tissue from the same patients with IgG4-ROD controlled for individual differences in gene expression, and normal lacrimal gland tissue controlled for tissue-specific gene expression. The results of the RNA-seq and pathway analysis suggest that IgG4-ROD is strongly related to the immune system and the up-regulation of MMP12 and SPP1. β (TGF-β) secreted by Tregs and CD4 + cytotoxic T cells (CD4 + CTLs) [The DEGs and pathways detected by RNA-seq for IgG4-ROD versus the lacrimal gland may provide insight into the pathology of IgG4-ROD. Fibrosis and class switches to IgG4 are characteristics of the pathology of IgG4-RD. Fibrosis in IgG4-RD is considered to be caused by transforming growth factor- + CTLs) . CD68 +  + CTLs) ,24,25.β , as well as multiple genes related to class switching  and Tfh , were up-regulated in IgG4-ROD. Thus, the DEGs detected by RNA-seq demonstrate the existence of multiple genes that are implicated in the pathology of IgG4-RD in previous reports, as well as novel DEGs that should be further investigated.Moreover, binding of the dendritic cells and T cells to B cells via BAFF and CD40 together with the activation of AID by various cytokines has been reported to induce class switching . FurtherIn the pathway analysis using DEGs detected in IgG4-ROD versus orbital MALT lymphoma, the pathways related to the complement system dominated. Some patients with IgG4-RD have hypocomplementemia , suggestNotably, IgG4 is regarded as an anti-inflammatory antibody because of its poor capacity to bind to Fc receptors , TherefoRLH is part of the spectrum of orbital lymphoproliferative disorders  and is cWhen common DEGs were extracted while excluding adjacent adipose tissue, 96 genes remained as DEGs. The genes obtained from this analysis are putatively involved in diseases involving IgG4 and IgE. However, there was no difference in the expression of these genes in comparison with the adjacent adipose tissue. This suggests that the surrounding tissues may also be affected by IgG4-ROD. However, the adipose tissue was a control tissue obtained from the same individuals, so the possibility of individual differences may be eliminated. Since there are no reports featuring analyses using different tissue samples collected from the same patient, the present findings could offer valuable data. In addition, although tissues from the lacrimal gland were also used in this study as a control, IgG4-ROD also occurs in tissues other than the lacrimal gland  and may β-induced fibrosis. This is inferred from our finding that SPP1 is strongly expressed in the fibrotic part of IgG4-ROD tissue.This study identified two novel genes involved in IgG4-ROD: MMP12 and SPP1. Up-regulation of these genes in IgG4-ROD relative to orbital MALT lymphoma was validated by real-time PCR, and increased expression at the protein level in the IgG4-ROD lesion compared to the control tissues was verified by immunostaining. SPP1 encodes osteopontin, which is involved in the attachment of osteoclasts to the mineralized bone matrix. MMP12 encodes an enzyme that decomposes the extracellular matrix. SPP1 was reported to be involved in fibrosis in various diseases ,40,41,42p values when IgG4-ROD was compared to each control tissue. It was also reported that M2 macrophages secrete MMP12, which is an elastase, and are involved in the pathological conditions of contact dermatitis [Schistosoma mansoni infection [In this study, commonly up-regulated DEGs among IgG4-ROD and control tissues, MMP12 showed low rmatitis . Generalrmatitis ,45,46 anrmatitis . The exhnfection  and may In our experiment, in which we identified the biopsy gene profile of RLH, we found that the gene profile of RLH was remarkably similar to that of IgG4-ROD and was characterized by decreased expression of MMP12 and SPP1 compared to IgG4-ROD . NotablyLimitations of this study include its retrospective design and relatively small number of cases collected from a single institution, which might have produced a selection bias and confounding bias. Due to the lack of comparable studies on IgG4-ROD including IgG4-RD with other diseases, it is not possible to compare the present findings with those of other studies. A prospective study with a large number of clinical samples from multiple centers is required to validate the present findings and further investigate other clinical manifestations of IgG4-ROD and other orbital tumors to diagnose clinical subtypes and elucidate their pathogeneses. In this study, we used real-time PCR to verify only two genes with enhanced expression in RNA-seq. However, it is necessary to carry out additional studies by quantifying other genes and analyzing their protein expression levels. Furthermore, MMP12 and SPP1 should be analyzed in larger samples of IgG4-ROD and other IgG4-RDs, and it is also necessary to examine the dynamics of these genes and proteins in IgG4-positive MALT lymphoma.To the best of our knowledge, this is the first report of a comprehensive mRNA analysis of IgG4-ROD using RNA-seq. We succeeded in detecting not only previously reported genes but also novel genes that that may serve as biomarkers or may be involved in the pathogenesis of IgG4-ROD. In addition, these results may provide novel insights for finding new therapeutic target molecules.


With the rise of location-based services and the rapidly growing requirements related to their applications, indoor localization based on channel state information–multiple-input multiple-output (CSI-MIMO) has become an important research topic. However, indoor localization based on CSI-MIMO has some disadvantages, including noise and high data dimensions. To overcome the above drawbacks, we proposed a novel method of indoor localization based on CSI-MIMO, named SICD. For SICD, a novel localization fingerprint was first designed which can reflect the time–frequency and space–frequency characteristics of CSI-MIMO under a single access point (AP). To reduce the redundancy in the data of CSI-MIMO amplitude, we developed a data dimensionality reduction algorithm. Moreover, by leveraging a log-normal distribution, we calculated the conditional probability of the naive Bayes classifier, which was used to predict the moving object’s location. Compared with other state-of-the-art methods, the results of the experiment confirm that the SICD effectively improves localization accuracy. The rapid development of location-based services and the rapidly growing requirements with regard to their applications have greatly facilitated the development of indoor localization. This has brought great changes to our lives. The WIFI fingerprint-based indoor localization method ,2,3,4 haThe key to indoor localization technology is obtaining accurate and reliable indoor locations. Channel state information (CSI) is sub-carrier level channel information. It can better describe the influence of the moving object on surrounding signals. Therefore, CSI has been proposed by researchers as a fingerprint for WIFI-based indoor localization in recent years. For example, Wang et al.  proposedFortunately, the indoor localization method based on CSI–multiple-input multiple-output (CSI-MIMO) can overcome the above shortcomings. In order to obtain more reliable and valuable CSI information for high-precision indoor localization, researchers have proposed a variety of localization methods based on CSI-MIMO. Chapre et al.  proposedTo overcome the above-mentioned shortcomings, we designed the SICD, a novel indoor localization scheme based on CSI-MIMO. In SICD, the localization fingerprint reflecting the time–frequency and space–frequency characteristics of CSI-MIMO under a single access point (AP) was first designed. Then, we developed a dimensionality reduction algorithm to map the high-dimensional CSI-MIMO amplitude data to a low-dimensional space. Finally, we leveraged the log-normal distribution to calculate the conditional probability of the naive Bayes classifier, which was used to predict the moving object’s location. Moreover, in order to verify the performance of SICD we conducted extensive experiments in a real indoor environment.The SICD scheme makes the following key contributions.(1)We proposed a method to construct localization fingerprints. The method collects MIMO-based CSI measurements under a single access point (AP), and extracts the amplitude information of the CSI-MIMO to construct a localization fingerprint.(2)We developed a dimensionality reduction algorithm based on locally linear embedding and low rank. The algorithm can map CSI-MIMO amplitude data from high-dimensional space to low-dimensional space and reduce the data redundancy.(3)We leveraged the log-normal distribution to calculate the conditional probability of the naive Bayes classifier, which can improve the classification performance of the classifier.(4)Single AP indoor localization based on CSI-MIMO with a dimensionality reduction method was proposed. The experiments were conducted in the laboratory, and the results show that the proposed method is superior to the state-of-the-art method.The remaining structure of the paper is demonstrated as follows. The related work of CSI and the truncated nuclear norm is briefly reviewed in the next section. In An indoor localization method based on WIFI signals can be divided into received signal strength (RSS)-based and CSI-based methods, which are briefly reviewed in this section.RSS is MAC (Media Access Control) layer information, which describes the attenuation of wireless signals during the propagation. It has been widely used in indoor localization. RSS-based localization methods mainly include geometric localization and fingerprint localization.For geometric localization methods, localization is performed by converting RSS measurements into the distance. In , MazuelaThere are two stages for the localization methods based on fingerprints: the training stage and the test stage. RSS measurements are collected at each location and converted into fingerprints during the training stage. The location is estimated by matching the target fingerprint with the offline fingerprint database during the test stage. In , Fang etHowever, there are some disadvantages of RSS-based localization methods. Firstly, RSS is sensitive to time-varied multipath fading, so it is easily affected by multipath and shadow fading. Secondly, RSS is a kind of coarse-grained channel information that averages the received signal amplitudes, making it difficult to obtain the accurate value.The disadvantages of RSS restrict its applications in the field of indoor localization. CSI is subcarrier-level channel information. It can better describe the influence of the moving object on surrounding signals. Therefore, CSI-based indoor localization has attracted more and more attention. Indoor localization based on CSI was proposed in ,19. In , Yang etIn this paper, we used the space and frequency diversity of a single AP to collect CSI. In order to overcome the high-dimensionality problem of CSI-MIMO data, we proposed a data dimensionality reduction algorithm. We used a Bayesian classifier based on the lognormal distribution for localization.R and S represent the signal vectors of the received and transmitted, respectively. H and N represent the matrix of CSI and the vector of Gaussian white noise, respectively. n is the index of sub-carriers. T represents transposition of a matrix. The CSI value of the i-th sub-carrier is expressed asi-th sub-carrier, respectively.CSI is fine-grained information from the physical layer, which can reflect the channel characteristics of the wireless communication link. It describes the channel quality between devices of the transmitted and received. When the CSI-MIMO system works on a flat fading channel, the signal collected by the received device is defined asj-th minimum singular value of Z.In , Hu et aZ is used to approximate the solution. Equation (4) can be rewritten asZ. Z, where r-order identity matrix.TNN is non-convex and is very difficult to directly solve (4). Therefore, singular value decomposition (SVD) of This section will show how to use the CSI collected from the network interface card (NIC) to estimate the moving object’s location in the indoor environment.To collect CSI data, we selected the TP-LINK wireless router with two antennas as the access point (AP) and the desktop computer with the NIC5300 as the terminal. Moreover, the wireless network card has three antennas. We placed the AP and terminal at two ends of the test area, respectively. However, the acquisition of CSI data must meet the hardware mentioned above conditions and requires the use of a software collection tool based on Linux 802.11n, namely, the CSI TOOL .The CSI TOOL is used to analyze the data which are received by the terminal. The data contains various types of information, e.g., the rate, the number of received and transmitted antennas, and the CSI. For simplicity, we selected CSI amplitude as the data feature. The traditional localization method based on CSI-MIMO requires multiple APs or terminals to construct the MIMO system ,30,31. Im data packets of CSI for each sub-carrier from every antenna pair, and then the CSI amplitude matrix of the i-th antenna pair is defined asg denotes the number of antenna pairs. We collect l of the m data packets to form a fingerprint, and arrange them into the form of a one-dimensional vector asIn order to facilitate the data dimensionality reduction processing in the following sub-section, we take In the indoor wireless environment, the CSI will be affected by noise. This means that the amplitude data of CSI-MIMO contains some outliers, which are not conducive to the extraction of features for localization from the amplitude data. However, outliers generally deviate significantly from the average. As shown in The After being processed, the amplitude data of CSI-MIMO are improved, with noise greatly suppressed, as shown in To reduce the redundancy in the CSI-MIMO amplitude data and improve localization accuracy, we designed a dimensionality reduction algorithm based on LLE and low-rank (DRLL). The general framework of the DRLL can be formulated asIn this paper, we use the least square loss function to evaluate the approximate error of data before and after dimensionality reduction. The loss function is defined asW by using the truncated nuclear norm to approximate the rank of the matrix [The rank function is non-convex, and its solution is NP -hard. Therefore, we impose a low-rank constraint on the projection matrix e matrix ,35,36,37k is the number of nearest neighbors, and o is the weight coefficient. I refers to an c-order identity matrix. O is the weight matrix, which is composed of the weight coefficient o.In order to keep the data structure after dimensionality reduction consistent with the raw data, we use locally linear embedding (LLE) as the regularization term.According to (16) and (18)–(20), the model of the DRLL algorithm can be expressed asIn order to solve the optimization problem of (21), we use the method of augmented Lagrangian multiplier ,39 to fiAt first, we convert (21) to the following equivalent problem asW, J, G, This problem can be solved by the augmented Lagrangian multiplier method, which aims to minimize the following augmented Lagrangian function ofJ, G, W byWhen The closed-form solution of optimal J, we first need to fix W, G, To optimize the operator .W, J, G is optimized byWith the Algorithm 1. Solution of the DRLL modelXInput: , C, D, k, t, e;Output:1 Initialize projection matrix W as the matrix with orthogonal column vectors, W = J = G, 0; 2 for T = 1:t 3 Optimize W by         4 Optimize J by  5 Optimize G by  6 Optimize  7 Optimize  8 end At each step, we optimize the Lagrangian multipliers bysolution .AlgoritThe device-free indoor localization based on CSI can be converted into a classification problem to obtain the moving object’s location information. To this end, we use the naive Bayes classification algorithm  in machii-th location point, and According to (32), the naive Bayes classifier in SICD is formulated asTo calculate the maximum value of The implementation and performance evaluation of SICD are introduced in this section. The environment of the experiment is first described. Then, we analyze the influence of setting different experimental parameters on the SICD. Finally, we compare the SICD with other off-the-shelf methods.In the performance verification experiment of SICD, we used a single TP-link wireless router as the access point. The model of the router was TL-WR840N, which carries two antennas. A Lenovo M736E desktop computer with the NIC5300 was used as the terminal. A ThinkStation P720 server with NIVIDA Quadro P2200 Graphic Card worked on predicting location. The CSI measurement experiments were conducted in the laboratory of Nanjing University of Posts and Telecommunications. The laboratory has some obstacles, such as chairs, tables, and computers. Simultaneously, we selected 10 locations to train and test in an experimental area of approximately To study the convergence of the DRLL algorithm, we calculated the value of (22) by leveraging the CSI amplitude data, as shown in In order to study the complexity of DRLL, we can decompose it into five equation: Equation (25), Equation (27), Equation (29), Equation (30), and Equation (31). For each iteration, Equation (25) needs to construct k nearest neighbors (KNN) in the LLE on the proposed SICD localization method, we conducted a specific experiment with different k values.In DRLL, we use LLE to ensure that the data structure after dimensionality reduction is consistent with the raw data. To verify the impact of the k = 9, SICD has the highest recognition rate at most locations. Thus, we set the number of nearest neighbors to nine.In the experiments, we used 3*2 MIMO technology and combined different antenna pairs of each received antenna according to (11). In order to evaluate whether the number of CSI data packets contained in each sample had an impact on the SICD localization method, we used the amplitude information carried by 30, 60, 90, 120, and 150 data packets to construct the respective fingerprint samples.In order to verify the impact of the dimensionality of the classification samples on the localization effect, we first leveraged the proposed DRLL algorithm to reduce the dimension of the CSI amplitude data to 60, 120, 180, and 240, respectively. Then, these dimensionality-reduced samples were input into the classifier for localization verification.When we calculate the conditional probability in the naive Bayes classifier, it needs to obey the log-normal distribution. In order to study the impact of the conditional probability distribution on the localization effect, we compared the localization accuracy of conditional probability which obeyed the log-normal distribution and Gaussian distribution, respectively.As shown in We verified the performance of our DRLL algorithm and compared it with three existing dimensionality reduction algorithms: LLE , PCA (PrWe verified the performance of our SICD method and compared it with three state-of-the-art CSI-based localization methods, ConFi , PICN 3, and MaLThe average localization accuracy of the four methods is presented in In this paper, we proposed the SICD, a single-access-point indoor localization method based on CSI-MIMO with dimensionality reduction. In the SICD we used a single access point to measure the CSI and constructed a fingerprint with rich localization information. In order to reduce the redundant information in fingerprint data, we designed an effective dimensionality reduction algorithm, namely the DRLL. In addition, we leveraged the log-normal distribution to calculate the conditional probability in the naive Bayes classifier, improving the classification performance of the classifier. Extensive experimental results showed that the SICD achieved a localization accuracy of 98.2% in the mess laboratory.In this paper, we provided a unique understanding of CSI-MIMO based localization and established a robust model. In the next step, we will focus on identifying the location of multiple moving objects indoors. Specifically, the multi-link of CSI-MIMO will be used to identify and extract the path reflection corresponding to each moving object. The location fingerprint of each moving object will be further constructed as if there was only a single object in the environment to promote the localization of multiple moving objects.


BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair. DNA helicases are ubiquitous motor proteins that couple the hydrolysis of nucleoside triphosphates (NTPs) to the unwinding of double-stranded DNA (dsDNA), providing the single-stranded DNA (ssDNA) required for many biological processes, including DNA replication, repair and recombination . Among tBloom syndrome protein (BLM) is one of the five RecQ family helicases that unwind DNA in a 3’−5’ direction . It is iIt has been widely appreciated that the BLM helicase interacts and collaborates with many protein partners to aid in cellular responses to replication stress and DNA damage in vivo . These iIn this study, by combining optical tweezers with confocal fluorescence microscopy, we examined BLM unwinding activities under various conditions at the single-molecule level. We found that, in the absence of hRPA, BLM at high concentrations can unwind dsDNA from a nick unidirectionally with a requirement of an external destabilizing force on the DNA template. Surprisingly, the presence of hRPA permits BLM’s unwinding in two opposite directions from a nick at low and zero forces. This stimulation demands free hRPAs in solution, suggesting that the coating of hRPA on the newly generated ssDNA is essential for enhancing BLM unwinding activity. These findings reveal a distinct SSB-enabled helicase unwinding mode that might facilitate the generation of the 3’ ssDNA tail in HR. Additionally, this study demonstrates various DNA unwinding activities by BLM that are regulated by tension on DNA, the concentration of BLM, and the presence of RPA, which provide a molecular insight into how BLM realizes its diverse biological functions.Gallus gallus BLM (gBLM). gBLM (hereafter referred to as BLM) has two similar RPA binding domains to the human BLM (hBLM) and the sequence of its helicase-core domain (core-gBLM) has an 80% identity with the one from hBLM. We combined dual-optical traps with confocal microscopy to monitor unwinding of a single DNA molecule by wild type (WT) We started the unwinding experiment at a force of 10 pN for ~1 min and increased it by 10 pN each time until dsDNA unwinding was detected. In the presence of 20 nM BLM, apparent dsDNA unwinding was not observed over a broad force range from 10 pN to 40 pN . In contAs nicks can be accidently generated along the long λ phage DNA template used in these assays, the observed dsDNA unwinding by BLM could be initiated either from a nick or from an intact duplex region melted internally, leading to distinct DNA intermediates during unwinding . To diffIn conclusion, the BLM helicase at a high concentration is indeed able to perform unidirectional unwinding from a nick, but this requires exerting an external destabilizing force (tens of pN) on the DNA template.Our data suggest that the BLM helicase unwinds dsDNA unidirectionally from a nick. This generates one intact strand under tension and one relaxed coiled strand in our experimental configuration. Since BLM unwinds by translocating on ssDNA in a 3’ to 5’ manner, we next sought to determine on which strand BLM prefers to translocate when initiating unwinding from a nick. This strand preference directly determines BLM’s unwinding polarity from a single nick . Thus, wSince RPA is a major protein partner that physically and functionally interacts with BLM, we next aimed to address how the human RPA  regulates dsDNA unwinding by BLM. We first examined whether the presence of RPA could enhance BLM unwinding activity by reducing the required destabilizing force on the DNA template. To do so, we monitored helicase unwinding on the λ phage DNA in the presence of BLM and RPA while discretely increasing the external force  until DNA unwinding was detected. Control experiments confirmed that RPA alone was incapable of unwinding dsDNA under these external forces . In the Next, we asked whether a high abundance of BLMs are necessary for dsDNA unwinding when RPA is present. We assayed DNA unwinding of BLM at a concentration of 20 nM with various concentrations of RPA. In contrast to the observations with 20 nM BLM alone where no DNA unwinding was monitored from 10 to 40 pN at all  and 3C, Collectively, we conclude that RPA stimulates DNA unwinding of limited BLMs by lowering the required force on the DNA template.We next sought to examine individual unwinding events in the presence of 50 nM RPA and 200 nM BLM and compare to those with 200 nM BLM alone. On average, there were 3.4 independent unwinding events monitored on a single DNA molecule, slightly higher than that without RPA . StrikinThis RPA-activated bidirectional unwinding by BLM could be initiated from either internally melted dsDNA or a nick. To determine that, we labeled the RPA with enhanced green fluorescent protein (eGFP) and repeated the unwinding experiments. This eGFP-RPA allowed for the direct visualization of the unwinding intermediates of ssDNA instead of dsDNA . If dsDNTo rule out that the observed bidirectional DNA unwinding was due to the heterologous combination of gBLM and hRPA, we also conducted the DNA unwinding experiments by using hBLM. When testing the homologous pair of hBLM and hRPA, results similar to the heterologous combination were obtained: hBLM alone initiates unidirectional unwinding from a nick and hRPA activates its bidirectional unwinding .Gallus gallus BLM that lacks major interaction domains with RPA (hereafter referred to as core-BLM)  . The WT ore-BLM) . When RPore-BLM) . These oore-BLM)  and the ore-BLM) . The ratore-BLM) . These fHaving demonstrated that RPA activates BLM’s bidirectional unwinding, we next aimed to gain mechanistic insights into this activation. A few potential mechanisms have been proposed to explain how RPA stimulates helicase unwinding of long duplex dsDNA Finding that only free RPA is necessary for BLM-mediated dsDNA unwinding once initiated raises a possibility that BLM might be needed to recognize a nick and bind to it. To test this hypothesis, we started the experiment by first placing the dsDNA tether in a buffer containing 20 nM BLM only where no dsDNA unwinding occurred . SequentIn summary, we conclude that once BLM recognizes a nick and initiates unwinding with RPA, only free RPAs are indispensable for its processive unwinding. These findings favor the model that ssDNA generated by BLM was immediately coated by free RPA, preventing the separated ssDNAs from reannealing.There are several lines of evidence supporting that unwinding activities of the RecQ family helicases can be regulated by the presence of SSB proteins . Herein,In the absence of RPA, BLM alone can only initiate unidirectional dsDNA unwinding from a nick where it translocates on the ssDNA with the assistance of an external destabilizing force on DNA . Since tA distinct feature of the bidirectional dsDNA unwinding by BLM and RPA is that the unwinding directionality is determined initially at a nick . The seqOur study also provided insights into the mechanisms by which BLM and RPA cooperate to facilitate end resection in HR. Recent studies support a model that the long-range end resection in HR initiates from a nick generated by the MRN/X complex once the DSB ends are coated by DNA adducts . This deE. coli is also able to promote dsDNA unwinding in both directions in the presence of E. coli SSB  and its helicase core mutant  were used in our experiments. Both WT BLM and core-BLM were expressed and purified as previously described  was transformed with the plasmid p11d-tRPA, permitting the co-expression of RPA70, RPA32, and RPA14. RPA was then purified over Affi-Gel Blue, Hydroxyapatite (Biorad), and Q-Sepharose chromatography columns as described previously . In brief, a 1,064 nm fiber laser and a water-immersion objective were used to create two orthogonally polarized optical traps. The trap separation was controlled using a piezo mirror for beam-steering one trap. Force measurements were performed by back-focal plane interferometry of the condenser top lens using a position-sensitive detector. A computer-controlled stage enabled rapid movement of the optical traps within a multiple-channel flow cell . This fl2, 0.1 mg/ml BSA, 2 mM ATP, 3 mM DTT and 50 nM Sytox Orange.As described previously , DNA molFluorescence microscopy was achieved by imaging the stained DNA on an EMCCD camera. Here, a 488 nm excitation laser and a 532 nm excitation laser were used for imaging eGFP-hRPA and Sytox Orange respectively. Kymographs were generated via a confocal line scan through the center of the two beads.http://www.nat.vu.nl/~iheller/download.html). Force and DNA extension data were taken at 50 kHz and filtered to 30 Hz. The kymograph was used to track the edge of the unwinding forks. The edges were marked manually by examining each pixel image and comparing to a set threshold value. Elasticity parameters of ssDNA were obtained from the DNA force-extension measurements for data conversion. The force-extension relation of ssDNA in our experimental condition was described as an extensible freely jointed-chain model .Thank you for submitting your article "RPA Activates BLM's Bidirectional DNA Unwinding from a Nick" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this study the authors performed optical tweezers-based single-molecule experiments combined with fluorescence microscopy to demonstrate that Bloom's syndrome (BLM) helicase, in conjunction with Replication Protein A (RPA), is able to perform bidirectional DNA unwinding starting from a nick located on one of the DNA strands.Essential revisions:The three reviewers and the reviewing editor came to an agreement that you have built a compelling case that in the presence of RPA BLM helicase can perform a "bidirectional" unwinding from a nick in the dsDNA, i.e. it can move on both, the relaxed strand and on the strand under tension. Combination of the force-extension and fluorescence measurements allowed you to quantify the rate, the frequency and directionality of the unwinding events. An impressive set of experimental conditions has been sampled to identify the circumstances under which the gBLM activity can be observed on the nicked DNA.The reviewers, however identified a number of issues with the paper that questions its suitability, at least in its current state.Major issues that require new experiments:1) The first significant problem lays in the use of heterologous BLM (from bird) and RPA (human). The observations are compared to what is known from the human system, but the gBLM itself does not seem to have a characteristic unwinding behavior of the human enzyme or other RecQ helicases, which is typically very dynamic with repetitive events and backtracking. Furthermore, the likely absence of the gBLM-hRPA interaction reduces the physiological significance of the authors' observations. While specific interactions between the two proteins may not be necessary for the observed reactions, the presence of such an interaction may alter the BLM behavior. The authors need to make a better case for the physiological relevance of their observations or to add an analysis of the cognate protein pair for at least the key experiments. The necessity of the experiments with human BLM is a major experiment that needs to be added to strengthen the conclusions of the manuscript. It has been requested by all three reviewers and the reviewing editor.2) A complementary biochemical in vitro experiment is necessary to establish that the bidirectional unwinding can indeed take place in the absence of force. Extrapolation of the rate of initiation dependence on force to zero force may be another way to address this question.Other important issues to address:3) It is unclear how many nicks are present on each λ DNA molecule, which complicates the analysis of the initiation rate.4) The authors should support their statements with statistical analyses , especially considering a relatively small number of events in each class. Essential revisions:[…] Major issues that require new experiments:1) The first significant problem lays in the use of heterologous BLM (from bird) and RPA (human). The observations are compared to what is known from the human system, but the gBLM itself does not seem to have a characteristic unwinding behavior of the human enzyme or other RecQ helicases, which is typically very dynamic with repetitive events and backtracking. Furthermore, the likely absence of the gBLM-hRPA interaction reduces the physiological significance of the authors' observations. While specific interactions between the two proteins may not be necessary for the observed reactions, the presence of such an interaction may alter the BLM behavior. The authors need to make a better case for the physiological relevance of their observations or to add an analysis of the cognate protein pair for at least the key experiments. The necessity of the experiments with human BLM is a major experiment that needs to be added to strengthen the conclusions of the manuscript. It has been requested by all three reviewers and the reviewing editor.2017) and biochemical studies with gBLM also showed similar results with hBLM . Thus, we chose to mainly use gBLM in our previous study to investigate how hRPA stimulates its unwinding.We greatly appreciate the comment and suggestion from the reviewers and the editor and would like to briefly comment on why we chose to use gBLM in our previous manuscript. First, gBLM is an ortholog of human BLM (hBLM) and shares high similarity with hBLM. gBLM has two similar RPA binding domains to the hBLM  and the sequence of the core gBLM has an 80% identity with the one from hBLM. Second, we have successfully over-expressed and purified gBLM with high purity .”2) A complementary biochemical in vitro experiment is necessary to establish that the bidirectional unwinding can indeed take place in the absence of force. Extrapolation of the rate of initiation dependence on force to zero force may be another way to address this question.We greatly appreciate the reviewers’ suggestion and would be more than happy to answer this question by conducting new experiments. We first tried to address whether BLM and RPA can unwind DNA in the absence of any externally applied force by employing an in vitro ensemble assay. However, as BLM and RPA can also initiate the DNA unwinding from the blunt-ended termini  which raises difficulties in the experimental design and data analysis. Instead, we designed a single-molecule assay to answer this question. In this assay, we first incubated a relaxed DNA tether (under zero force) in a BLM and eGFP-hRPA channel for a short period time. Following that, we transported the DNA tether to a buffer channel wherein no unwinding occurred because of the absence of BLM, RPA and ATP. In this channel, we examined the fluorescence signal of eGFP-hRPA along the DNA tether under 10 pN. We found the existence of the unwound ssDNA on the tensioned DNA as well as one or two bright spots at the end of the newly generated ssDNA. This results strongly suggest that BLM and RPA can indeed unwind dsDNA from a nick in the absence of external force. We have added this set of data as Figure 3—figure supplement 2 and also incorporated this information in the revised manuscript by stating:“Moreover, we demonstrated that BLM/RPA-mediated DNA unwinding can even take place in the absence of any externally applied force .” and “Notably, bidirectional DNA unwinding events were also observable under low and zero external forces .”Other important issues to address:3) It is unclear how many nicks are present on each λ DNA molecule, which complicates the analysis of the initiation rate.We apologize for not being explicit on describing our experiment. For the experiments with λ DNA, we can detect unwinding events along the template but not the number of nicks on it. In other words, the nicks that were not used to initiate dsDNA unwinding cannot be detected. Thus, it is impossible to analyze the initiation rate by using this DNA template. However, we can indeed calculate the initiation rate by the experiments with the 6.4 kbp DNA template which contains a single nick. In the presence of BLM alone, 25 traces in all 29 examined DNA tethers (86%) were found to be unwound from the single nick. In the presence of BLM and RPA, we found that nearly all examined DNA molecules (26 out of 27 examined traces) were unwound from the nick. This information was provided in the revised manuscript in the subsection “RPA activates BLM’s bidirectional unwinding from a nick”.4) The authors should support their statements with statistical analyses , especially considering a relatively small number of events in each class.We thank the reviewer for the suggestion. We have incorporated them in the updated manuscript and provided source data as well. Please note that p-values have been provided in the revised figure legends.


We describe the case of a gentleman with pleural thickening. On follow-up imaging, dilatation of the main pancreatic and common biliary ducts was noted and an initial diagnosis of pancreatic malignancy was made. During his preoperative workup for pancreatic head malignancy, a PET-CT was performed, which demonstrated increased uptake in the pancreas, in the pleura and in the prostate gland. This raised the possibility of immunoglobulin G4-related disease (IgG4-RD), which was effectively treated with oral steroids. IgG4-RD is a well-described cause of autoimmune pancreatitis but can affect other regions, including the pleura and prostate. It is essential that radiologists are aware of the imaging findings in IgG4-RD and can direct clinicians towards this important multisystem diagnosis. A 76-year-old male presented to his general practitioner (GP) with a 4-week history of dry cough. He was a lifelong non-smoker, with a past medical history of non-muscle invasive bladder cancer and sigmoid cancer. He was a carpenter and had previously worked with asbestos sheets. His GP requested a chest X-ray, which demonstrated pleural calcification and a new rounded density in the right mid-zone that was not present on the previous imaging . A CT scThe patient was treated with antibiotics, seen in respiratory clinic and scheduled for CT surveillance of his pulmonary consolidation and pleural disease. Three-month, six-month and twelve-month follow-up CT scans demonstrated stable pleural disease, however prior to the 12-month scan the patient had developed deranged liver function tests and the abdomen was included on the subsequent CT. The CT, acquired in the portal venous phase, demonstrated dilatation of the proximal portion of the pancreatic duct within the head of pancreas, and a normal calibre duct more distally. There was associated intra-hepatic and extra-hepatic biliary dilatation, which were normal on previous imaging . The panFollowing discussion at the UGI multidisciplinary team meeting and an initial diagnosis of pancreatic malignancy, an MR pancreas and staging PET-CT were arranged to further investigate and evaluate disease extent. MR demonstrated pancreatic and biliary duct dilatation , with a Fluoro-deoxyglucose (FDG) PET-CT was performed , which dThe differential diagnosis for this patient’s pleural thickening was a pleural malignancy, especially in light of the patient’s previous asbestos exposure, while inflammatory causes and infection were the other main differentials. The patient’s pancreatic and biliary duct dilatation were suspicious for malignancy and the main differential was a pancreatic head or ampullary tumour; IgG4-related pancreatitis was considered a differential. The diffuse changes seen in the prostate gland were unusual for malignancy, in that the peripheral zone was spared, and so prostatitis was the main differential.−1, while total IgG was also elevated at 221.8 mg dl−1. Histology demonstrated inflammatory changes only and the samples were IgG4-positive on immunohistochemistry.Serum IgG4 levels were requested and endoscopic retrograde cholangio-pancreatography (ERCP) was performed. Endoscopic ultrasound demonstrated a mass in the pancreatic neck and body, and biopsies of the ampullary region were performed. A cholangiogram demonstrated a common bile duct stricture with intrahepatic regions of alternating strictures and dilatation. A stent was placed in the common bile duct and brushings were taken. The patient’s serum IgG4 was raised at 160 mg dlSteroid treatment was commenced with oral prednisolone 30 mg once daily. A repeat PET-CT was undertaken 10 weeks following steroid therapy, illustrated in 1 It is characterized histologically by IgG4-positive plasma cell infiltration, obliterative phlebitis and fibrosis.2IgG4-related disease (IgG4-RD) is a multisystem disorder of auto-immune aetiology. It can affect a number of different organ systems and may mimic infection, inflammatory disease or malignancy.3 The most common manifestations of IgG4 are autoimmune pancreatitis and sialadenitis; however the disease can affect a number of different organs including the kidneys, lungs, liver, thyroid and prostate.1It is predominantly a disease of elderly males, with a male-to-female ratio of 3:1; however, the disease can also affect children.4 Autoimmune pancreatitis was originally linked to IgG4 by Hamano et al (2001), who demonstrated higher serum concentrations of IgG4 in 20 patients with autoimmune pancreatitis, while Kamisawa et al (2003) demonstrated IgG4-positive plasma cell infiltrates on immunohistochemistry in eight patients with histologically confirmed autoimmune pancreatitis.5,6The pathophysiological mechanism underlying IgG4 disease has not been elucidated; however, it is thought that IgG4 is not the driver of the disease, as IgG4 is unable to activate the complement cascade.7 Despite this, serology plays a large role in the diagnosis of this condition. Tissue immunohistochemistry remains the gold standard for the diagnosis.1 The diagnostic criterion for IgG4-RD focuses on the triumvirate of organ-specific clinical findings, serum IgG4 level >135 mg dl−1 and histological findings of IgG4-positive plasma cell infiltration.8Elevated serum IgG4 levels are neither sensitive nor specific for the diagnosis of IgG4-RD.9 The pancreas is uniformly enlarged and there is diffuse loss of the lobulated margin of the pancreas, resulting in a smooth featureless appearance.10 Focal autoimmune pancreas is less common but can pose a significant diagnostic dilemma. Focal autoimmune pancreatitis can present as a mass that may be difficult to differentiate from pancreatic malignancy and care must be taken to avoid unnecessary surgery.11 There are a number of important imaging findings which point to a diagnosis of autoimmune pancreatitis rather than pancreatic malignancy. A low attenuation or low intensity capsule-like rim evident on CT and MRI, respectively, has been demonstrated to be a reliable sign, while other findings include a smooth tapering of the upstream pancreatic duct, the so-called “icicle sign”.12,13 On the other hand, imaging appearances, which favour a diagnosis of pancreatic malignancy, include an abrupt cut-off in the main pancreatic duct and heterogeneous enhancement within the mass on CT.10,12 The presence of a peritumoural cyst has been recently demonstrated to be a specific finding for pancreatic malignancy.14The archetypal disease process in IgG4-RD is autoimmune pancreatitis, which can be diffuse or focal. The more common, diffuse pattern of autoimmune pancreatitis is classically demonstrated on imaging as a oedematous pancreas, which can be described as sausage-shaped.15 The diagnostic criteria for IgG4-related pulmonary disease make specific reference to pleural thickening,8 which was the initial finding in our patient’s case.The lung manifestations of IgG4-RD are characterized by inflammatory lung pseudotumour, central airway disease, interstitial pneumonia and pleuritis.16 Due to the increased risk of prostate malignancy in elderly males, prostatic uptake should be followed-up either with repeat imaging or PSA monitoring.Prostatitis is also a recognized manifestation of IgG4-RD, with IgG4 plasma cell infiltration on immunohistochemisty, and has been shown to demonstrate increased FDG uptake on PET-CT.17–19 In 21 patients from the French IgG4 registry, all patients demonstrated increased uptake on FDG PET-CT in typical organ locations, while there was also a good correlation between PET-CT findings and treatment response.17 In a prospective study of 35 patients, increased FDG uptake was seen in all patients pre-treatment, and there was multisite involvement in 97% of patients.18 Following initial steroid therapy in the same cohort, repeat PET-CT demonstrated complete remission in 72% of patients. Although there is a good response to steroid therapy in most patients, there are a proportion of patients with poor responses and a proportion who relapse and require treatment escalation.3 Therefore, PET-CT can play an important role in the identification of these patients and allow for the optimization of treatment.PET-CT has been demonstrated to be effective in the diagnosis, evaluation of the extent of disease and evaluation of treatment response in patients with IgG4 disease.1 Although, the long-term prognosis of IgG4-RD is unknown, especially with regard to newly recognized manifestations of the disease, in particular those of the lung.15The treatment of IgG4-RD is oral steroids in the acute setting as standard, with the use of steroid sparing agents, such as azathioprine and methotrexate, in the long term.In the present case, our patient was due to undergo surgical excision of the head of pancreas for a presumed malignancy. A PET-CT performed prior to this demonstrated increased uptake throughout the pleura, pancreas and prostate that led to the diagnosis of IgG4-related disease and the patient responded promptly to steroid therapy. The UGI team have become familiar with IG4-RD in the form of auto-immune pancreatitis; however, our respiratory and urology teams have less experience in these rarer sites of involvement. The radiologist has a role to play in guiding teams towards this multisystem diagnosis.The recognition of IgG4-RD as a disease process is in its infancy. An increased awareness of this entity can lead to an earlier diagnosis for patients, thereby leading to improved outcomes. It is eminently treatable with a good response to steroids, and so early diagnosis can lead to early effective treatment and the avoidance of unnecessary interventions. Therefore, an awareness of IgG4-related disease among radiologists is essential.The pancreas, biliary ducts and salivary glands are the most commonly affected organs in IgG4-related disease.The disease can also affect the orbit, thyroid, kidney, retroperitoneum, lungs, aorta, prostate, musculoskeletal system and the skin.The characteristic image findings of pancreatic IgG4-related disease is an oedematous pancreas with a halo of low density or low signal intensity. This may be associated with pancreatic or biliary dilatation and strictures.There is an evolving role for PET-CT in the diagnosis, staging and evaluation of treatment response in patients with IgG4-related disease.IgG4-related disease is an important differential in patients with multisystem disease. As a highly treatable condition, awareness of IgG4-RD is essential to ensure effective treatment can be initiated promptly.Written informed consent was obtained from the patient(s) for publication of this case report, including accompanying images.


Reversible phase-change behaviors of Ge–Sb–Te based superlattices (GST-SL) were studied by ab initio molecular dynamics (AIMD) simulations based on three models containing Ge/Sb intermixing, namely the Petrov-mix, Ferro-mix, and Kooi-mix models. The flipping behavior of Sb atoms was found in all the three GST-SL models in the melting process. Among them the Kooi-mix model exhibited the best stability, and the analyses of bond length distribution and electron localization function provided a better explanation on the phase transition of GST-SL. Finally, we proposed a fast switching model for GST-SL based on Sb flipping. Recentlmaterial , etc., iThe Ge atoms switching mechanism in GST-SL superlattice was firstly proposed by Simpson et al. . Then, sThe simulation was performed using the VASP codes . Perdew–Generally, the amorphization (RESET) process of phase change materials is achieved by melting at a constant high temperature, and the melting process is usually accompanied by a rapid increase in free energy . AccordiIn the AIMD simulation, free energy of an atomic model will increase due to the absorption of latent heating. This property is usually employed to trace the melting process . To compIn order to investigate crystallization properties of GST-SL, we performed the annealing simulation at 700 K for 100 ps based on RESET structures obtained in the melting process. 2Sb2Te5 alloy [According to the melting and crystallization simulation above, we can see that Kooi-mix model exhibited the best performance in stability and crystallization velocity. Hence, the Kooi-mix model is considered helpful to reveal the phase-change mechanism of GST-SL. e5 alloy ,18. Besie5 alloy ,10,19. A1–Te1, Te1–Sb1–Te2, Te2–Ge2–Te3, Ge3–Te3) are stronger than the other bonds according to the value of ELF. These strong bonds provide the stability of vdW gap of the Kooi-mix model in the melting process at elevated temperature. Interestingly, the bonds connecting the inner Te atoms  with the Sb1 atom is weaker than those of Ge1, Ge2, and Ge3 atoms. Hence, this is reason why the Sb atom is more likely to enter the vdW gap when annealing. Besides, it should be noted that the ELF of the atoms within the block is relatively symmetric, implying the occurrence of the resonant bond [ant bond ,21 in phWe proposed a phase-change mechanism about the GST-SL based on the Kooi-mix model shown in . In this−5, energy cut off was 380 eV, convergence criterion of the force acting on each atom was −0.015, tetrahedron method with Blöchl corrections, K points used a 2 × 2 × 2 Gamma point grid. Finally, we performed energy band calculation and charge density analysis, GST-SL sampling was done using a 2 × 2 × 2 grid, all atoms were fully relaxed until the forces on each atom were smaller than 0.015 eV/Å, the Energy Convergence Standard was 10The amorphization and crystallization processes of three GST-SL atomic models and GST models were simulated through first-principles molecular dynamics. In the simulation of the heating process, it was found that the layer structure of GST-SL makes its thermal stability better than that of GST. Additionally, from the free energy curve of GST-SL, it was found that its structure changes greatly at 1400 K. The amorphization simulations of three GST-SL models all showed the phenomenon of Sb entering and exiting a Te–Te paradigm layer before the layer structure was scattered, and the Kooi-mix model was the most stable structure in the GST-SL model. The Ferro-mix model cannot return to the original crystalline structure. In addition, the crystallization time of the GST model was greater than 150 ps. Therefore, the excellent crystalline properties of GST-SL come from its stable layer structure. Then, it was found in the crystallization simulation that both the crystallization time of the Kooi-mix and Petrov-mix models only take a few picoseconds. What is more, structural characteristics of the Koo-mix model and the reason why the Sb atom jumps into the paradigm layer was explained by analyzing the bond length distribution and the electronic local function. Based on the simulation results of amorphization and crystallization of GST-SL, this paper proposes a phase transition mechanism based on the Kooi-mix model, which is completely different from a phase transition mechanism of the highly recognized Ge atom jumping in the Te–Te paradigm layer ,24: afteIn this work, in order to explore the phase transition mechanism of GST-SL, we used AIMD simulation to study three phase transition characteristics of GST-SL models based on Ge/Sb mixture. The results revealed that in the melting process, the Kooi-mix model had a longer melting time than Petrov-mix and Ferro-mix models. (30 ps vs. 8 ps), compared to the other two models, the Ge–Te bonds in the Kooi-mix model were stronger. Therefore, Kooi-mix model has the best structural stability compared with the Petrov-mix and Ferro-mix models, the Kooi-mix model is more in line with the phase transition mechanism of GST-SL. The structure transition characteristics of GST-SL are explained by the key distribution and electronic positioning function. The phase transition caused by the jumping of Sb atoms required only 0.5 eV of phase change activation energy, which was much lower than the activation energy of the Ge atom jumping mechanism, and Sb atoms more easily jumped into the paradigm layer than Ge atoms, and this was also verified by electron density calculation and energy band calculation. In light of these results, we propose a new a phase transition mechanism for GST-SL based on Sb flipping. The current findings help to study the phase transition mechanism of GST-SL and optimize the performance of PCM.


In “ColistinDose, a Mobile App for Determining Intravenous Dosage Regimens of Colistimethate in Critically Ill Adult Patients: Clinician-Centered Design and Development Study” :e20525) the authors noted one error due to an editing issue.In the Abbreviations section, two abbreviations were listed incorrectly as follows:CVVD: continuous veno-venous hemofiltrationCVVH: continuous veno-venous hemofiltrationThese abbreviations have been corrected to:CVVH: continuous veno-venous hemofiltrationCVVHD: continuous veno-venous hemodialysisThe correction will appear in the online version of the paper on the JMIR Publications website on December 18, 2020, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories.


Herpesvirus of turkey (HVT) is commonly used as a vaccine to protect chickens against Marek’s disease. Following vaccination, HVT infects feathers where it can be detected in all chicken lines examined. Unlike the parental Brown line (BL), Smyth line (SL) chickens develop vitiligo, due to autoimmune destruction of melanocytes in feathers. Previous reports showed a strong inflammatory response in Smyth chickens’ feathers at vitiligo onset, that subsided once melanocytes were destroyed, and depigmentation was complete. Here, we questioned whether the local autoimmune response in the Smyth model influences HVT infection and persistence in feathers. For this, one-day-old SL and BL chickens were vaccinated with Newcastle disease (rHVT-ND). Vitiligo was scored and HVT loads in pigmented and non-pigmented growing feathers were quantified regularly over 20 weeks. Chickens of both lines showed moderate HVT loads in feathers. At the onset of active vitiligo, the HVT load was significantly higher in SL compared to BL feathers. However, no difference in HVT loads was noticed between pigmented and non-pigmented feathers from SL chickens. Therefore, surprisingly, the inflammatory response in feathers of SL chickens did not inhibit HVT infection and persistence, but on the contrary, temporarily promoted HVT infection in feathers. Meleagrid herpesvirus (MeHV), commonly named herpesvirus of turkey (HVT), is extensively used in the poultry industry worldwide to protect against Marek’s disease (MD) [Mardivirus genus in the Alphaherpesvirus sub-family is genetically and serologically related to Marek’s disease virus (MDV) or gallid herpesvirus type 2. For the past two decades, this virus is also increasingly used as a platform vector to develop recombinant vaccines against other major viral chicken diseases, like infectious bursal disease and Newcastle disease (ND) [Mardivirus persistence in feathers and how to eradicate it are major issues for poultry health, sustainability of poultry production, and for more effective control of environmental contamination by pathogens. HVT is a good virus model to identify factors that modulate Mardivirus infection and persistence in feathers.The ase (MD) ,2. Indeease (ND) ,4,5,6. Aase (ND) ,8). HVT ase (ND) . In the ase (ND) . In thisase (ND) ,12,13. Iase (ND) ,15. In case (ND) . In partMardivirus infection in feathers. Chickens from this line spontaneously develop vitiligo-like loss of feather pigmentation due to melanocyte-specific autoimmune responses that result in the apoptotic death of epidermal melanocytes in growing feathers (GFs) [Mardivirus connection in SLV development, the availability of a parental control, namely the Brown line (BL) from which the SL originates, further underlines the suitability of this animal model for exploring HVT infection and persistence in GFs. Vitiligo susceptibility is maintained in BL chickens, however, less than 2% of the BL population exhibit pigmentation loss even with HVT vaccination at hatch [B locus, B101/101), and hence are expected to mount comparable immune responses to HVT as they do when immunized with sheep red blood cells [The Smyth line (SL) of chickens is an interesting chicken model to examine rs (GFs) ,18,19. Lrs (GFs) . Melanocrs (GFs) ,22. Melars (GFs) . Therefors (GFs) ,23,24,25rs (GFs) . In addiat hatch . SL and od cells .The vitiligo-prone SL and parental BL chickens constitute an excellent opportunity to examine whether vitiligo development and the associated local T cell-mediated inflammatory response in GFs can reduce HVT infection of feathers and/or modulate its replication kinetics as well as persistence. Using a recombinant HVT-ND vaccine, HVT infection and persistence in GFs from vitiligo-prone SL and normally pigmented BL chickens were monitored every 3 weeks starting at 2 weeks of age (2 weeks post-vaccination) and throughout the 20 weeks when SLV develops. Additionally, levels of NDV antibodies were examined when the chicks were 13 weeks of age to confirm immunogenicity and success of the vaccination.® HVT-ND, CEVA Animal Health, LLC, Lenexa, KS serial number 372-1004, 4000 pfu-dose) as recommended by the manufacturer. Note: All chicks came from dams that were vaccinated with live HVT ; hence, chicks may have had maternal antibodies to HVT, but not to NDV. Chicks were placed in floor pens and raised using conventional rearing conditions on wood shaving litter with food and water available ad libitum.The University of Arkansas Institutional Animal Care and Use Committee (IACUC) approved all protocols and procedures involving animals used in this trial . Chicks used in this study were offspring from the annual breeder replacement pedigree hatch of SL and BL populations maintained by G. F. Erf at the Arkansas Agricultural Experiment Station Poultry Farm at the University of Arkansas, Fayetteville. At hatch, all chicks were individually tagged for identification and vaccinated (s.c.) with a single dose of live rHVT-ND vaccine  was negative for NDV antibodies and did not develop SLV; hence, she was considered not HVT-ND vaccinated and excluded from all analyses. Prior to sampling, all chicks were visually scored for signs of depigmentation, indicative of vitiligo onset. Scoring was done by close examination of the most proximal end (newest growth of GFs). Scores were assigned as follows: 1  to 5 (100% of GFs depigmented—complete amelanosis). In a few cases, feather re-pigmentation was observed and scored “RepigYes”, in contrast to “RepigNo”. Both scoring and GF sampling of the 20 birds  began at 2 weeks of age and continued every 3 weeks until the chickens were 20 weeks of age. Week 20 was chosen to stop the experiment because at this age mature plumage is established, only few regenerating feathers are present and SLV development ends because epidermal melanocytes are only present in GFs . For SL iNOS cellular gene. All HVT loads in feathers and spleens were expressed as HVT genome copy number per million cells.All protocols were previously described . BrieflyTo verify that the birds were correctly vaccinated, NDV antibody titers in the plasma were determined at week 11 post-vaccination by ELISA . Titrations were generously performed by CEVA BIOMUNE, Lenexa, KS, USA.p-values for pairwise comparisons was used [https://www.R-project.org/) were used for plots and computing.The variables are: the chicken line , the date of sampling . Two data points were missing, corresponding to uninterpretable qPCR, and re-imputated . Re-imputation was made for two data points by replacing the missing data by the mean of the two values that framed the missing data . Because of small sample sizes and the impossibility to test normality of distributions, only non-parametric tests were used. For HVT loads, all tests were performed after decimal logarithm transformation. Permutation tests were used on numerical values when homogeneity of variances was verified, otherwise on ranks. Comparisons of proportions were performed by Fisher’s exact test. For comparisons of independent samples  or dependent samples , permutation methods for ANOVA were used, either exact, if made possible by the sample size, or by Monte Carlo approximation. For analysis of variance in the case of mixed models , a non-parametric ANOVA-like test for non-parametric analysis of longitudinal data in factorial experiments using ranks and adjusted was used ,30. GrapFeather depigmentation was observed in week 8 in both lines, with all 11 SL (100%) and 1 BL  chickens developing severe vitiligo (score 4–5) by 20 weeks . The onsp = 0.6698), indicating that both lines mounted a similar antibody response to HVT-ND vaccine.The HVT-ND uptake was verified by measuring NDV antibody titers in plasma in week 11, by ELISA. All 20 chickens (BL and SL) had NDV antibody titers, with 90% above 10,000, indicating effective vaccine uptake and antibody response to the HVT-ND vaccine B. A Mannp = 0.00083; SL > BL), the time at which vitiligo was first observed in SL chickens. A boxplot representation of HVT loads in the two lines over time . Therefore, HVT infection was faster and/or higher in SL than in BL around vitiligo onset.HVT load (genome copy number per million cells) in pigmented and non-pigmented feather samples was determined using qPCR. Note: for birds with vitiligo scores of 2 to 4, both pigmented and non-pigmented GFs were collected. Of 140 expected measures (63 for BL and 77 for SL), all were obtained after two imputations .Examination of the time course profiles of HVT load in feathers established for each SL and BL chicken revealed two types of profiles : chickenn = 30); 893 for non-pigmented feathers  . Altogether, these results indicate that the stable depigmentation state of feathers had no influence on HVT replication efficacy.To determine whether active vitiligo in a feather influences HVT replication, we compared HVT loads in all pigmented and non-pigmented SL feathers from week 8 (the time vitiligo was first recorded) to week 20. The feather load medians in both types of feathers were of moderate level and numerically close: 1088 for pigmented feathers  A. In add n = 33) B. With tFour birds showed re-pigmentation event(s) of their feathers . In all cases, these re-pigmentation events were incomplete (vitiligo score ≥ 2) and transient, as all four birds had stable vitiligo with a score of 5 at week 20. We did not notice any peculiarity for these birds in terms of re-pigmentation events and HVT loads (not shown).p-value = 0.7995). This result indicates that the genetic defect associated with SLV expression in SL chickens does not influence HVT persistence in the spleen.MD vaccines are known to persist for several weeks in the spleen of vaccinated birds. To determine whether the SL phenotype affects this feature, we analyzed HVT loads in the spleen of seven birds from each line at 15.5 months of age. The median HVT loads were 212 in BL and 507 in SL . A permuMardivirus infection and persistence in feathers in the presence and absence of local T cell-mediated immune responses. Specifically, both SL and BL chickens are genetically similar and share the same MHC genotype (B101/101), however, SL chickens predictably develop a high incidence of autoimmune loss of melanocytes (vitiligo) while BL chickens rarely express this disorder. Mardivirus infection and the melanocyte-specific autoimmune response share the same target tissue—the feather. Moreover, expression of SLV is strongly associated with live HVT administration at hatch. In order to better decipher the mutual interactions between HVT and vitiligo development in chickens, HVT infection of feathers in SL chickens was measured through qPCR HVT-specific genome loads over 20 weeks, before and throughout vitiligo development, and compared to HVT infection in the parental BL controls. The HVT loads in feathers of SL and BL were moderate, rarely exceeding 104 HVT genome copies per million cells, with medians of 774 and 425, respectively. These viral loads were in accordance with the ones we reported in feathers in four different lines vaccinated at hatch with the same HVT-ND vaccine [Chickens from the vitiligo-prone Smyth line (SL) and the parental Brown line (BL) provide a unique opportunity to examine enotype B1/101, ho vaccine ,31. AltoWe observed some feather samplings with no detectable HVT genomes at some time points. We reported similar results earlier . A chickIt is important to question whether HVT genome copies measured in feathers are associated with HVT replication and/or correspond to the presence of latently infected T cells. In a previous theoretical calculation for a normal chicken, we estimated that 20 copies per million pulp cells may come from latently infected CD4+ T cells . As featWe found that SL chickens exhibited higher HVT loads in feathers than BL at 8 weeks post-vaccination, although HVT feather loads were not significantly different in SL compared to BL chickens when examined across all sample times. Week 8 coincides with the first recording of vitiligo that had developed in 36.4% birds between the 5- and 8-week samplings. The age of vitiligo onset with HVT-ND is in accordance with our previous observations of vitiligo development between 6 and 16 weeks of age in SL chickens vaccinated with classical HVT . It has Depigmented feathers, corresponding to feathers in which melanocytes were eliminated, had similar HVT loads than pigmented feathers. This result indicates that melanocytes do not play an essential role in HVT infection and persistence in feathers and that melanocytes are not a reservoir for HVT in feathers, as we hypothesized earlier .Based on earlier immunological studies in BL and SL chickens, we wonder here if a high level of IFN-γ locally influences HVT infection/replication in feathers. IFN-γ is a pleiotropic molecule well known to support Th1 response and to activate dendritic cells, macrophages, and NK cells . If antiDespite the presence of a strong local T cell-mediated melanocyte-specific inflammatory response, Smyth chickens developing vitiligo do not better control HVT infection, replication, and persistence in feathers than non-vitiliginous parental chickens. They even exhibit higher HVT loads at vitiligo onset. There is therefore a mutual positive effect between HVT infection and vitiligo development.


This data article provides a series of 492 stress-strain curves and compressive strength values obtained under the uniaxial compression of concrete samples fabricated from three different normal-weight concrete mixtures with four different cylindrical sample sizes ranging from 40 × 80 mm to 160 × 320 mm. These data are related to two research articles: “Revisiting statistical size effects on compressive failure of heterogeneous materials, with a special focus on concrete”   The first one contains the analyzed data of: (i) different characteristic properties of hardened concrete samples ; and (ii) the microstructural characteristics of our undamaged concrete samples. The data of point (i) are reported in ••••••••••••••The values of the elastic properties and several characteristic parameters of hardened concrete samples, as well as the values of the compressive strength failure strength for all concrete samples are presented in 14 (fourteen) tables as follows:22.1To obtain the dataset presented in this paper, several types of experiments were performed.The first set of experiments consisted in non-destructive tests (i.e. Acoustic Emission (AE) measurements) performed to determine the velocity of P-waves through hardened concrete samples. This velocity was then used to calculate the dynamic elastic modulus of concrete samples. These P-wave tests were performed by using a MISTRAS-2001 AE system manufactured by Physical Acoustics Corporation (PAC) to record AE signals artificially generated from the breaking of a Pencil lead (Hsu-Nielsen source) at the ends of concrete cylindrical samples. Full details on the measurement procedure can be found in ref. The second type of experimental data was obtained from image analysis of internal sections. This work aimed at investigating the microstructural characteristics of hardened concrete samples: the microstructural characteristics length scales  and 0.63 m3/m3 for the F-concrete. All the concrete samples were cylinders with a constant height-to-diameter ratio, For our experimental investigations, 539 concrete specimens of 3 different concrete mixtures and 4 different sizes were fabricated. Twelve (12) of these concrete samples were selected for the microstructural characterization from image analysis  and the 2.3(.txt) files of stress-strain data provided as supplementary files. Each text file provides uniaxial compressive stress and strain values recorded during the loading test of each concrete sample. The names of this text file and of the “Sample ID” in the 12 tables (from txt files and for the identification of the concrete samples (i.e. Sample ID) is XY-Z-K. “X” indicates the concrete mixture and can be either F, M, or C (see above). “Y” is the diameter of the concrete cylindrical sample (in cm) and can be either 4, 7, 11 or 16. “Z” is related to the batch number. For each concrete mixture, all the 40 × 80- and 70 × 140 mm samples were produced from a single batch. Hence, “Z” is always B1 for these sample sizes. However, for the 110 × 220- and 160 × 320 mm samples it can be either B1, B2, B3, or B4. Indeed, as the result of a limited capacity of the concrete mixer, it was not possible to prepare all the large samples (110 mm and 160 mm in diameter) from a single batch. Instead, four batches were required to prepare all the 110 mm samples, as well as for the 160 mm samples. These different batches can be considered a priori as a source of strength variability for each concrete group, and this has been taken into account in our analysis in Ref. As mentioned above, the dataset herein consists of the text les from  are the The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Participant-reported outcomes are important. Prior MOVE UP reports show ≥5% weight loss was not significantly associated with depressive symptoms but was associated with positive SPPB physical function and the Physical Component Score of the SF-36 HRQOL scale. We examined the SF-36 subscales that showed, a priori, clinically meaningful +5.0-point increases over 13 months, the change in subscales per 5% weight loss, and variability by race. Among all participants (n =240) several subscales show significant pre-post changes [mean (SD)] but only Vitality [+5.6 (15.4)] and Physical Function [+5.0 (16.7)] meet the criterion. Blacks (n = 60) compared to Whites (n = 172) had higher baseline scores on these subscales, were less likely to lose ≥5% (31.7% vs. 59.9%), but mixed regression models indicate that those who did demonstrated a larger change on Vitality  than Whites . Studying weight loss and HRQOL associations in larger minority samples is needed.