With the broad application of high-throughput sequencing and its reduced cost, simple sequence repeat (SSR) genotyping by sequencing (SSR-GBS) has been widely used for interpreting genetic data across different fields, including population genetic diversity and structure analysis, the construction of genetic maps, and the investigation of intraspecies relationships. The development of accurate and efficient typing strategies for SSR-GBS is urgently needed and several tools have been published. However, to date, no suitable accurate genotyping method can tolerate single nucleotide variations (SNVs) in SSRs and flanking regions. These SNVs may be caused by PCR and sequencing errors or SNPs among varieties, and they directly affect sequence alignment and genotyping accuracy.Here, we report a new integrated strategy named the accurate microsatellite genotyping tool based on targeted sequencing (AMGT-TS) and provide a user-friendly web-based platform and command-line version of AMGT-TS. To handle SNVs in the SSRs or flanking regions, we developed a broad matching algorithm (BMA) that can quickly and accurately achieve SSR typing for ultradeep coverage and high-throughput analysis of loci with SNVs compatibility and grouping of typed reads for further in-depth information mining. To evaluate this tool, we tested 21 randomly sampled loci in eight maize varieties, accompanied by experimental validation on actual and simulated sequencing data. Our evaluation showed that, compared to other tools, AMGT-TS presented extremely accurate typing results with single base resolution for both homozygous and heterozygous samples.https://amgt-ts.plantdna.site:8445 and https://github.com/plantdna/amgt-ts, respectively.This integrated strategy can achieve accurate SSR genotyping based on targeted sequencing, and it can tolerate single nucleotide variations in the SSRs and flanking regions. This method can be readily applied to divergent sequencing platforms and species and has excellent application prospects in genetic and population biology research. The web-based platform and command-line version of AMGT-TS are available at The online version contains supplementary material available at 10.1186/s12859-021-04351-w. Simple sequence repeats (SSRs), also named microsatellites or short tandem repeats (STRs), can be widely found in eukaryotic genomes . The seqAll applications of SSRs are based on accurate SSR genotyping methods, and less accuracy may have serious consequences , 10. MorHere, we developed a new open-source microsatellite genotyping strategy that includes an accurate microsatellite genotyping tool based on targeted sequencing (AMGT-TS) and a user-friendly web-based version. AMGT-TS can quickly perform precise SSR genotyping with ultradeep coverage and high locus throughput, and it includes a broad matching algorithm (BMA) that can handle situations with nucleotide variations in the SSR and flanking regions. We also performed a comprehensive assessment of AMGT-TS using internal laboratory testing and simulated data testing. The results showed that AMGT-TS could achieve nearly 100% typing accuracy. Although AMGT-TS developed on plants, which is the focus of our current work, the new method is generic and can be used as a new tool for many biological fields. All completed codes, sample data, and documentation have been submitted to GitHub.bona fide loci according to the reference sequences; then, the SSR regions are determined by the loci’s flanking information; and finally, AMGT-TS obtains accurate SSR genotyping results based on the dissection of read information, such as read number and primary SSR typing.The process of AMGT-TS has three main steps to obtain accurate genotyping information Fig. . For eachttp://hannonlab.cshl.edu/fastx_toolkit/) to remove low-quality data. Then, we perform the "Alignment to loci" processing step with bwa-mem [https://broadinstitute.github.io/picard/) is used to group reads in the same locus together. At the same time, SAMtools [https://github.com/lh3/seqtk) to “Adjust direction,” which flips reverse sequences into forward sequences. After that, we use the BLAST tool [In detail, after obtaining raw sequencing data , we use FASTX  to generate the alignment figure for the reads. The platform grouped the read files to align the heap sequences in Fig. Here, as an example of the AMGT-TS processing workflows, two actual experimental datasets are provided for result 1 and 2 in Fig. j represents the index of the locus, k represents the typing index, and Ramgt represents the reads of the k-th typing of the j-th locus from AMGT-TS.The typing error can be measured in two ways. One is the false-positive rate of SSR typing and the other is the rate of the error reads found for the correct typing result. Equation  can be oSra represents the sum of reads from artificial data and Er represents the error of the reads. Equation  has the same first step as the precise matching algorithm. However, in the second step, BMA directly processes the information of BAM files and uses the Concise Idiosyncratic Gapped Alignment Report (CIGAR) information for each read to mask the classification information, which makes it compatible with the variation within a certain error range and results in better fault-tolerant classification information. As shown in Fig. Class A: We created the SSR_Region according to 3 repeats of the motif of s17883. We then added 35 bp from the left and right flanking sequences of the SSR_region of the reference sequence as Flank_L and Flank_R, respectively. Finally, we added one SNP on the left flank and one SNP on the right flank. There were 2000 artificial reads for this dataset.Class B: We created the SSR_Region according to 5 repeats of the motif (AGCT) and 6 repeats of the motif of s423645. We then added 35 bp from the left and right flanking sequences of the SSR_region of the reference sequence as Flank_L and Flank_R, respectively. There were 1000 artificial reads for this dataset.Class C: We created the SSR_Region according to 4 repeats of the motif (CGCAT) and 3 repeats of the motif (CGCAT) + CACAT + 2 repeats of motif s566749. We then added 35 bp from the left and right flanking sequences of the SSR_region of the reference sequence as Flank_L and Flank_R, respectively. There were 2000 artificial reads for this dataset.Class D: In the SSR_Region of this type, Flank_L and Flank_R are also random bases. With the addition of Random_L and Random_R, the total length was randomly extended to 180 ~ 220 bp. There were 1000 artificial reads for this dataset.Class E: The rule is the same as for Class A, but There were 2000 artificial reads for this dataset. Random_L and Random_R are random bases and the total length was randomly extended to 180 ~ 220 bp.To better simulate different situations, each read was divided into five parts , representing the artificial sequence. The second segment is category information, using 1, 2, 3, 4, and 5 to represent A, B, C, D, and E, respectively. The third is the ordinal number, starting from 1 in each category and ending with the maximum number of entries in the current category. After 10,000 reads were created, they were randomly distributed into FASTQ files.AMGT-TS was verified on Ubuntu Server 14.04.4 LTS and 18.04.2 LTS. AMGT-TS relies on various tools including Bamtools (v2.5.0) , BLAST tAMGT-TS runs on Linux, and Ubuntu 18.04 has been tested. After downloading the code from GitHub, the user needs to install the dependent components as described in the README.md file. The ENV_FILE variable in the launch.sh specifies the location of the configuration file. In the configuration file, the user must configure the corresponding component location. The targeted sequencing sample files are placed in the “working/00_fastq” directory. Under the directory “REF_DIR” of the configuration file is the reference sequence file information for each locus. Once this information is configured, the user can execute the launch.sh file to run the tool. Before running the program, the user can specify the different algorithms: precise or broad. When the tool is finished running, a log file will be generated. In the “working/04_reads” directory, the locus typing information of the whole sample is present. In the directory of each locus is the typing information of the current loci and reads corresponding to each type. For each subheap read file, the user can use the Reads Alignment tool for a graphical presentation.Currently, no suitable genotyping method can achieve tolerance of single nucleotide variations (SNVs) in the SSRs and flanking regions, which may be caused by PCR and sequencing errors or SNPs among varieties and can directly affect the sequence alignment and genotyping accuracy. As shown in Fig. To address this issue, in this study, we developed a broad matching algorithm (BMA) that can quickly and accurately achieve SSR typing for ultradeep coverage and high-throughput loci with SNV compatibility and grouping of the typed reads for further in-depth information mining. We also designed the AMGT-TS tool incorporating the BMA for targeted microsatellite genotyping. Below, we tested the AMGT-TS tool using both experimental data and simulated data. We also compared AMGT-TS with other SSR-typing tools, as well as the popular commercial SSR-typing software, NextGENe.We used three genetically related samples to map the genotyping information of 50 loci  from B73 , using three sets of Ion Torrent sequencing data . The remaining genotypes tend to be caused by amplification stutter or sequencing error. Take two loci as examples, as shown in the Additional file Usually, for ultradeep sequencing, the prominent peak(s) will be considered the Previous tools based on targeted sequencing can only identify consecutive SSR motifs. They cannot deal with cases where there is variation in the SSR region  , 24. In In conclusion, the BMA and AMGT-TS tools provide an integrated strategy for accurate microsatellite typing for ultradeep coverage and high-throughput analysis of loci with SNV compatibility and grouping the typed reads for further in-depth information mining. With the broader application of next-generation sequencing techniques and the current application of AMGT-TS to divergent sequencing platforms and species, we expect that AMGT-TS will have excellent application prospects in genetic and population biology research in the future.Additional file 1 : Figure S1. Artificial read composition design; Figure S2. Comparison of targeted sequencing results and resequencing results (8 samples/21 loci). This figure shows a comparison of targeted sequencing results analyzed by AMGT-TS and resequencing results (8 samples/21 loci). The abscissa is the name of each sample. The orange ordinate represents the number of loci that were compared. The green ordinate represents the same number of compared loci. Loci with missing or incomplete data were not compared. In the figure above, refer to Table S1 for the corresponding resequencing data. For the corresponding data of targeted sequencing results analyzed by AMGT-TS, please refer to Table S2. For the loci information, please refer to Table S5; Figure S3. The situation with SNP in the SSR and flanking regions; Figure S4. Comparison of SSR genotyping results between AMGT-TS and NextGENe; Figure S5. SSR typing results of two representative loci by AMGT-TS; Table S1. Genotyping information for three example loci; Table S2. This table shows the results of genotyping of 50 loci from Figure 3; Table S3. Data of Figure S2 - Resequencing data of 8 samples; Table S4. Data of Figure S2 - Targeted sequencing results analyzed by AMGT-TS; Table S5. Analysis results of simulated data typed by the precise and broad algorithm; Table S6. Locus information from Maize B73 reference genome for simulation; Table S7. Four simulated situations to test SSR-typing tools.Additional file 2 : Table S8. SSR typing results of 484 evaluated SSR loci in B73 by AMGT-TS; Table S9. SSR typing results of 484 evaluated SSR loci in B73 by NextGENe; Table S10. SSR typing results of 484 evaluated SSR loci in Jing724 by AMGT-TS; Table S11. SSR typing results of 484 evaluated SSR loci in Jing724 by NextGENe; Table S12. SSR typing results of 484 evaluated SSR loci in Jingke968 by AMGT-TS; Table S13. SSR typing results of 484 evaluated SSR loci in Jingke968 by NextGENe.


Complete atrioventricular block (AVB) is a life-threatening condition that usually occurs in elderly people with organic heart disease. We herein describe a rare case of complete AVB in a young man with hypertrophic obstructive cardiomyopathy (HOCM) complicated by cholecystitis and cholangitis. Both cardio-biliary reflex and alcohol septal ablation (ASA) can cause conduction block, but the latter is often irreversible. However, their simultaneous occurrence in a patient has not been reported.A 31-year-old man presented with acute cholecystitis and cholangitis and complete AVB, which had been diagnosed at a local hospital on the third day after onset. On the fourth day, he was transferred to the emergency department of our hospital because of persistent complete AVB, although his abdominal pain had been partially relieved. An echocardiogram showed a remarkably elevated left ventricular outflow tract (LVOT) gradient (105.2 mmHg) despite the performance of ASA 9 years previously. The abdominal pain gradually disappeared, and normal sinus rhythm was completely recovered 11 days after onset. We determined that cardio-biliary reflex was the cause of the AVB because of the absence of other common causes. Finally, the patient underwent implantation of a permanent pacemaker to reduce the LVOT obstruction and avoid the risk of AVB recurrence.Cholecystitis is a rare cause of complete AVB, which is a difficult differential diagnosis when complicated by HOCM after ASA. Clinicians should be alert to the possibility of cholecystitis in patients with abdominal pain and an unknown cause of bradycardia, complete AVB, or even sinus arrest.The online version contains supplementary material available at 10.1186/s12872-021-02165-5. Complete atrioventricular block (AVB) is characterized as independent atrial and ventricular activity due to anatomic or functional disorders of the conduction system. Complete AVB may have physiologic, pathophysiologic, or iatrogenic etiologies, some of which are reversible . Acute cThis is the first report of 9 days of complete AVB complicated by biliary tract infection after ASA. In this mixed situation, a thorough clinical history and examination were important for accurate diagnosis and treatment.A 31-year-old man presented with acute postprandial abdominal pain, vomiting, and progressive dyspnea until he was unable to walk on 5 January 2020 , neutrophil percentage of 65.7%, alanine aminotransferase level of 257 IU/L , aspartate aminotransferase level of 110 IU/L , total bilirubin level of 54 µmol/L , brain natriuretic peptide level of 2717 pg/mL , C-reactive protein level of 19 mg/L , and procalcitonin level of 0.18 ng/mL . Serum electrolytes, troponin I, thyroid hormone, multiple blood cultures, antinuclear antibody, anti-dsDNA, complement C3/C4, rheumatoid factor, and anti-streptolysin O were all normal or negative. ECG showed complete AVB with junctional escape at 40 beats/min  of complete AVB complicated by acute biliary tract infection and HOCM after ASA. Cholecystitis causes multiple ECG abnormalities, and close monitoring and management are necessary to avoid misdiagnosis and possible complications. Further research is needed to elucidate the mechanism of the cardio-biliary reflex.Additional file 1: Figure S1.Cardiac magnetic resonance imaging.Additional file 2: Figure S2.Chest radiograph.Additional file 3: Figure S3.Electrocardiogram in 2011.


Suboptimal interaction with patient data and challenges in mastering 3D anatomy based on ill-posed 2D interventional images are essential concerns in image-guided therapies. Augmented reality (AR) has been introduced in the operating rooms in the last decade; however, in image-guided interventions, it has often only been considered as a visualization device improving traditional workflows. As a consequence, the technology is gaining minimum maturity that it requires to redefine new procedures, user interfaces, and interactions. The main contribution of this paper is to reveal how exemplary workflows are redefined by taking full advantageof head-mounted displays when entirely co-registered with the imaging system at all times. The awareness of the system from the geometric and physical characteristics of X-ray imaging allows the exploration of different human-machine interfaces. Our system achieved an error of 4.76 ± 2.91 mm for placing K-wire in a fracture management procedure, and yielded errors of 1.57 ± 1.16° and 1.46 ± 1.00° in the abduction and anteversion angles, respectively, for total hip arthroplasty (THA). We compared the results with the outcomes from baseline standard operative and non-immersive AR procedures, which had yielded errors of  and , respectively, for wire placement, and abduction and anteversion during THA. We hope that our holistic approach towards improving the interface of surgery not only augments the surgeon’s capabilities but also augments the surgical team’s experience in carrying out an effective intervention with reduced complications and provide novel approaches of documenting procedures for training purposes. I.Interventional image guidance is widely adopted across multiple disciplines of minimally-invasive and percutaneous therapies –4]. Des. Des4]. Surgical navigation and robotic systems are developed to support surgery with localization and execution of well-defined tasks –8]. Tho. Tho8]. As the expectations from surgical outcomes increase with more advanced technologies introduced in the ORs, the communication between the surgeon, crew, and the information becomes an important concern. Ineffective communication leads to increase of surgery time, radiation, and frustration to a point where in fluoroscopy-guided procedures, instead of the X-ray technician, the surgeons may reposition the scanners to ensure the task-defined views are optimal , 13]..13].i.e. surgeon, anesthesiologist and staff to communicate and access information. This demands user-centric designs that can also accommodate fluid movement of information and surgical inference across the entire team.To bridge the inefficiency gaps in surgical workflows, researchers have investigated the importance of human factor considerations in improving the usability of surgical data –16]. Re. Re16]. Augmented reality (AR) solutions have gained popularity in computer-integrated surgeries, as they can provide intuitive visualizations of medical data directly at patients’ site. Early works on surgical AR focused largely on multi-modal fusion of information and provided display-based overlays –21]. Su. Su21]. The emergence of commercially available optical see-through head-mounted display (HMD) systems has led to development of AR solutions for various image-guided surgical disciplines, including percutaneous vertebroplasty, kyphoplasty, lumbar facet joint injection, orthopedic fracture management, bone cancer treatment, total hip arthroplasty (THA), interlocking nailing, cardiovascular surgeries, and surgical education , 26]–[3–[326]–33.et al. suggested a flexible marker-based surgical AR methodology which only required the marker to appear in the X-ray beam during the image acquisition, and was removed immediately after ..et al. si.e. K-wire placement in fracture care surgery, and acetabular cup placement in THA.This manuscript introduces the methodology and usability of a novel spatially-aware concept that enables immediate interaction with the medical data and promotes team approach where all stake-holders share a unified AR experience and communicate effectively. Our methodology exploits the viewing frustum of the imaging devices and human observers in the operating room , and proII.Our main contributions are the collaborative AR concepts using spatiotemporal-aware flying frustums  that enaA.Visual data from cameras contain a wealth of information that can be used for simultaneous localization and mapping (SLAM). Visual SLAM is an important ingredient in our AR-based interaction recipe that enables co-localization of augmented users and the imaging device, which we will refer to as imaging observer, in a shared operating room environment. Marker-free co-localization will enable the 3D information to easily propagate through different bodies and be spatially-registered for all HMD users.α and β using the environment map M can be estimated by minimizing the following reprojection function:Iα and Iβ, and P is the projection operator. In direct SLAM, the features include all pixels, and in indirect SLAM, the features are a sparse set of keypoints in the image. In α and β using only the features present in the images. This step, also known as environment tracking, has become an standard part of most AR applications.In the first step, each AR user is localized within the environment. The relative pose between two frames i.e. OR coordinate system, and every other member of the AR session will share their pose in a master-slave configuration with respect to this OR frame  mm.2) per surgeon, whereas 14 fluoroscopic images with a dose of 1.96 cGY(cm2) were acquired during SOP. With the AR system, the average errors were 1.57° and 1.46° for the abduction and anteversion angles, respectively. Based on the 95% confidence interval, the mean error for abduction and anteversion are between [0.72° – 2.44°] and [0.72° – 2.20°], respectively. Under SOP the respective angles were 4.76° and 4.78°. p = 0.05. We tested the results of our immersive AR system against the results from the NI-AR system and SOP for both the K-wire insertion procedure and the acetabular cup placement. We did not test the number of acquired X-rays, since in these experiments they turned out to be a constant value for each procedure.Correspondingly, we present the outcome for the acetabular cup placement procedure with the immersive AR system in IV.i) the placement of K-wires through tubular structures for fracture repair tasks, and ii) placement of acetabular components into the hip socket for total hip arthroplasty. We selected these two high volume procedures among the many other applications which can be enabled by our interactive AR system, as they each represent a class of common orientational alignment and localization tasks that are prevalent across different fields of image-guided surgery.We evaluated our spatially-aware AR system in two clinically relevant procedures, For the K-wire insertion procedure, the immersive AR system performed significantly faster than the conventional SOP, yielding less than a fifth of the time . Table I2), compared to the  significantly lower dose of 0.255 cGY(cm2), which was emitted during the AR procedures. The RGBD-CBCT system in [2).With the AR system every surgeon used exactly 2 X-ray images, which were the 2 images required for procedure planning. Despite explicitly allowing them to take as many radiographs as they desire, no one of the surgeons requested additional X-ray images. As mentioned above, during SOP, surgeons inserted the K-wires with an average number of 40.86 fluoroscopic images and with an average dose of 4.43 cGY, coFinally, evaluating the outcome of the procedure with regard to the drilling error, AR (4.76 mm) outperforms RGBD-CBCT (5.13 mm), both being marginally worse than SOP (4.61 mm). The differences in accuracy were not statistically significant. Considering that we only instructed the surgeons to drill through the tube and not precisely through the center of the tube, we regard these difference as negligible. It is important to note that, our AR system performed similar to the conventional X-ray method in terms of accuracy, while reducing time by a factor of 5, number of fluoroscopic acquisitions by a factor of 20, and the radiation dose by a factor of 17.We observed that, in addition to the planning information, the surgeons took multiple other considerations into account while deciding on the insertion path. One of which was direct visualization of the X-ray images that they acquired for planning. Observing the C-arm pose with respect to the patient, and the visualization of the images within their viewing frustum, assisted them in better localizing the target structure. In our setup, tactile feedback did not play a significant role; however, in reducing real fractures, haptic feedback further assists the surgeon in identifying whether the wire is inserted in specific anatomy or not.et al. [The SLAM-based error for HMD is dominantly along the principal axis of the viewer, i.e., direction pointing away from the user to the scene. When the users are presented with a trajectory to follow, we observed that they naturally place their heads in the direction of the target trajectory for optimal visualization and ease of alignment . This coet al. .2), which is significantly lower than with SOP, where the surgeons used an average of 13.75 radiographs with an average dose of 1.96 cGY(cm2), and lower than with NI-AR where one pre-procedure CBCT lead to a dose of 1.83 cGY(cm2). The objective of this procedure was to achieve abduction and anteversion angles of 40° and 15°, respectively, which lie in the clinical safe-zone [A similar trend to the K-wire experiment is observed with the measurements for the placement of the acetabular cup, demonstrating the effectiveness of our AR system, we compare it against SOP and a NI-AR system as presented in . As showafe-zone . The resP2, P6, and P8, who had previous experience with AR HMDs and were familiar with our system performed faster than other users, especially compared to P1 and P7, who had no prior exposure to HMDs. In order to reduce the disparity in acquaintance with the technology, each participant completed a short training in which they were familiarized with the headset and interaction techniques used in our software. Despite the same training session, the differences mentioned above still seem related to experience with AR. We expect the performances to level after users gain more experience with the system. These outcomes are not surprising as any new technology requires time and experience to exploit its full potential.Concerning the planning and execution time, some participants such as “see the planning annotations”. However, if the system wanted to be adapted to other fields, such as interventional neuroradiology — the suitability of resolution, contrast, brightness, frame-rate, etc. may need to be further investigated.In almost all orthopedic procedures, we rarely use live fluoroscopy and rely almost exclusively on still images. Therefore, the frame-rate for these procedures appeared entirely adequate. The resolution also seemed satisfactory for the experiments that we performed. No users complained about not being able to For both procedures the deployment of our AR system lead to a comparable or higher accuracy, fewer X-ray images with a consequently lower radiation dose. For the total time, it has to be noted that our planning time does not include the recording of the X-ray images that were necessary to plan the procedure. This step however can be fully automated, resulting in an immediate availability of the fluoroscopic images.V.We presented the embodiment of a novel interaction concept based on spatiotemporal-aware AR. In our work, we aimed to provide meaningful registration and visualization without the need for tracking patients or tools with outside-in navigation hardware. Instead, we brought intuition to visualization by connecting the viewing frustums of the scanner with the surgical team. For the two orthopedic use cases presented in this manuscript, our immersive AR system demonstrated improvements in time, number of X-ray acquisitions, radiation dose, and outcome during cup placement.The most significant source of error is from the localization of the AR HMD using SLAM. This error is present both during calibration and application. If additional sensors such as IMUs and depth cameras would constraint the tracking algorithm in the future, we can expect improved tracking and smaller drifts.As computer vision methodologies enter the medical and AR domains, the accuracy and robustness of medical AR systems will also increase. This work presents a general end-to-end workflow for integration of AR into orthopedic surgery, which will also observe dramatic improvement as such progress occurs.e.g. The spatiotemporal awareness inherent in AR overhauls the ill-posed communication between the surgeon, staff, and information; A significant advantage of our system is that it is based on 2D C-arm fluoroscopy —- and therefore does not diverge from the standard workflow. The standard C-arm is always present in the OR to take confirmatory images. Consequently, the surgeons are not required to solely rely on the AR system during a procedure. Rather, its value lies in guiding the surgeon to a narrow area of interest and more importantly, to align them with the right trajectory. The surgeon can then seamlessly switch between standard fluoroscopic images and the AR view to guarantee accurate drilling. We believe this is useful, translatable, safe, and novel.“surgical replay”, where the residents can review the surgery, accompanied with its temporal and spatial information including all the X-ray acquisitions and optical point-clouds from the patient site. This enables the medical trainees to identify distinct actions that were taken by the experienced surgeon based upon each image. Access to such 3D post-operative analysis has the potential to dramatically improve the quality of surgical education.An application of AR outside the OR is In a training environment, such as in most academic centers with residents and fellows — this ability for all users to wear the HMD and view the same annotations and AR guidance would be helpful. The more experienced user could ensure that the anatomy had been annotated appropriately and that the AR guidance trajectories are at the correct location.Direct visualization of X-ray images within their corresponding viewing frustums delivers intuition that effectively unites the content of the 2D image with the 3D imaged anatomy. In this setting, images from various perspectives can be grouped within their frustums to form multi- or extended-view representations of the anatomy. The interlocked frustums shown in In this work, we demonstrated that two elements share the AR experience; the first one was the augmented surgeon, and the second one was the imaging observer which was the C-arm scanner that used a calibrated HMD attached to it. With the same design, any number of other users can connect to the same AR session, collaborate, and share an experience. Though assessment of each of these concepts presented in Despite that our solution delivers spatial awareness, it should not be regarded as a surgical navigation system. This is because marker-less tracking, currently, cannot deliver the level of accuracy achieved by marker-based surgical navigation or robotic systems. It is important to note that, as most AR systems display the data anchored to the surrounding environment, tracking with respect to the environment has become an inherent component in modern AR systems. In an optical see-through HMD, the integrated camera sensors are used to run SLAM in the background, construct a dynamic map from the environment, and localize the HMD within that environment. Despite that this marker-less mapping and localization cannot deliver the same level of accuracy as the external navigation systems with explicit tracking nature, it delivers spatial awareness for the visualization of information. Hence, we regard our solution as a spatially-aware advanced visualization platform that enhances the interaction across the surgical ecosystem and promotes effective collaboration and team approach.i) create semantic understanding from the surgical environment and augment surgeon’s intelligence, and ii) enhance the spatial mapping and co-localization, thus improving the stability of marker-less AR systems. Lastly, future integration of eye tracking systems into HMDs can circumvent the internal eye-to-display calibration, adjust the rendering based on each user, and replace unnecessary gestures with gaze-based interaction.The widespread adoption of human-centerd AR in interventional routines requires careful considerations regarding surgeons’ experience. The interaction with the virtual content using intuitive hand gestures, and resolving the perceptual ambiguities between real and virtual that occur due to vergence-accommodation conflict, can greatly contribute to the acceptance of AR technologies. Furthermore, development of artificial intelligence strategies can Regarding sterilization, since the HMD on the user’s head does not enter the operating field, it could be viewed in a similar way to surgical loupes, surgical hoods, headlamps — all of which are used daily and do not need to be sterilized.Our solution that promotes the team approach, also demands the entire crew to get trained appropriately and be able to interact with the system comfortably. Therefore, time, money, and effort need to be spent to prepare the surgical team for such a digital transformation. From a hardware standpoint, other than HMDs, no additional hardware is required in the OR. Therefore, the device cost is lower than complex navigation-based or robotic solutions.As shown in this paper, the introduction of new technology requires a user-centric design for its full integration into the clinical workflow. The ultimate goal may be the discovery of new surgical workflows enabled by the introduction of novel technology. This goal could only be achieved with a close and extended partnership between surgeons and technical experts, as well as the full integration of the advanced technology in the broad spectrum of surgical teaching, training, planning, workflow, and documentation.


In the progression of castration-resistant prostate cancer (CRPC), the androgen receptor (AR) that serves as a transcription factor becomes the most remarkable molecule. The transcriptional activity of AR is regulated by various coregulators. As a result, altered expression levels, an aberrant location or activities of coregulators promote the development of prostate cancer. We describe herein results showing that compared with androgen-dependent prostate cancer (ADPC) cells, AR nuclear translocation capability is enhanced in androgen-independent prostate cancer (AIPC) cells. To gain insight into whether AR coregulators are responsible for AR translocation capability, we performed coimmunoprecipitation (CO-IP) coupled with LC-MS/MS to screen 27 previously reported AR cofactors and 46 candidate AR cofactors. Furthermore, one candidate, myosin heavy chain 9 (MYH9), was identified and verified as a novel AR cofactor. Interestingly, the distribution of MYH9 was in both the cytoplasmic and nuclear compartments yet was enriched in the nucleus when AR was knocked down by AR shRNA, suggesting that the nuclear translocation of MYH9 was negatively regulated by AR. In addition, we found that blebbistatin, an inhibitor of MYH9, not only promoted AR nuclear translocation but also enhanced the expression of the AR target gene PSA, which indicates that MYH9 represses nuclear AR signaling. Taken together, our findings reveal that MYH9 appears to be a novel corepressor of AR plays a pivotal role in the progression of CRPC. Despite decreasing rates of some cancers amenable to early detection, prostate cancer (PCa) is the second deadliest cancer and the incidence remains stubbornly the highest in the American for over two decades . AndrogeIn the progression of CRPC, AR that serves as a transcription factor, becomes the most remarkable molecule. Interestingly, AIPC cells continuously rely on AR and its signaling cascades, whereas other pathways such as alternative pathways also promote PCa to a more aggressive stage . The abe2+ leading to a significant enhancement of growth in PCa cells , which can either enhance or reduce AR transactivation. AR coactivators, mainly including the p160 steroid receptor coactivator (SRC) family , chaperone proteins, filamentous actin (f-actin)-binding proteins, DNA repair proteins and ubiquitin ligases, were crucial for PCa progression as well as AIPC conversion , 13. OneCa cells . In addiCa cells . AlthougCa cells , 16, theTo mimic patients undergoing ADT treatment, we had already generated two androgen-independent cell lines, LNCaP-AI cells and LNCaP-AI+F cells derived from androgen-sensitive LNCaP cells cultured under androgen-depleted conditions . Our pre2, while LNCaP−AI cells were grown in phenol red−free Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12) medium with 10% dextran−charcoal stripped FBS . Another cell line, LNCaP-AI+F, was induced as LNCaP-AI cells but was maintained in phenol red−free DMEM/F12 with 10% CS−FBS containing 10 μM hydroxyflutamide for more than 100 passages and maintain throughout all experiments. DHT , hydroxyflutamide tablets  and (-)-blebbistatin  were commercially obtained. DHT was dissolved in alcohol at a concentration of 10 mM as a stock solution and was kept in a light-resistant container. Hydroxyflutamide tablets were also dissolved in alcohol at a concentration of 10 μM. Blebbistatin was dissolved in DMSO at a final concentration of 10 μM.LNCaP cells, a typical androgen-dependent prostate cancer cell line, were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences ; LNCaP-AI cells, an androgen-independent cell line, were generated as previously described . LNCaP cTotal RNA was extracted from 10 nM dihydrotestosterone (DHT)-treated or equivalent ethanol-treated cells for the indicated times using TRIzol Reagent  according to the manufacturer’s instructions. For reverse transcription, 1 μg of total RNA was used with a Transcriptor First Strand cDNA Synthesis Kit  according to the manufacturer’s procedure. mRNA expression was analyzed by quantitative reverse transcription-PCR (RT-qPCR) using FS Universal SYBR Green Master Mix . The following primers were applied in this study:AR Forward: 5-’GCCACTCAGACCCACTTAGC-3’AR Reverse: 5-’CCTCACTCTTCGTCCACATCG-3’PSA Forward: 5-’CCTAGATGAAGTCTCCATGAGCTAC-3’PSA Reverse: 5-’GGGAGGGAGAGCTAGCACTTG-3’MYH9 Forward: 5-’ACAGGTCGTCGGACCGAGAA-3’MYH9 Reverse: 5-’TAGGAGCTGACTTCGCGGT-3’GAPDH Forward: 5-’CTGAGCACCAGGTGGTCTC-3’GAPDH Reverse: 5-’AGGGACTCCCCAGCAGTGAG-3’-ΔΔCt relative quantitative analysis.ABI 7500 Real-Time PCR system  was used to perform qPCR. Three independent samples were collected in triplicate and analyzed using 2Collected cells were rinsed twice with precooled PBS and extracted by NE-PER™ Nuclear and Cytoplasmic Extraction Reagents  strictly in accordance with the instructions. After lysing in CERII combined with proteinase inhibitor cocktail , the samples were centrifuged at 16,000 rpm for 10 min, and the supernatant was collected as the cytoplasmic fraction. Likewise, the nuclear extract was collected after NER was added.Three PCa cell lines were seeded for 24 h in FBS (LNCaP cells) or CS-FBS (LNCaP-AI cells) or CS-FBS added with 10 μM hydroxyflutamide (LNCaP-AI+F cells) before 10 nM DHT or equal amount of ethanol treatment and harvested at the indicated time points . Five hundred micrograms of total protein extracted by RIPA lysis buffer  were immunoprecipitated with AR or MYH9 antibodies. Control immunoprecipitation was performed using rabbit normal IgG (Millipore Corporation). The antigen-antibody mixtures protected by proteinase inhibitor cocktail were incubated with gentle rotation overnight at 4°C. The antigen-antibody complexes were incubated with PureProteome Protein A Magnetic Beads  (2 μl of beads to 1 μg of antibody) at 4°C for 2 hours with gentle rotation and then separated by a magnetic frame. After washing three times with 500 µl of 1×PBS (pH 7.4) containing 0.1% Tween-20, the pull-down complexes were eluted by 60 μl of citrate buffer  and neutralized by 5 µl of glycine buffer  for LC/MS/MS detection or were eluted by boiling the beads in 5× loading buffer  and were subjected to western blotting or Coomassie brilliant blue staining .Proteins were prepared from (1) whole-cell lysates of LNCaP or LNCaP-AI cells with or without 10 nM DHT treatment; (2) cytosolic or nuclear fraction extracts; and (3) CO-IP elution. Proteins were diluted with 5× loading buffer. Thirty microliters of each diluted sample were boiled for five minutes, separated on 10% SDS-PAGE gels, and transferred to PVDF membranes . The membranes were blocked with 5% bull serum albumin for two hours at room temperature, incubated with primary antibodies recognizing AR , MYH9 , or GAPDH  overnight at 4°C, and finally detected with anti-rabbit IgG-HRP  or anti-mouse IgG-HRP . The protein bands were visualized using chemiluminescent HRP substrate (Millipore), followed by exposure to ChemiDoc (Bio-Rad).https://www.matrixscience.com) for protein identification. Search parameters for MS/MS database were as follows: one missed cleavage in the trypsin digests was allowed and a mass tolerance of 0.05 Da (the tolerance was “± 0.05 Da”) was permitted for intact peptide masses and ±0.1 Da for fragmented ions. Potential variable modifications were Gln → pyro-Glu (N-term Q), Oxidation (M), Deamidated (NQ) and Carbamidomethyl (C) as fixed modifications. Charge distribution of the peptides were set to +2, +3 and +4. Qualitative data analysis was performed with MASCOT using a 99% confidence interval. We report protein scores as Mascot score (-10*LOG10(P), where P is the absolute probability). For example, a 1% probability that the peptide spectrum match is a random event would translate into a Mascot score of 20. Cluster of orthologous groups of proteins , a system for automated detection of homologues among the annotated genes of several completely sequenced eukaryotic genomes, were applied for functionally annotation and classification of all the identified proteins. We mapped the cofactor-interacting proteins to the protein-protein interaction (PPI) network from the String (https://string-db.org/) website and extracted all cofactor-protein interactions. All proteins in the MS results were screened by text mining for known AR cofactors and possible AR cofactors.The eluate mentioned above was electrophoresed by SDS-PAGE, digested in gel and eluted before being loaded for liquid chromatography coupled with tandem mass spectrometry (LC−MS/MS)  detection. MS/MS spectra were processed and queried against the NCBInr protein database with taxonomy filter set for Homo sapiens (91464 sequences) using the MASCOT algorithm . Finally, immunofluorescence was observed with a fluorescence microscope .Cells on coverslips  were washed briefly in PBS and fixed for 10 min in cold 4% paraformaldehyde. Each step was followed by three washes with PBS throughout the procedure. Cells were treated with 0.1% Triton X-100 (Biosharp) for 10 min at room temperature. Blocking was performed for 60 min at room temperature in 10% normal goat serum in PBS, followed by incubation overnight at 4°C with AR or MYH9 antibody at 1:200 in 1.5% goat serum in PBS. The secondary antibody was DyLight 488 goat anti-rabbit IgG conjugate  or DyLight 594 anti-mouse IgG conjugate  at 1:1,000 in 1.5% goat serum in PBS and incubated in the dark for 60 min at room temperature. Coverslips were then rinsed three times with PBS and incubated in DAPI  at 1:1,000 in PBS for 5 min in the dark. The coverslips were carefully removed, rinsed briefly with ddHAR RNA interference assays were performed as previously described . BrieflyThe values were expressed as the means ± S.D. for triplicate experiments. Date analysis was performed using one-way ANOVA followed by Dunnett’s test for more than three groups or LSD (least significance difference) test for three groups and Student’s two-tailed t-test. Statistical analysis was performed by using SPSS 22.0  and a value of P<0.05 or 0.01 was accepted statistically significant.Colocalization and quantification of fluorescence together with density of protein bands were analyzed with ImageJ 1.52i software . Spots of poor quality in fluorescence pictures were removed from further analysis by visual inspection. Pearson’s R value (above threshold) was applied as colocalization level for AR and MYH9.Our previous study demonstrated that LNCaP-AI cells undergone long-term androgen deprivation exhibited higher proliferation rate and more aggressiveness than LNCaP cells . AdditioUpregulated expression of PSA indicates that AR signaling pathways are activated, suggesting that not AR expression but AR nuclear translocation is potentially enhanced in LNCaP-AI cells. We consequently analyzed whether AR translocation activity was enhanced in LNCaP-AI cells compared to LNCaP cells. The cells were subcultured in media supplemented with CS−FBS for 24 h (0 h) then treated with DHT or ethanol as a vehicle control for the indicated time  before AR was observed by both immunofluorescence microscopy  annotations were performed. The top 5 amount of proteins in COG classes were as follows: A-energy production and conversion; B-translation, ribosomal structure and biogenesis; C-posttranslational modification, protein turnover, and chaperones; D-general function prediction only; and E- cytoskeleton Figure 4http://string.embl.de/) website, 73 different target proteins were identified in accordance with the protein score, molecular function, and metabolic pathways. These proteins were further categorized according to their function as heat shock proteins, cytoskeletal proteins, UBSs, RNA polymerase II-related proteins, transcription factors and other proteins that regulate the stability, translocation and transcription of AR. The proteins are listed in In addition, based on published studies together with protein-protein interaction analysis on the String (Since MYH9 obtains the highest protein score (1914.82) in MS identification among the 46 candidates of AR interactors, we suggest that MYH9 might be a novel binding protein of AR. On the other hand, as shown in To further determine the interaction between AR and MYH9, lysates of LNCaP-AI cells were pulled down by CO-IP assay and stained with Coomassie Brilliant Blue. As We have verified that MYH9 is a novel AR cofactor; therefore, it is necessary to determine whether AR and MYH9 regulate each other, especially in AR nuclear translocation, and what role MYH9 plays in AR signaling pathways. We previously constructed AR-knockdown LNCaP-AI cells with AR shRNA lentivirus vectors (LNCaP-AI-I) or scrambled lentiviral particles (LNCaP-AI-NC). For the purpose of understanding MYH9 expression in ADPC and AIPC cells, both mRNA and protein levels were tested. MYH9 mRNA was systematically upregulated in LNCaP-AI-I cells compared with that in LNCaP-AI-NC cells, and MYH9 mRNA was decreased in both LNCaP-AI-NC cells and LNCaP-AI-I cells but not in LNCaP cells when stimulate with 10 nM DHT , and MYH) and reduced MYH9 protein expression , and Zhejiang Medical and Health Science and Technology Project (2019RC039).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hyperglycemia is a common complication of Cushing syndrome (CS).We aimed to determine the impact of curative procedure on hyperglycemia and its management in patients with CS.1c (HbA1c), intensity of hyperglycemia therapy, and improvement of hyperglycemia.This retrospective longitudinal cohort study took place 2000 to 2019 in a referral center among adults with endogenous CS and hyperglycemia. Main outcome measures included glycated hemoglobin A1c was 6.9% , with 41 (24%) patients not on any therapy for hyperglycemia, 93 (52%) on oral medications, and 64 (37%) on insulin . Following CS remission, at the end of follow-up (median 10.5 months), 37 (21%) patients demonstrated resolution of hyperglycemia, 82 (47%) demonstrated improvement, and 55 (32%) had no change or worsening in hyperglycemia. At the end of follow-up, HbA1c decreased by 0.84% (P < .001) and daily insulin dose decreased by a mean of 30 units (P < .001). Biochemical hypercortisolism severity score , and CS subtype , but not type of hyperglycemia (diabetes vs prediabetes: OR of 2.1 [0.9-4.9]) were associated with hyperglycemia improvement at the end of follow-up.In 174 patients with CS , baseline median HbATwo-thirds of patients with CS and hyperglycemia demonstrate resolution or improvement of hyperglycemia after a curative procedure. Close monitoring during CS recovery is needed to ensure appropriate therapy modification. Cushing syndrome (CS) develops in patients with chronic exposure to endogenous or exogenous cortisol excess . EndogenDiabetes mellitus (DM) has been reported in up to 20% to 48% of patients with CS, and hyperglycemia (DM and prediabetes) in up to 38% to 84% of patients with CS , 6. In pThe goal of our study was to determine the effect of curative procedure on the extent of hyperglycemia and its management in patients with CS. We further aimed to determine the baseline factors  that are associated with the improvement of hyperglycemia at follow-up.2 body surface area for subgroup analysis. Eligible patients were identified through review of medical records of patients with the diagnosis of CS  were collected. Based on the available clinical and laboratory data, each patient was assigned a biochemical and clinical severity of CS classification score, as previously described  (Supplem1c [HbA1c]) and medications used for management of DM. Data were collected before the curative procedure (baseline), and at any available postoperative time points for each individual patient. We collected hyperglycemia data during glucocorticoid taper, with the final postoperative workup when the patient was off glucocorticoids completely, or on stable glucocorticoid chronic replacement therapy after bilateral adrenalectomy.Data on hyperglycemia included biochemical data , without any need for antihyperglycemic therapy. Improvement was defined as one of the following: 1) improvement in HbA1c and/or fasting glucose concentrations at the end of follow-up while on stable antihyperglycemic therapy; or 2) the decision to decrease the dose and/or number of antihyperglycemic medications/insulin by the treating physician from whom quantitative data were not available .Our primary end point was improvement or resolution of hyperglycemia following remission of CS with subgroup analyses based on the CS subtype. Resolution of hyperglycemia was defined as the absence of hyperglycemia (based on HbA1c (Δ HbA1c) and the intensity of hyperglycemia management  during follow-up compared to baseline. Subgroup analysis for Δ HbA1c included 1) patients on stable management for hyperglycemia, 2) patients who needed a decrease in the intensity of hyperglycemia management, and 3) patients who needed an increase in the intensity of hyperglycemia management during the follow-up that followed the curative procedure.Secondary end points included changes in HbAP less than .05.Data were analyzed using JMP 14.1.0 software (SAS Inc). All continuous data were summarized as median and ranges, while categorical data are summarized as a number (percentage). Associations between variables were assessed using the Kruskal-Wallis test for continuous variables and the chi-square test for categorical variables, as appropriate. Multivariable analysis was performed to determine independent predictors of improvement in hyperglycemia. Intraindividual differences were assessed using the matched pair analysis for continuous variables. Statistical significance was defined as P = .001) and included more women . Patients with pituitary CS also reported the longest duration of CS-related symptoms before diagnosis  (see P ≤ .001), the clinical severity score was highest in patients with pituitary CS  , and 127 (73%) were women. Diagnosis was pituitary CS in 106 patients (60.9%), ectopic CS in 25 patients (14.4%) and adrenal CS in 43 patients (24.7%) . Patient001) see . WhereasP = .02) (P = .03) . A higher number of patients with pituitary CS were treated with oral medications  , prediabetes , and type 1 DM . Duration of hyperglycemia before presentation with CS was highest in adrenal CS  . Patient.03) see . At base.04) see . No diffIn patients treated with oral medications (n = 93), metformin was the most used , followed by sulfonylurea drugs  (Supplementary Table 5 ). Other At the time of surgery, an inpatient specialist diabetes consulting service was used in the care of 97 patients (55.7%), more commonly if patients had ectopic CS, in those with more severe hyperglycemia, and those treated with insulin .Twelve patients (6.9%) had a significant program change that intensified their discharge DM regimen ; the specialist consulting service was involved in 11 of these cases (91.7%). Other changes of intensified pharmacotherapy noted at discharge included 5 patients (2.9%) who had their noninsulin medication dosage(s) increased, and 5 patients (2.9%) who had their insulin doses increased. All other patients did not have a program change (whether on medications or not) or had their medication dosage(s) reduced at the time of discharge.2 body surface area was 8.1 (interquartile range = 0-13.4) at the time of final follow-up.Patients were followed for hyperglycemia management for a median of 10.5 months  after achieving CS remission. At the time of final follow-up, 102 patients (58.6%) were either no longer treated with glucocorticoids or reached a stable glucocorticoid replacement dose. Median hydrocortisone mg/m1c had decreased by a mean of 0.84%; P ≤ .001 (1c decrease (Δ HbA1c) was associated with weight loss (Δ body mass index [BMI]) (P ≤ .001) (P ≤ .001) P = .02).Subtype of hyperglycemia, biochemical severity of hypercortisolism, and subtype of hypercortisolism were associated with improvement of hyperglycemia during follow up . When coP ≤ .001 . The degx [BMI]) . In 60 p ≤ .001) . In 42 p1c decreased gradually during follow-up (1c decreased by a mean of –0.5% (median of –0.3%) by the end of follow-up (1c decreased by a mean of –1.2% (median of –1.0%) (1c of +0.2% (median of +0.1%) (1c was 6.9% (5.5%-10.7%). Univariate and multivariable analysis did not demonstrate a significant factor in this group that was associated with the need to increase therapy (data not shown). Notably, no differences in the initial postoperative daily glucocorticoid dose, in the number of patients still treated with glucocorticoids, or in the median daily hydrocortisone dose at the end of follow-up were found between the groups based on the intensity of hyperglycemia management  (Table 4). Overall, HbAollow-up . In patiollow-up . In 71 pf –1.0%) . Only 17f +0.1%) . This co Table 6 ).2 body surface area, no differences in the decrease of HbA1c or the proportion of patients with improved hyperglycemia were observed . In ano Table 9 ).On univariate analysis, age, sex, and BMI were not associated with improvement of hyperglycemia following CS remission . In contIn this study, we characterized the presentation at baseline and the evolution of hyperglycemia in patients with CS following the curative procedure and CS remission. We showed that two-thirds of patients demonstrated resolution or improvement of hyperglycemia during follow-up, and more than half were able to decrease the dose or the number of medications used for hyperglycemia. Patients with ACTH-dependent CS and those with a higher biochemical severity score were more likely to have improved hyperglycemia during follow-up.1c or fasting glucose were found between the CS subtypes; however, patients with pituitary CS were more commonly treated for hyperglycemia and with a higher number of hyperglycemia agents than patients with other CS subtypes. Potential explanations for this finding include a higher duration of hypercortisolism in patients with pituitary CS before diagnosis, along with a high proportion of patients with severe biochemical and clinical CS severity scores. Patients with pituitary CS also had the highest BMI, further contributing to the severity of hyperglycemia at baseline. While several studies reported on the differences in the prevalence of hyperglycemia among CS subtypes [We found that at the time of diagnosis, no differences in baseline HbAsubtypes , 6, 15, 1c and/or a decrease in the dose or number of medications used to treat hyperglycemia. Applying this definition, 47% of our patients demonstrated improvement in hyperglycemia by the end of follow-up. We showed that the overall HbA1c decreased by 0.84%, and only a minority of patients needed to intensify their hyperglycemia regimen after achieving CS remission.We found that almost half of patients in our study demonstrated improvement in hyperglycemia, and another 21% experienced resolution of hyperglycemia following the curative procedure for CS. Several small studies in patients with CS reported resolution of hyperglycemia in 20% to 79% of patients , 19-22. We identified several factors that were associated with either resolution or improvement of hyperglycemia. These included the severity of CS , type of CS, and severity of hyperglycemia at the time of diagnosis. ACTH-dependent CS and severe biochemical score were independent predictors of hyperglycemia improvement on multivariable analysis. These data suggest that in patients with milder forms of CS, hypercortisolism may play a smaller role in hyperglycemia at the time of diagnosis, and a subsequent curative procedure is less likely to improve it.Several clinical implications arise from our study. First, as improvement in hyperglycemia is seen in most patients, counseling and close monitoring of those treated with insulin or other medications is needed to modify therapy and avoid hypoglycemia. Second, patients with milder CS  should be treated for more severe hyperglycemia rather than adopt an expectant attitude because hyperglycemia in these cases is less likely to improve with CS remission. Notably, not all patients achieved an optimal glucose control during follow-up, suggesting that intensification of therapy rather than an expectant attitude is warranted in some patients with CS.1c measurements in a patient not treated with pharmacotherapy for hyperglycemia. Duration of follow-up varied, and at the time of last follow-up not all patients recovered adrenal function. Some of the subgroup analyses were limited by the small sample size.Our study has several strengths. We include a large sample size, with appropriate patient representation regarding CS subtype and severity, and type of hyperglycemia. Availability of a detailed medical record system allowed us to perform an in-depth analysis both of baseline and follow-up variables. This is the first study to analyze the quantitative changes, based on the time from the curative surgery, to assess the changes in the intensity of hyperglycemia therapy and identify predictors for hyperglycemia improvement. The limitations of our study include the retrospective design with selection, referral, and information biases. Not all patients with CS treated at our institution were followed for hyperglycemia after surgery for the whole duration of follow-up. Management of hyperglycemia was not standardized and differed among clinicians. While the initial diagnosis of hyperglycemia was made based on the American Diabetes Association guidelines, resolution of hyperglycemia was based on multiple factors, such as glucose log, normal fasting glucose, and HbA1c, and can be treated with less insulin and fewer noninsulin agents. Patients with more severe hyperglycemia, ACTH-dependent CS, and more severe CS are more likely to improve after surgery. Patients with hyperglycemia need appropriate monitoring and medication adjustment after surgery for CS.In conclusion, we demonstrated that almost 70% of patients with CS demonstrate either resolution or improvement in hyperglycemia following CS remission. As a group, patients demonstrate a decrease in HbA


Androgen receptor (AR) is an important prognostic marker and therapeutic target in luminal androgen receptor triple-negative breast cancer (LAR TNBC) and prostate cancer (PCa). Endoplasmic reticulum (ER) stress may activate the unfolded protein response (UPR) to regulate associated protein expression and is closely related to tumor growth and drug resistance. The effect of ER stress on AR expression and signaling remains unclear. Here, we focused on the regulation and underlying mechanism of AR expression induced by ER stress in LAR TNBC and PCa. Western blotting and quantitative RT-PCR results showed that AR expression was markedly decreased under ER stress induced by thapsigargin and brefeldin A, and this effect was dependent on PERK/eIF2α/ATF4 signaling activation. Chromatin immunoprecipitation-PCR and luciferase reporter gene analysis results showed that ATF4 bound to the AR promoter regions to inhibit its activity. Moreover, ATF4 overexpression inhibited tumor proliferation and AR expression both in vitro and in vivo. Collectively, these results demonstrated that ER stress could decrease AR mRNA and protein levels via PERK/eIF2α/ATF4 signaling in LAR TNBC and PCa. Targeting the UPR may be a treatment strategy for AR-dependent TNBC and PCa. Upon binding of its androgen ligand, the complex is translocated to the nucleus, where it binds to DNA and stimulates the transcription of androgen-responsive genes7. Interestingly, some studies have shown that AR is also highly expressed in up to 70–90% of all breast cancer types, including as much as 30% of triple-negative breast cancers (TNBC) that are deficient in the expression of estrogen receptor α (ERα), progesterone receptor (PR) and HER2. Furthermore, studies have shown that AR-directed therapy induces tumor response10. A luminal androgen receptor (LAR) subtype of TNBC (LAR TNBC) is dependent on AR signaling and AR has become increasingly important as a prognostic marker and potential therapeutic target in precision medicine for this TNBC subtype11. Therefore, AR-targeted drugs have the potential to be used in AR+ PCa and LAR TNBC-specific precision treatments.Breast cancer and prostate cancer (PCa) are typical hormone-dependent malignant tumors with high heterogeneity. AR is the primary factor driving all stages of PCa. Therefore, targeted suppression of AR expression is the primary treatment for PCa via surgical or medical castration with anti-androgens or luteinizing hormone-releasing hormone analogs (agonists or antagonists)12. Cells exposed to ER stress trigger the unfolded protein response (UPR)13. In mammals, the UPR is initiated through the activation of three ER-resident sensors: inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6 (ATF6), and protein kinase R (PKR)-like ER kinase (PERK)15. These signaling pathways clear misfolded proteins and restore ER homeostasis by weakening protein translation and transcription to relieve ER load, upregulating the expression of genes that are involved in increasing ER protein folding capacity , and activating the ER-associated degradation pathway if the protein cannot be refolded correctly17. Thus, ER stress response dysfunction is associated with various diseases including neurodegeneration, chronic obstructive pulmonary disease, fatty liver, diabetes, and cancers17. Moreover, the UPR has been reported to be involved in the resistance of breast cancer cells to radiotherapy and chemotherapy. Conversely, the UPR can promote breast cancer cell death if ER stress is excessive or maintained for a prolonged period12.The endoplasmic reticulum (ER) is an essential cellular organelle for the production, processing, and transport of membrane-bound or secretory proteins and lipids. The accumulation of misfolded proteins in the ER disrupts ER homeostasis resulting in ER stressIn this study, we investigated the regulation and underlying mechanism of AR expression induced by ER stress in LAR TNBC and PCa. We hope to clarify whether targeting UPR could be a strategy for the treatment of AR-dependent TNBC and PCa.We first examined whether ER stress influences AR protein expression. LAR TNBC cell lines MDA-MB-453, HCC2185, and MFM-223, and PCa cell lines LNCap and 22RV1 were treated with ER stress inducers thapsigargin (TG), brefeldin A (BFA), and tunicamycin (TM) for 24 h, respectively. The levels of ER stress markers, immunoglobulin heavy chain-binding protein (BiP), and CCAAT/enhancer-binding protein homologous protein (CHOP) were markedly increased after treatment Fig. . The expWe examined the effects of ER stress on AR mRNA expression in LAR TNBC and PCa cell lines. TG and BFA increased the mRNA expression of BiP and CHOP  also showed that there were binding sites with a relative profile score threshold of >75% in the AR promoter region from −2813 to −2486 nt and from −2084 to −1742 nt. These results indicated that the nuclear transcription factor ATF4 could bind to the AR promoter region. Luciferase assays were performed to verify the binding of ATF4 to the AR promoter. MDA-MB-453 and LNCap cells were transfected with different reporters and then treated with or without TG. The results showed that in the absence of TG, the luciferase activities of the reporters pmir-GLO-AR-3000 and pmir-GLO-AR-2084 which contain AR gene regions from 0 to −3000 nt and 0 to −2084 nt, respectively, were significantly higher (p < 0.01) than those of the pmir-GLO-Basic -PCR experiments were conducted. Chromatin DNA from MDA-MB-453 cells was extracted and purified after immunoprecipitation with anti-ATF4. The enriched DNA was detected using 11 primer pairs containing the AR promoter region utilizing PCR and then sequenced. The results showed that the PCR products  were found in the ATF4 immunoprecipitated group Fig. . In addisic Fig. , supportsic Fig. , supportsic Fig. . The abohttp://gepia.cancer-pku.cn/), we compared the mRNA expression of AR and ATF4 in clinical samples of breast cancers (n = 1085) and PCas (n = 492) available in the datasets. The results indicated that the expression levels of AR were higher in breast invasive carcinoma (BRCA) than in normal breast tissues, whereas the expression levels of ATF4 were lower in the former than in the latter .Using the Gene Expression Profiling Interactive Analysis (GEPIA) datasets , the duration of response varies from months to years and the disease inevitably progresses to a lethal stage called castration-resistant PCa (CRPC)21. Although second-generation AR antagonists, such as abiraterone and enzalutamide, are both approved for the treatment of CRPC, the inherent or acquired resistance of CRPC to these drugs remains a major clinical obstacle25. Therefore, the development of novel agents targeting the AR pathway is urgently needed, and downregulation of AR expression is a promising alternative treatment strategy.PCa is the most common cancer and the leading cause of cancer-related deaths in men worldwide26. However, in ERα-negative/AR-positive cells, they accelerate tumor growth28. In a phase II study, androgen deprivation by abiraterone acetate combined with prednisolone had some responses in AR-positive/metastatic TNBC30. Preclinical and clinical data showed that targeted inhibition of AR could be a potential therapy for AR-positive/ER-negative breast cancer patients. LAR TNBC is an ERα-negative/AR-positive breast cancer and anti-androgens may be effective in its management. Therefore, AR-targeted therapies require further evaluation for LAR TNBC.Some studies have reported that androgens mainly inhibit tumor growth in ERα-positive breast cancer21. AR signaling can be indirectly inhibited by decreasing circulating androgen levels through ADT.25 Moreover, AR expression can be directly blocked by treatment with AR antagonists, such as bicalutamide and enzalutamide31. However, some studies have reported that there are many problems with the direct or indirect methods associated with the resistance to AR-targeted therapies in PCa because of amplification or overexpression of AR, ligand-binding domain mutations, ligand-independent activation, and the expression of active AR splice variants. Therefore, promoting AR degradation or inhibiting AR synthesis may serve as a novel strategy for inhibiting AR signaling33.The AR signaling cascade can be inhibited by several treatment approaches34. ER stress may activate the UPR to regulate associated protein expression and may be closely related to tumor growth and drug resistance. Some studies have shown that ER stress plays a role in regulating AR signaling. For example, riluzole induces AR protein degradation via ER stress pathways to exert its anti-PCa effects35; rosemary extracts modulate ER stress to promote AR degradation in PCa36; TG analogs containing amino acids deplete AR protein via synthesis inhibition and induce the death of PCa cells37. However, the detailed mechanisms by which ER stress regulates AR response in PCa are still not fully understood, and the relationship between ER stress and AR expression in LAR TNBC remains unknown. Our results showed that treatment with different ER stress inducers  significantly decreased the AR levels in LAR TNBC and PCa cell lines. In addition, TG and BFA decreased AR mRNA and protein levels in LAR TNBC and PCa cell lines. Furthermore, we found that ER stress did not promote AR mRNA and protein degradation based on the results of the experiments using the protein synthesis inhibitor CHX and the general transcription inhibitor ActD. These results indicate that ER stress inhibits AR transcription and reduces AR expression.ER stress triggers the UPR, which results in a temporary blockade of global mRNA translation to alleviate the accumulation of misfolded proteins in the EROur results indicate that the PERK pathway, but not the IRE1α and ATF6 pathways, participates in the ER stress-induced AR decrease. Further mechanistic investigations demonstrated that ATF4 can bind to the AR promoter region from −2813 to −2486 nt and from −2084 to −1742 nt, and that TG and ATF4 overexpression inhibited AR promoter activity. Therefore, we conclude that the PERK/eIF2α/ATF4 pathway is responsible for the ER stress-induced decrease in AR expression.In our study, an orthotopic xenograft mouse model was used to evaluate AR expression and its inhibitory effects on tumor growth in vivo by lentivirus-mediated delivery of ATF4. Our results showed that overexpression of ATF4 inhibited tumor growth and AR expression in a CAL-148 cell xenograft mouse model. The above results establish that ER stress inhibits AR expression via the PERK/eIF2α/ATF4 pathway activation. The in vivo results were consistent with those obtained in vitro.In conclusion, our findings demonstrate that AR is a new ER stress response gene and that the PERK/eIF2α/ATF4 signaling pathway participates in this response. Our findings may lead to a better understanding of ER stress activation and AR expression control in AR-dependent TNBC and PCa. Targeting UPR may be a strategy for the treatment of AR-dependent TNBC and PCa.TG, BFA, TM, 4μ8c, GSK2656157, CHX, and ActD were purchased from MedChemExpress . Female nude mice  were obtained from Beijing HFK Bioscience Co.,Ltd. . TNBC tissue microarray was obtained from Avilabio Biotechnology Co., Ltd. . Matrigel was purchased from BD Biosciences . Antibodies against PERK (Cat#5683), eIF2α (Cat#5324), p-eIF2α (Cat#3398), ATF4 (Cat#11815), ATF6 (Cat#65880), IRE1α (Cat#3294), CHOP (Cat#2895), XBP1s (Cat#12782), Bip (Cat#3177) and AR (Cat#5153) were obtained from Cell Signaling Technology . The Ki-67 antibody (Cat#AF0198) was obtained from Affinity Biosciences , and the β-actin antibody was obtained from Santa Cruz Biotechnology . Cell Counting Kit-8 (CCK-8) was purchased from Bimake . RIPA lysis buffer, BCA protein assay kit, and dual-luciferase reporter assay kit were obtained from Beyotime . ChIP assay kit was purchased from Thermo Fisher Scientific . PERK siRNA, ATF4 siRNA, CHOP siRNA, and control siRNA were purchased from Santa Cruz Biotechnology ; ATF6 siRNA was obtained from RiboBio ; IRE1α siRNA, IRE1α-overexpressing lentivirus (LV-IRE1α), ATF4-overexpressing lentivirus (LV-ATF4) and negative control lentivirus (LV-GFP) were purchased from GeneChem .2. All the cells were verified for mycoplasma contamination free.Human breast cancer cell lines  and human PCa cell lines  were obtained from the American Type Culture Collection. MDA-MB-453 cells were cultured in Leibovitz’s L-15 medium and the other cell lines were cultured in DMEM-high glucose medium supplemented with 10% fetal bovine serum. Cells were grown at 37 °C within a humidified atmosphere containing 5% COCCK-8 assay was used to measure cell viability. Cells were seeded in a 96-well plate. Then, the cells were directly treated with 10 or 12 μM CCT020312 or 1 μM TG for 24 h. Subsequently, 10 µL of CCK-8 solution was added to each well, and the cells were subsequently incubated for 1 or 2 h at 37 °C. Absorbance was measured at 450 nm using a full-wavelength microplate reader (Thermo Fisher Scientific).TM II Kit. The primers used for qPCR are listed in Supplementary Table −ΔΔCT method was used to analyze the gene expression data.Total RNA was extracted from cells using the TRIzol reagent. Next, 2 μg of total RNA was reverse-transcribed into cDNA using Superscript III (Invitrogen) following the manufacturer’s instructions. qPCR was performed using SYBR Premix Ex Taq38. Briefly, the transferred PVDF membranes were blocked by incubation with 5% non-fat milk in TBST buffer for 1 h, and then incubated with primary antibody (diluted in 5% non-fat milk or 5% BSA in TBST buffer) at 4 °C overnight. The dilution for the primary antibodies was 1:1000 unless otherwise indicated. After washing four times with TBST buffer, the membranes were incubated with the HRP-conjugated secondary antibodies for 1 h. Then, the membranes were washed four times and incubated with the enhanced chemiluminescence (ECL) substrate. The signals on the membranes were captured by the ChemiDoc MP imaging system. All blots derived from the same experiment and were processed in parallel.Total cell protein was extracted using the RIPA lysis buffer, and the protein concentration was measured using a BCA Protein Assay Kit. Subsequently, the equivalent amount of proteins was separated using SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Immunoblotting was performed as previously describedTumor specimens were embedded in paraffin and cut into 5 μm-thick sections. Samples were incubated with a specific primary antibody at 4 °C overnight and incubated with HRP-conjugated secondary antibodies at 25 °C for 2 h. The signal was visualized with DAB reagent and examined under a light microscope.The TNBC tissue microarray was incubated with a specific primary antibody at 4 °C overnight. The cells were then incubated for 1 h with Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 647-conjugated donkey anti-rabbit secondary antibodies (Molecular Probes). Nuclei were counterstained with 0.1 μg/mL DAPI (Sigma). Total fluorescence intensity was examined and analyzed using the Pannoramic MIDI scanner from “3DHISTECH”.6/mL) were seeded in 10 cm cell culture dishes and treated the following day with 1 μM TG for 24 h. The ChIP assay was performed according to the manufacturer’s instructions (Thermo Fisher Scientific). Anti-ATF4 antibodies were used for immunoprecipitation, and normal mouse IgG was used as a control in ChIP analysis. DNA was amplified using PCR with a TaKaRa Ex Taq DNA polymerase kit. PCR has performed under the following reaction conditions: denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 60 s (30 cycles). The PCR products were identified using 1.0% agarose gel electrophoresis and ethidium bromide staining. Images of the gels were obtained using a Tanon 3200 Gel Imaging System. The primers used for the PCR are listed in Supplementary Table Cells (3 × 10BglII-NheI restriction sites. The AR-overexpressing plasmid was a gift from Dr. Yuanzhong Wang from the Beckman Research Institute of the City of Hope. All inserts were verified using DNA sequencing.To generate the ATF4-overexpressing plasmid (pMG-ATF4), the DNA fragment encoding ATF4 was generated using PCR with RNA from MCF7 cells using the following primers: 5′-CTGAGATCTCACCATGACCGAAATGAGCTTCC-3′ and 5′-GGGGCTAGCCTAGGGGACCCTTTTCTTC-3′. The coding sequence of ATF4 was cloned into the mammalian expression vector pMG-H2 using 4 cells/well for 24 h. The cells in each well were transfected with 1 μL of Lipofectamine 2000 and 0.2 μg of reporter plasmids in 50 μL of Opti-MEM. Six hours after transfection, 500 μL of cell culture medium was added to each well. After 48 h of incubation, the cells were washed once with PBS. A luciferase reporter assay was performed using a dual-luciferase reporter assay system according to the manufacturer’s protocol. The relative luciferase activity was calculated. The data are expressed as the means ± SD.Cells were seeded in 24-well plates at a density of 5 × 10Cells were transfected with siRNA or plasmids using RNAiMax or Lipofectamine 3000 and Opti-MEM I reduced serum medium for 6–12 h. Then, the cells were replaced with the normal growth medium and subjected to further treatment as indicated.For lentivirus transduction, cells were cultured to 50% confluence and transduced with IRE1α lentivirus (LV-IRE1α), ATF4 lentivirus (LV-ATF4), or control lentivirus (LV-GFP) according to the protocol in the Lentivirus Operation Manual. The culture was renewed in a regular medium for 72 h, and the cells were subjected to further treatment.http://gepia.cancer-pku.cn) is a newly developed interactive web server for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from TCGA and GTEx projects, using a standard processing pipeline. GEPIA provides customizable functions such as tumor/normal differential expression analysis, profiling according to cancer type or pathological stage, patient survival analysis, similar gene detection, correlation analysis, and dimensionality reduction analysis39. For analyzing relative expression of AR or ATF4 between breast tumors and normal breast tissues or between PCa and normal prostate tissue, Single Gene Analysis under GEPIA (http://gepia.cancer-pku.cn) was chosen. After entering the analyzed gene name (ATF4 or AR), “Boxplot” was clicked, and BRCA (breast invasive carcinoma) or PRAD (prostate adenocarcinoma) was selected as the datasets for analysis accordingly. We kept the others as the default setting ; log scale: Yes; Jitter Size: 0.4; Match TCGA normal and GTEx data. After clicking “Plot”, the plot box results of relative expression of AR or ATF4 between breast tumors and normal breast tissues or between PCa and prostate normal tissue were generated. For generating expression correlations between AR and ATF4 in breast cancer and in PCa, Multiple Gene Analysis under GEPIA was chosen and Correlation Analysis was selected. After entering “AR” as Gene A and “ATF4” as Gene B, we chose “Pearson” as Correlation Coefficient and selected “BRCA Tumor” or “PRAD” under TCGA Tumor as the analysis datasets. After clicking “Plot”, the plot results for correlation analysis results were generated. During the whole analysis, no accession numbers or codes are needed or generated.Gene Expression Profiling Interactive Analysis  stably expressing LV-GFP or LV-ATF4 mixed with Matrigel (1:1) was established in nude mice (five mice/group). When tumors grew to ~40–50 mm3, tumor growth in the mice was measured every third day. The tumor volume (V) was measured using a slide caliper and calculated using the following formula: V (mm3) = 0.5 × ab2, where a and b represent the long diameter and perpendicular short diameter (mm) of the tumor, respectively.Female nude 5 weeks old mice weighing 18–22 g were obtained from Beijing HFK Bioscience Co., Ltd.  and housed in a pathogen-free facility with a 12 h artificial light–dark cycle in Chongqing Medical University. The experiments were performed in accordance with the National Guidelines for Animal Care and Use and approved by the Animal Care and Use Committee of Chongqing Medical University. An orthotopic implantation model at the eighth breast with CAL-148 cells . Comparisons among multiple groups were performed using one-way ANOVA. The criterion for statistical significance was set at p < 0.05.The data were analyzed using GraphPad Prism 8.0 software . All data are expressed as mean ± SD. The student’s Further information on research design is available in the REPORTING SUMMARYSupplementary


Allergic rhinitis (AR) is characterized by IgE-mediated mucosa response after exposure to allergens. Extracellular vesicles (EVs) are nano-size vesicles containing biological cargos for intercellular communications. However, the role of plasma EVs in pathogenesis of AR remains largely unknown.+T cells and their effects on CD4+T cell proliferation, respectively.Plasma EVs from patients with AR were isolated, quantified, and characterized. The expression of Der p 1 and antigen-presenting molecules on EVs was determined by Western blot, flow cytometry, or ELISA. PKH26- and CFSE (carboxyfluorescein succinimidyl ester)-stained AR-EVs were used to determine the uptake of EVs by CD4+ T-cell proliferation.Plasma EVs in healthy control (HC) and AR patients were similar in the concentration of particles, expression for specific EV markers, and both had structural lipid bilayer. However, the levels of Der p 1 on plasma EVs from both mild and moderate-severe AR patients were significantly higher than that on HC. The levels of antigen-presenting molecules on plasma EVs were similar from three subjects. Moreover, levels of Der p 1 on EVs in plasma, but not nasal secretion, were significantly associated with the symptom score of AR patients and level of plasma IL-13. Additionally, plasma EVs from patients with AR promoted the development of Th2 cells, while no effect was found on CD4Plasma EVs derived from patients with AR exhibited antigen-presenting characteristics and promoted differentiation of Th2 cells, thus providing novel understanding of the pathogenesis of AR. Allergic rhinitis (AR) is one of the most common allergic diseases that globally affects 10% to 40% of the population across all age groups , leadingExtracellular vesicles (EVs) are nano-vesicles that are substantially released by almost all cells and extensively involved in physiological and pathological processes in human bodies . Based o+ T cells, which are regarded as the key players in allergic responses  and patients with moderate AR (M-AR-EVs) or moderate-severe AR (S-AR-EVs) by differential ultracentrifugation, as previously described with minor modifications . The detConcentration and size distribution of plasma EVs were determined by nanoparticle tracking analysis using a NS300 Nanosight instrument . The detailed methods are presented in Morphologies of plasma EVs were observed using a TEM instrument  as we previously reported . The detTo determine levels of specific EV markers (CD9/CD63/CD81/Alix/TSG101) and molecules associated with antigen presentation (Der p 1/CD40/CD80/CD86/HLA-ABC/HLA-DR) in plasma EVs, Western blot analysis was conducted as we previously reported . The detThe isolated plasma EVs were incubated with 20 nM CFSE for 1 h at room temperature, and the CFSE-stained plasma EVs were further isolated by ultracentrifugation. The CFSE-stained plasma EVs were significantly characterized by flow cytometry analysis  following manufacturer’s instructions.Human PBMCs were isolated from buffy coats of anonymous donors provided by Guangzhou Blood Center by using density centrifugation with LSM™ Lymphocyte Separation Medium . Human CD4+ T cells were seeded at a density of 5 × 105 cells in 96-well plates and cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum , 10 ng/ml IL-2  and penicillin/streptomycin  in the presence or absence of HC-EVs or AR-EVs at a concentration of 5 × 108 particles/ml for 3 days. The cells were collected for Th1, Th2, and Th17 examination by flow cytometry analysis. The isolated CD4+ T cells were treated with CFSE-stained EVs and evaluated their uptake for EVs using flow cytometry or immunofluorescence.The PBMCs or CD4+ T cells, PBMCs were stained with CFSE, then stimulated with 5 mg/ml phytohemagglutinin (PHA), and treated with HC-EVs and AR-EVs as described above. The cells were then detected by flow cytometry analysis.For determination of the effects of plasma EVs on proliferation of CD4The detailed methods are presented in + T cells treated with CFSE-stained EVs were analyzed as previously reported with minor modifications . All values were expressed as mean ± SEM for each group. Normal distribution was performed by using Kolmogorov-Smirnov test. For data with normal distribution, statistical significance was assessed by using one-way ANOVA with Tukey’s correction for multiple comparisons. For data with abnormal distribution, statistical significance was assessed by Mann-Whitney We first isolated plasma EVs from HC and AR patients that were sensitive to HDM by differential centrifugation, and we found no significant difference on the concentration of plasma EVs derived from HC, M-AR, and S-AR as determined by NTA (+), B cells (CD19+), NK cells (CD56+), monocytes (CD14+), and granulocytes (CD66b+) were almost undetectable, while the expression of HLA-DR+ on plasma EVs were significantly detected  and S-AR-EVs compared to HC-EVs , Guangdong Basic and Applied Basic Research Foundation (grant no. 2020A1515110794), and China Postdoctoral Science Foundation (grant no. 2021M693639).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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