Augmented reality (AR) has gained enormous popularity and acceptance in the past few years. AR is indeed a combination of different immersive experiences and solutions that serve as integrated components to assemble and accelerate the augmented reality phenomena as a workable and marvelous adaptive solution for many realms. These solutions of AR include tracking as a means for keeping track of the point of reference to make virtual objects visible in a real scene. Similarly, display technologies combine the virtual and real world with the user’s eye. Authoring tools provide platforms to develop AR applications by providing access to low-level libraries. The libraries can thereafter interact with the hardware of tracking sensors, cameras, and other technologies. In addition to this, advances in distributed computing and collaborative augmented reality also need stable solutions. The various participants can collaborate in an AR setting. The authors of this research have explored many solutions in this regard and present a comprehensive review to aid in doing research and improving different business transformations. However, during the course of this study, we identified that there is a lack of security solutions in various areas of collaborative AR (CAR), specifically in the area of distributed trust management in CAR. This research study also proposed a trusted CAR architecture with a use-case of tourism that can be used as a model for researchers with an interest in making secure AR-based remote communication sessions. Augmented reality (AR) is one of the leading expanding immersive experiences of the 21st century. AR has brought a revolution in different realms including health and medicine, teaching and learning, tourism, designing, manufacturing, and other similar industries whose acceptance accelerated the growth of AR in an unprecedented manner . AccordiThe adoption of AR in its various perspectives is backed up by a prolonged history. This paper presents an overview of the different integrated essential components that contribute to the working framework of AR, and the latest developments on these components are collected, analyzed, and presented, while the developments in the smart devices and the overall experience of the users have changed drastically . The traOnce the points of reference are collected after tracking, then another important factor that requires significant accuracy is to determine at which particular point the virtual objects have to be mixed with the real environment. Here comes the role of display technologies that gives the users of augmented reality an environment where the real and virtual objects are displayed visually. Therefore, display technologies are one of the key components of AR. This research identifies state-of-the-art display technologies that help to provide a quality view of real and virtual objects. Augmented reality displays can be divided into various categories. All have the same task to show the merged image of real and virtual content to the user’s eye. The authors have categorized the latest technologies of optical display after the advancements in holographic optical elements (HOEs). There are other categories of AR displays, such as video-based, eye multiplexed, and projected onto a physical surface. Optical see-through has two sub-categories, one is a free-space combiner and the other is a wave-guide combiner ,12. The To develop these AR applications, different tools are used depending on the type of application used. For example, to develop a mobile-based AR application for Android or iOS, ARToolKit  is used.After going through a critical review process of collaborative augmented reality, the research has identified that some security flaws and missing trust parameters need to be addressed to ensure a pristine environment is provided to the users. Hackers and intruders are always active to exploit different vulnerabilities in the systems and software, but the previous research conducted on collaborative augmented reality did not depict reasonable efforts made in this direction to make secure collaboration. To address the security flaws and to provide secure communication in collaborative augmented reality, this research considered it appropriate to come up with a security solution and framework that can limit danger and risks that may be posed in the form of internal and external attacks. To actualize the secure platform, this study came up with an architecture for presenting a secure collaborative AR in the tourism sector in Saudi Arabia as a case study. The focus of the case study is to provide an application that can guide tourists during their visit to any of the famous landmarks in the country. This study proposed a secure and trustful mobile application based on collaborative AR for tourists. In this application, the necessary information is rendered on screen and the user can hire a guide to provide more information in detail. A single guide can provide the services to a group of tourists visiting the same landmark. A blockchain network was used to secure the applications and protect the private data of the users ,17. For Observing the fact that AR operates in an integrated fashion that combines different technologies including tracking technologies, display technologies, AR tools, collaborative AR, and applications of AR has encouraged us to explore and present these conceptions and technologies in detail. To facilitate researchers on these different techniques, the authors have explored the research previously conducted and presented it in a Venn diagram, as shown in Contribution: This research presents a comprehensive review of AR and its associated technologies. A review of state-of-the-art tracking and display technologies is presented followed by different essential components and tools that can be used to effectively create AR experiences. The study also presents the newly emerging technologies such as collaborative augmented reality and how different application interactions are carried out. During the review phase, the research identified that the AR-based solutions and particularly collaborative augmented reality solutions are vulnerable to external intrusion. It is identified that these solutions lack security and the interaction could be hijacked, manipulated, and sometimes exposed to potential threats. To address these concerns, this research felt the need to ensure that the communication has integrity; henceforth, the research utilizes the state-of-the-art blockchain infrastructure for the collaborating applications in AR. The paper further proposes complete secure framework wherein different applications working remotely have a real feeling of trust with each other .Outline: This paper presents the overview of augmented reality and its applications in various realms in People, for many years, have been using lenses, light sources, and mirrors to create illusions and virtual images in the real world ,23,24. HEns et al.  review tARToolKit tracking library  aimed toCortes et al.  introducThe AR industry was tremendously increasing in 2015, extending from smartphones to websites with head-worn display systems such as Google Glass. In this regard, Agati et al.  propose AR for Tourism and Education: Shukri et al.  aim to iSarkar et al.  present Hadar et al.  scrutiniit combines virtual and the real content;it is interactive in real time;is is registered in three dimensions.Tracking technologies introduce the sensation of motion in the virtual and augmented reality world and perform a variety of tasks. Once a tracking system is rightly chosen and correctly installed, it allows a person to move within a virtual and augmented environment. It further allows us to interact with people and objects within augmented environments. The selection of tracking technology depends on the sort of environment, the sort of data, and the availability of required budgets. For AR technology to meet Azuma’s definition of an augmented reality system, it must adhere to three main components:Determination of the position and orientation of the viewer relative to the real-world anchor: registration phase;Upgrading of viewer’s pose with respect to previously known pose: tracking phase.The third condition of being “registered in three dimensions” alludes to the capability of an AR system to project the virtual content on physical surroundings in such a way that it seems to be part of the real world. The position and orientation (pose) of the viewer concerning some anchor in the real world must be identified and determined for registering the virtual content in the real environment. This anchor of the real world may be the dead-reckoning from inertial tracking, a defined location in space determined using GPS, or a physical object such as a paper image marker or magnetic tracker source. In short, the real-world anchor depends upon the applications and the technologies used. With respect to the type of technology used, there are two ways of registering the AR system in 3D:In this document, the word “tracking” would define both phases as common terminology. There are two main types of tracking techniques which are explained as follows  used in inertial tracking to evaluate the velocity and orientation of the tracked object. An inertial tracking system is used to find the three rotational degrees of freedom relative to gravity. Moreover, the time period of the trackers’ update and the inertial velocity can be determined by the change in the position of the tracker.Advantages of Inertial Tracking: It does not require a line of sight and has no range limitations. It is not prone to optical, acoustic, magnetic, and RE interference sources. Furthermore, it provides motion measurement with high bandwidth. Moreover, it has negligible latency and can be processed as fast as one desires.Disadvantages of Inertial Tracking: They are prone to drift of orientation and position over time, but their major impact is on the position measurement. The rationale behind this is that the position must be derived from the velocity measurements. The usage of a filter could help in resolving this issue. However, the issue could while focusing on this, the filter can decrease the responsiveness and the update rate of the tracker . For thevisible light tracking;3D structure tracking;infrared tracking.Vision-based tracking is defined as tracking approaches that ascertain the camera pose by the use of data captured from optical sensors and as registration. The optical sensors can be divided into the following three categories:In recent times, vision-based tracking AR is becoming highly popular due to the improved computational power of consumer devices and the ubiquity of mobile devices, such as tablets and smartphones, thereby making them the best platform for AR technologies. Chakrabarty et al.  contribuMoreover, Krishna et al.  explore Ranging from smartphones to laptops and even to wearable devices with suitable cameras located in them, visible light tracking is the most commonly used optical sensor. These cameras are particularly important because they can both make a video of the real environment and can also register the virtual content to it, and thereby can be used in video see-through AR systems.Chen et al.  resolve Three-dimensional structure information has become very affordable because of the development of commercial sensors capable of accomplishing this task. It was begun after the development of Microsoft Kinect . SyahidiRambach et al.  present The objects that emitted or reflected the light are some of the earliest vision-based tracking techniques used in AR technologies. Their high brightness compared to their surrounding environment made this tracking very easy ,72. The The three-dimensional tracking of real-world objects has been the subject of researchers’ interest. It is not as popular as natural feature tracking or planner fiducials, however, a large amount of research has been done on it. In the past, tracking the three-dimensional model of the object was usually created by the hand. In this system, the lines, cylinders, spheres, circles, and other primitives were combined to identify the structure of objects . Wuest eIn addition, Gao et al.  explore It provides additional robustness .Enables spatial tracking and thereby is able to be operated in open environments .For variable and complex environments, greater robustness was required. Therefore, they introduced the concept of keyframes  in additThe combination of edge-based tracking and natural feature tracking has the following advantages:Figen et al.  demonstrDeveloping and updating the real environment’s map simultaneously had been the subject of interest in model-based tracking. This has a number of developments. First, simultaneous localization and map building (SLAM) was primarily done for robot navigation in unknown environments . In augmOskiper et al.  propose This technology refers to the positioning of outdoor tracking with reference to the earth. The present accuracy of the GPS system is up to 3 m. However, improvements are available with the advancements in satellite technology and a few other developments. Real-time kinematic (RTS) is one example of them. It works by using the carrier of a GPS signal. The major benefit of it is that it has the ability to improve the accuracy level up to the centimeter level. Feiner’s touring machine  was the Yang et al. , in ordeMoreover, Bach et al.  introducImproving the accuracy of the tracking system.Coping with the weaknesses of the respective tracking methods.Adding more degrees of freedom.Hybrid tracking systems were used to improve the following aspects of the tracking systems:Low drift of vision-based tracking.Low jitter of vision-based tracking.They had a robust sensor with high update rates. These characteristics decreased the invalid pose computation and ensured the responsiveness of the graphical updates .They had more developed inertial and magnetic trackers which were capable of extending the range of tracking and did not require the line of sight. The above-mentioned benefits suggest that the utilization of the hybrid system is more beneficial than just using the inertial trackers.Gorovyi et al.  detail tIn addition, Mao et al.  propose The GPS tracking system alone only provides the positional information and has low accuracy. So, GPS tracking systems are usually combined with vision-based tracking or inertial sensors. The intervention would help gain the full pose estimation of 6DoF . MoreoveFiducial Tracking: Artificial landmarks for aiding the tracking and registration that are added to the environment are known as fiducial. The complexity of fiducial tracking varies significantly depending upon the technology and the application used. Pieces of paper or small colored LEDs were used typically in the early systems, which had the ability to be detected using color matching and could be added to the environment . If the This section provides comprehensive details on tracking technologies that are broadly classified into markerless and marker-based approaches. Both types have many subtypes whose details, applications, pros, and cons are provided in a detailed fashion. The different categories of tracking technologies are presented in For the combination of a real and the virtual world in such a way that they both look superimposed on each other, as in Azuma’s definition, some technology is necessarily required to display them.Methods or procedures required for the merging of the virtual content in the physical world include camera calibration, tracking, registration, and composition as depicted in It is a procedure or an optical model in which the eye display geometry or parameters define the user’s view. Or, in other words, it is a technique of complementing the dimensions and parameters of the physical and the virtual camera.In AR, calibration can be used in two ways, one is camera calibration, and another is optical calibration. The camera calibration technique is used in video see-through (VST) displays. However, optical calibration is used in optical see-through (OST) displays. OST calibration can be further divided into three umbrellas of techniques. Initially, manual calibration techniques were used in OST. Secondly, semi-automatic calibration techniques were used, and thirdly, we have now automatic calibration techniques. Manual calibration requires a human operator to perform the calibration tasks. Semi-automatic calibration, such as simple SPAAM and display relative calibration (DRC), partially collect some parameters automatically, which usually needed to be done manually in earlier times by the user. Thirdly, the automatic OST calibration was proposed by Itoh et al. in 2014 with the model of interaction-free display calibration technique (INDICA) . In videFurthermore, these geometric calibrations lead to perceptual errors while converting from 3D to 2D . To counSome sort of technology is required to track the position and orientation of the object of interest which could either be a physical object or captured by a camera with reference to the coordinate plan (3D or 2D) of a tracking system. Several technologies ranging from computer vision techniques to 6DoF sensors are used for tracking the physical scenes.Registration is defined as a process in which the coordinate frame used for manifesting the virtual content is complemented by the coordinate frame of the real-world scene. This would help in the accurate alignment of the virtual content and the physical scene.Now, the accuracy of two important steps, i.e., the accurate calibration of the virtual camera and the correct registration of the virtual content relative to the physical world, signifies the right correspondence between the physical environment and the virtual scene which is generated on the basis of tracking updates. This process then leads to the composition of the virtual scene’s image and can be done in two ways: Optically  or digitally. The physical or digital composition depends upon the configuration and dimensions of the system used in the augmented reality system.The combination of virtual content in the real environment divides the AR displays into four major types, as depicted in These kinds of displays use the optical system to merge the real scenes and virtual scene images. Examples of AR displays are head-up display HUD systems of advanced cars and cockpits of airplanes. These systems consist of the following components: beam splitters, which can be of two forms, combined prisms or half mirrors. Most beam splitters reflect the image from the video display. This reflected image is then integrated with a real-world view that can be visualized from the splitter. For half mirrors as a beam splitter, the working way is somewhat different: the real-world view is reflected on the mirror rather than the image of the video display. At the same time, the video display can also be viewed from the mirror. The transport projection system is semi-transparent optical technology used in optical display systems. Their semi-transparent property allows the viewer to witness the view at the back of the screen. Additionally, this system uses diffused light to manifest the exhibited image. Examples of semi-display optical systems are transparent projection film, transparent LCDs, etc. Optical combiners are used for the combination of virtual and real scene images. Optical see-through basically has two sub-categories, one is a free-space combiner and the other is a wave-guide combiner . AdditioPapers related to free space combiners are discussed here. Pulli et al.  introducThe waveguide combiner basically traps light into TIR as opposed to free-space, which lets the light propagate without restriction ,105,106.These displays execute the digital processes as their working principle . To rephFirst, they track the user’s viewpoint.Secondly, they track the physical object.Then, they impart the interactive augmentation .Real models  and wallMostly, these displays have a projector attached to the wall or a ceiling. This intervention has an advantage as well as a disadvantage. The advantage is that this does not demand the user to wear something. The disadvantage is that it is static and restricts the display to only one location of projection. For resolving this problem and making the projectors mobile, a small-sized projector has been made that could be easily carried from one place to another . More reIn eye-multiplexed AR displays, the users are allowed to combine the views of the virtual and real scenes mentally in their minds . RephrasThe division of the displays on the basis of the position of the display between the real and virtual scenes is referred to as the “eye to world spectrum”.Head-attached displays are in the form of glasses, helmets, or goggles. They vary in size from smaller to bigger. However, with the advancement of technology, they are becoming lighter to wear. They work by displaying the virtual image right in front of the user’s eye. As a result, no other physical object can come between the virtual scene and the viewer’s eye. Therefore, the third physical object cannot occlude them. In this regard, Koulieris et al.  summarizThe most commonly used displays in AR research are head-mounted displays (HMDs). They are also known as face-mounted displays or near-eye displays. The user puts them on, and the display is represented right in front of their eyes. They are most commonly in the form of goggles. While using HMDs, optical and video see-through configurations are most commonly used. However, recently, head-mounted projectors are also explored to make them small enough to wear. Examples of smart glasses, Recon Jet, Google glass, etc., are still under investigation for their usage in head-mounted displays. Barz et al.  introducPreviously, the experimentation with handheld display devices was done by tethering the small LSDs to the computers ,178. HowIn today’s world, computer tablets and smartphones are powerful enough to run AR applications, because of the following properties: various sensors, cameras, and powerful graphic processors. For instance, Google Project Tango and ARCore have the most depth imaging sensors to carry out the AR experiences. Chan et al.  discuss Micro projectors are an example of a mobile phone-based AR display. Researchers are trying to investigate these devices that could be worn on the chest , shouldeSpatial displays are used to exhibit a larger display. Henceforth, these are used in the location where more users could get benefit from them i.e., public displays. Moreover, these displays are static, i.e., they are fixed at certain positions and can not be mobilized.The common examples of spatial displays include those that create optical see-through displays through the use of optical beamers: half mirror workbench ,194,195 In addition to visual displays, there are some sensors developed that work with other types of sensory information such as haptic or audio sensors. Audio augmentation is easier than video augmentation because the real world and the virtual sounds get naturally mixed up with each other. However, the most challenging part is to make the user think that the virtual sound is spatial. Multi-channel speaker systems and the use of stereo headphones with the head-related transfer function (HRTF) are being researched to cope with this challenge . DigitalThis section presented a comprehensive survey of AR display technologies. These displays not only focused on combing the virtual and real-world scenes of visual experience but also other ways of combining the sensory, olfactory, and gustatory senses are also under examination by researchers. Previously, head-mounted displays were most commonly in practice; however, now handheld devices and tablets or mobile-based experiences are widely used. These things may also change in the future depending on future research and low cost. The role of display technologies was elaborated first, thereafter, the process of combining the real and augmented contents and visualizing these to users was elaborated. The section elaborated thoroughly on where the optical see-through and video-based see-through are utilized along with details of devices. Video see-through (VST) is used in head-mounted displays and computer vision techniques such as cameras are used for registration of real environment, while in optical see-through (OST), VST calibration techniques cannot be used due to complexity, and cameras are replaced by human eyes. The optical see-through is a trendy approach as of now. The different calibration approaches are presented and analyzed and it is identified after analysis, the results show that recycled INDICA is more accurate than other common techniques presented in the paper. This section also presents video-based AR displays. The effectiveness of AR technologies depends on the perception of distance of users from both real and virtual objects ,215. MikIn addition to the tracking and display devices, there are some other software tools required for creating an AR experience. As these are hardware devices, they require some software to create an AR experience. This section explores the tools and the software libraries. It will cover both the aspects of the commercially available tools and some that are research related. Different software applications require a separate AR development tool. A complete set of low-level software libraries, plug-ins, platforms, and standalones are presented in Low-level software development tools: needs high technological and programming skills.Rapid prototyping: provides a quick experience.Plug-ins that run on the existing applications.Standalone tools that are specifically designed for non-programmers.Next generation of AR developing tools.In some tools, computer vision-based tracking see  is prefeLow-level software and frameworks make the functions of display and core tracking accessible for creating an AR experience. One of the most commonly used AR software libraries, as discussed in the previous section, is ARToolKit. ARToolKit is developed by Billing Hurst and Kato that has two versions . It workResolving inaccurate pattern matching by preventing the false positive matches.Enhancing the functioning in the presence of the imbalanced lightening conditions.Making the occlusion more invariant.ARTag is designed to resolve the limitations of ARToolkit. This system was developed to resolve a number of issues:However, ARTag is no longer actively under development and supported by the NRC Lab. A commercial license is not available.This is also a web-based authoring tool for creating mobile-based AR applications. It allows the utilization of computer vision-based technology for the registration of the real world. Several types of media such as animation and 3D models can be used for creating an AR scene. One of the important features of Wikitude is that the developed mobile AR content can be uploaded not only on the Wikitude AR browser app but also on a custom mobile app . WikitudStandalone AR tools are mainly designed to enable non-programmer users to create an AR experience. A person the basic computer knowledge can build and use them. The reason lies in the fact that most AR authoring tools are developed on a graphical user interface. It is known as a standalone because it does not require any additional software for its operation. The most common and major functions of standalone are animation, adding interactive behaviors, and construction. The earliest examples of the standalone tools are AMIRE  and CATOThis standalone AR authoring tool has the advantage of quickly adding to the development of the AR experience. BuildAR has important characteristics. This allows the user to add video, 3D models, sound, text, and images. It has both arbitrary images and the square marker for which it provides computer vision-based tracking. They use the format of proprietary file format for saving the content developed by the user. BuildAR viewer software can be downloaded for free and it helps in viewing the file. However, BuildAR has no support available and the exe file is not available on their website.Limitation: It does not support adding new interactive features. However, Choi et al.  have proActivate their camera.The AR markers could be viewed in a camera.The virtual content could be overlaid and loaded on the tracked image.In order to cope with the limitation of low-level libraries, another more fast and more rapid AR application development tool is required. The major idea behind the development of rapid prototyping was that it rapidly shows the user the prototype before executing the hard exercise of developing the application. In the following paragraphs, a number of different tools are explained for developing rapid prototyping. For the creation of multimedia content, Adobe Flashis one of the most famous tools. It was developed on desktop and web platforms. Moreover, the web desktop and mobile experiences can be prototyped by it. Flash developers can use the FLARManager, FLARToolKit, or any other plug-ins for the development of AR experience. Porting the version of ARToolKit over the flash on the web creates the AR experience. Its process is so fast that just by writing a few lines, the developer can:FLARToolkit is the best platform for creating AR prototyping because it has made it very easy for being operated by anyone. Anyone who has a camera and flash-enabled web browser can easily develop the AR experience. Alternatives to Flash: According to the website of Adobe, it no longer supports Flash Player after 31 December 2020 and blocked Flash content from running in Flash Player beginning 12 January 2021. Adobe strongly recommends all users immediately uninstall Flash Player to help protect their systems. However, some AR plug-ins could be used as an alternative to Flash-based AR applications. For instance, Microsoft Silverlight has the SLARToolKit. HTML5 is also recently used by researchers for creating web-based AR experiences. The major benefit of using HTML5 is that the interference of the third-party plug-in is not required. For instance, the AR natural feature tracking is implemented on WebGL, HTML5, and JavaScript. This was developed by Oberhofer and was viewable on mobile web browsers and desktops. Additionally, the normal HTML, with few web component technologies, has been used by Ahn  to develAR tracking and visualization functions for the existing authoring tools.It depends on the content authoring function supplied by the main authoring tool.For the creation of AR experiences, the software libraries require tremendous programming techniques. So, plug-ins could be used as an alternative. Plug-ins are devices that could be plugged into the existing software packages. The AR functionality is added to the software packages that to the existing two-dimensional or three-dimensional content authoring tools. If the user already knows the procedure of using authoring tools that are supported by plug-ins, then AR plug-ins for the non-AR authoring tools are useful. These tools are aimed at:The AR plug-in of the Vuforia tracking library can be used with Unity 3D. This integration will help Vuforia in the creation of AR applications for the android or iOS platform.Similarly, the ARToolkit for Unity also provides marker-based experiences. It provides both image and marker-based AR visualization and tracking.There are certain tools available as plug-ins and standalone through which AR applications can be built comparatively simply. These are commercial and some of them are freely available. As discussed earlier, Vuforia can be installed as a plug-in in Unity  and alsoThe need for proprietary software could arise for the content produced by the authoring tool. The design provided by the authoring tools could restrict the user’s interactive and interface designs.Moreover, the authoring tools can also restrict the configurations of hardware or software within a certain limit.In such integrations, the highly interactive experiences are created by the normal Unity3D scripting interface and visual programming. Limitations of AR plug-ins: The following are the limitations accrued with the AR plug-in:Moreover, Nebeling et al.  reviewedThis section presents a detailed survey of different software and tools required for creating an AR experience. The section outlines hardware devices used in AR technology and various software to create an AR experience. It further elaborates on the software libraries required and covers bother the aspects of the commercially available tools. In general, collaboration in augmented reality is the interaction of multiple users with virtual objects in the real environment. This interaction is regardless of the users’ location, i.e., they can participate remotely or have the same location. In this regard, we have two types of collaborative AR: co-located collaborative AR and remote collaborative AR. We mention it further in hlActive Discussion: A face-to-face discussion including all participants.Single Shared view: The participants focus on a single device.Disjoint and Shared View: Two to three participants focus on a single device.Disjoint and Distributed View: One to two people focus on their devices while the others are discussing.Distributed View: Participants focus on their devices with no discussion.Distributive View with Discussion: Participants focus on their devices while discussing in the group.In this type of collaborative AR, the users interact with the virtual content rendered in the real environment while sharing the same place. The participant are not remote in such case. In this regard, Wells et al.  aim to dIn this paper, the author did not contribute to the technology of co-located collaborative AR, but rather performed analysis on the effectiveness of different collaborative AR.Grandi et al.  target tKim et al.  propose Tseng et al.  present In this type of collaborative AR, all the users have different environments. They can interact with virtual objects remotely from any location. A number of studies have been done in this regard. Billinghurst et al.  introducMunoz et al.  present Lien et al.  present Irlitti et al.  explore This section provides comprehensive details on collaborative augmented reality which is broadly classified into co-located collaborative AR, where participants collaborate with each other in geographically the same location, and remote collaboration. The applications of both approaches are presented as well. Co-located collaborative AR is mostly adopted in learning realms for sharing information, for example, in museums. On the other hand, in remote collaborative AR the remote user can explore the scene regardless of the local user’s camera position. The applications of this technology are mostly found in education.The interaction and input technologies are detailed in this section. There are a number of input methods that are utilized in AR technologies. First, multimode and 3D interfaces such as speech, gesture and handheld wands. Second, the mouse, and keyboard traditional two-dimensional user interfaces (UI). The type of interaction task needed for the interface defines which input method would be utilized in the application. A variety of interfaces have been developed: three-dimensional user interfaces, tangible user interfaces, multimedia interfaces, natural user interfaces, and information browsers.Wikitude and Navicam are one of the most popular examples of AR information browsers. The only problem with AR browsers is that they cannot provide direct interaction with the virtual objects.3D motion tracking sensors are one of the most commonly used devices for AR interaction. The motion tracking sensors allow the following functions: tracking the parts of the user’s body and allow pointing as well as the manipulation of the virtual objects [A three-dimensional user interface uses the controllers for providing the interaction with virtual content. By using the traditional 3D user interface techniques, we can directly interact with the three-dimensional object in the virtual space. There are a number of 3D user interface interaction techniques as follows:  objects . Haptic  objects ,297,298.The tangible user interface is one of the main concepts of human–computer interface technology research. In this, the physical object is used for interaction . It bridRecently, more accurate gesture and motion-based interactions for AR and VR applications have become extensively available due to the commercialization of depth cameras such as Microsoft Kinect and technical advances. Bare-hand interaction with a virtual object was made possible by the introduction of a depth camera. It provided physical interaction by tracking the dexterous hand motion. For instance, the physical objects and the user’s hands were recognized by the use of Kinect Camera, designed by the Microsoft HoloDesk . The virIn addition to speech and gesture recognition, there are other types of voice recognition are being investigated. For example, the whistle-recognition system was developed by Lindeman  in mobilThe physical object.The virtual image to be developed.An interface to create an interaction between the physical world and the virtual objects.The previous section detailed the wide range of different AR input and interaction technologies; however, more rigorous research is required to design the AR experience. This section explores the interface patterns and design guidelines to develop an AR experience. The development of new interfaces goes through four main steps. First, the prototype is demonstrated. Second, interaction techniques are adopted from the other interface metaphors. Third, new interface metaphors are developed that are appropriate to the medium. Finally, the formal theoretical models are developed for modeling the interaction of users. In this regard, Wang et al.  employ udesign patterns could be an alternative technique to develop the AR interface design. These design patterns are most commonly used in the fields of computer science and design interface. Alexander has defined the use of design patterns in the following words: “Each pattern describes a problem that occurs over and over again in our environment, and then describes the core of the solution to that problem in such a way that you can use this solution a million times over, without ever doing it the same way twice” [Use of y twice” ,310. They twice” . This idy twice” . This apy twice” . DWARF iy twice” , in desiy twice” ,316. Howy twice” . Radu, iy twice” .Security is very important in augmented reality, especially in collaborative augmented reality. While using collaborative AR applications, the data are exposed to external attacks, which increases concerns about security relating to AR technologies. Moreover, if the users who share the same virtual collaborative environments are unknown to each other, it also elevates these issues. In , the bastrust management is too important a factor to consider because there are unknown parties that participate in remote activities to interact with each other and as such, they are unknown to each other as well [In this paper, the authors have reviewed the literature extensively in terms of tracking and displays technology, AR, and collaborative AR, as can be seen in  as well ,337,338. as well ,339,340. as well . To addr as well ,342,343. as well . A modelThe commercialization efforts of companies have made AR a mainstream field. However, for the technology to reach its full potential, the number of research areas should be expanded. Azuma has explained the three major obstacles in the way of AR: interface limitation, technological limitations, and the issue of social acceptance. In order to overcome these barriers, the two major models are developed: first, Roger’s innovation diffusion theory  and the This document has detailed a number of research papers that address certain problems of AR. For instance, AR tracking techniques are detailed in


Published by Oxford University Press on behalf of Nanjing Agricultural University. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 20 June 2022; Revised: 14 July 2022; Accepted: 20 July 2022
 Dear Editor,CsTSI in the roots of different tea plant cultivars was not obviously correlated with the theanine contents; while the expression of CsAlaDC was shown to be highly correlated with both EA contents and theanine contents in the roots [CsAlaDC expression is critical for adjusting the activity of theanine synthesis in tea plants. However, the mechanism underlying the regulation of CsAlaDC expression is largely unknown in tea plants. Uncovering the mechanism will be critical for improving theanine biosynthesis in tea plants.Theanine, one of the most important tea quality components, confers the ‘umami’ taste and anti-stress benefits of tea infusion . Theaninhe roots . These rCsAlaDC. To verify our hypothesis, we first performed yeast one-hybrid assay, and the result suggested that TEA011475.1 and TEA003248.1 bound to the promoter of CsAlaDC , we identified 16 MYB transcription factors which were highly correlated with the expression of theanine pathway genes under various conditions [ CsAlaDC .3+-N (1.43 mM), EA-N (1.43 mM), NH4+-N (1.43 mM), or NO3+ plus NH4+-N (1.43 mM) for 10 days [CsAlaDC, with CsAlaDC as the hub gene, to identify potential transcription factors regulating CsAlaDC transcription  as a reporter  with CsAlaDC pro:Luc, the activity of CsAlaDC promoter was not obviously different from the no-effector control (CsAlaDC pro:Luc). These results demonstrated that CsHHO3 represses, and CsMYB40 activates, the expression of CsAlaDC in planta.To test the regulatory role of CsHHO3 and CsMYB40 in reporter . The respromoter . In contCsAlaDC promoter, we conducted an electrophoretic mobility shift assay (EMSA) with recombinant CsHHO3 and CsMYB40 proteins. The binding sites were first screened by testing the binding of CsHHO3 and CsMYB40 with the 12 fragments (200 bp each) of the promoter   . By lookCsAlaDC expression in response to N levels, we grew tea plants in hydroponic solution with various concentrations of N  for 18 days. Compared with the 0 N, 1 N, and 10 N significantly increased theanine contents in the roots , the National Key R&D Program of China  and the Base of Introducing Talents for Tea Plant Biology and Quality Chemistry (D20026).Z.Z., X.W., J.G., Q.C., and L.L. conceived the study and designed the experiments; J.G., B.Z., Y.C., S.L., S.Q., and F.M. carried out the experiments; J.G., B.Z., S.L., and T.Y. analysed data and organize figures; J.G., Z.Z. and X.W. wrote the manuscript. All authors reviewed and approved the final manuscript.Data are available upon request to the corresponding author.The authors declare that they have no conflict of interest.


Mucuna pruriens has been identified to have fertility-enhancing and anti-lipid peroxidation properties. Based on these properties of M. pruriens, this study aimed to investigate the efficacy of M. pruriens seed extract in reducing sodium arsenite-induced testicular impairment and hepato-renal histopathology in rats.A significant cause of arsenic poisoning is polluted groundwater. Arsenic poisoning results in the suppression of spermatogenesis and the liver and kidneys are vulnerable to the toxic effects as well. M. pruriens  orally once daily. Subgroups 8 and 9 received a combination of NAC and aqueous seed extract  orally once daily. Following the treatment, animals were sacrificed and sperm parameters and DNA damage were evaluated. Testis, liver, and kidneys were analyzed for histopathology.The study was divided into two groups; short-term (45 days) and long-term (90 days) treatment groups and each group was divided into nine subgroups. Subgroups 1 and 2 served as normal and N-acetyl cysteine (NAC) controls, respectively. Subgroups 3–9 received sodium arsenite in the drinking water (50 mg/L). Subgroup-4 received NAC (210 mg/kg body weight [BW]) orally once daily. Subgroups 5–7 received aqueous seed extract of M. pruriens seed extract ameliorated the damaging effects of sodium arsenite with respect to tissue architecture and sperm parameters when coadministered.Sodium arsenite-induced a significant reduction in sperm parameters and increase in the abnormal architecture of spermatozoa. Histology revealed tissue necrosis. The Mucuna pruriens has beneficial effects against the deleterious effects of sodium arsenite on various tissues. Thus, M. pruriens (530 and 700 mg/kg BW) supplementation would reduce the adverse changes observed with sodium arsenite exposure. Arsenic is an all-pervading metalloid found in the earth’s crust and exists in elemental, organic, and inorganic forms. Contaminated groundwater is the major cause of arsenic poisoning . ArsenicMucuna pruriens is a leguminous plant that possesses antidiabetic and aphrodisiac effects  and 530 mg/kg BW [p < 0.01]) exhibited an increase in sperm count when compared to the sodium arsenite-only treatment group (Tables-There was a significant reduction (p < 0.001) in the sperm count after treatment with sodium arsenite compared to the normal control group. Co-administration of p Tables- and 3.M. pruriens  along with sodium arsenite significantly increased (p < 0.01) the number of motile sperm in comparison to the sodium arsenite-only treated group. Sperm motility significantly increased in the animals treated with a combination of sodium arsenite + NAC along with M. pruriens (350 and 530 mg/kg BW [both p < 0.01]) in comparison to the sodium arsenite-only treated group (Tables-The number of motile sperm significantly decreased (p < 0.001) in the sodium arsenite-only treated group compared to the normal control. Co-administration of p Tables- and 3.M. pruriens  along with sodium arsenite significantly decreased the number of abnormal sperms in comparison to the sodium arsenite only treatment group. Animals treated with sodium arsenite + NAC + M. pruriens (350 [p < 0.05] and 530 [p < 0.001] mg/kg BW) showed a significant decrease in the number of abnormal sperms when compared to sodium arsenite + NAC treatment group (Tables-Exposure to sodium arsenite significantly increased (p < 0.001) the number of abnormal sperm in comparison to the normal control. Co-administration of p Tables- and 3.M. pruriens  and co-administration of sodium arsenite + NAC+ M. pruriens (350 and 530 mg/kg BW [p < 0.001]) exhibited a significant decrease in the seminiferous tubular diameter when compared to sodium arsenite only treated group  in the seminiferous tubular diameter, indicating a distortion of the tubule when compared to the normal controls. In the long-term treatment group, co-administration of sodium arsenite + p Tables- and 3.M. pruriens 700 mg/kg BW had a significantly increased (p < 0.001) seminiferous epithelial height when compared to the sodium arsenite-only treatment group. Animals treated with sodium arsenite + NAC + M. pruriens (350 mg/kg BW) showed a significant increase (p < 0.001) in seminiferous epithelial height when compared to the sodium arsenite-only treated group. In the long-term treatment group, co-administration of sodium arsenite + M. pruriens  and co-administration of sodium arsenite + NAC + M. pruriens (350 and 530 mg/kg BW [p < 0.001]) exhibited a significant increase in the seminiferous epithelial height when compared to sodium arsenite only treated group (Tables-Sodium arsenite administration caused a significant reduction (p < 0.001) in the seminiferous epithelial height when compared to the normal control group. In the short-term treatment group, animals treated with sodium arsenite + p Tables- and 3.M. pruriens (530 and 700 mg/kg BW [p < 0.001]) along with sodium arsenite showed a reduction in the spermatozoa containing damaged DNA in comparison to the sodium arsenite-only group. The spermatozoa from the groups treated with a combination of NAC and M. pruriens (350 and 530 mg/kg BW [p < 0.001]), along with sodium arsenite, also exhibited a significant reduction in the percentage of DNA damage  in the percentage of spermatozoa carrying DNA damage when compared to normal control was observed. Animals treated with a combination of A damage .M. pruriens (350 and 530 mg/kg BW) along with sodium arsenite minimized the toxic effect of arsenic on the testis. The combination of NAC + M. pruriens (350 and 530 mg/kg BW) along with sodium arsenite showed improved structural recovery with minimal damage and intact cellular structures with an improved cell count  showed a significant reduction in seminiferous tubule diameter, seminiferous tubule epithelial height, and increased tubular lumen. Sloughed germ cells with no maturation stages were observed and the tubular basement membrane was irregular or interrupted. The cell lining of Sertoli cells was damaged and in general, there were fewer spermatozoa observed in the central core of the tubule. Co-administration of  Figures- and -2.M. pruriens (530 and 700 mg/kg BW) showed improved cellular structure. Clear sinusoidal regions were evident. Inflammatory changes were reduced. The combination of M. pruriens and + NAC showed reduced inflammatory and degenerative changes due to sodium arsenite  with sodium arsenite reduced the toxic effects of arsenic. There was damage to the cellular lining of the duct system. Higher doses of M. pruriens (530 and 700 mg/kg BW) showed minimal damage and an improved histological structure in comparison to the lower dose. The kidney tubules exhibited acidophilic cytoplasm. The combination of M. pruriens (350 and 530 mg/kg BW) + NAC showed results that were similar to sodium arsenite + M. pruriens (700 mg/kg BW)  with sodium arsenite was found to attenuate the adverse changes observed with arsenic exposure. The study suggests that a diet supplemented with M. pruriens can ameliorate the undesirable changes in rats exposed to sodium arsenite.Excessive arsenic ingestion leads to significant damage to various body tissues , making the individual prone to further complications. Sodium arsenite-exposed animals showed impaired hepatic and renal function and reproductive toxicity. The supplement of PLC, KLB, and APR: Contributed to the conception and design of the study. PLC: Performed the experiment and wrote the manuscript. PLC, KLB, and APR: Statistical analysis. KLB and APR: Reviewed the manuscript. All authors have read and approved the final manuscript.


Results: RP differed from controls by HRF-ER, HRF-ON and EZ-length (p < 0.001). D-A and D-V were narrower and A-SO2 and V-SO2 were higher in RP (p ≤ 0.001). Within RP, significant interactions were found between the HRF-ER* group and: BCVA, EZ length, D-A, A-SO2 and A-V SO2 (p ≤ 0.018). The HRF-ONL* group interactions were significant for: BCVA, EZ length, D-A, A-SO2 and A-V SO2 (p ≤ 0.014). Conclusion: The present study highlights the presence of HRF to reflect the vascular, morphologic and metabolic alterations in RP. These biomarkers seem to be associated with remodeling and apoptosis that occur with the progression of degeneration.Background: The presence of hyperreflective foci (HRF) in retinitis pigmentosa (RP) is a potentially new finding. We investigated the presence of HRF in SD-OCT images in eyes with RP and its relation to vascular, morphologic and metabolic findings in RP. Methods: The study was performed on 42 RP patients and 24 controls. Using SD-OCT, we calculated the amount of HRF within the entire retina (HRF-ER) and the outer nuclear layer (HRF-ONL). Retinal vessel diameters (μm) and oxygen saturation (%) values were measured using Oxymap T1. We evaluated the mean diameter in retinal arterioles (D-A) and venules (D-V), the corresponding oxygen saturation values (A-SO The most apparent and frequently recognized form of inherited retinal disease is retinitis pigmentosa (RP). Its prevalence is accounted to be approximately 0.025% and is nowadays one of the leading causes of blindness within the working population ,2,3. RP Typical signs in patients with RP include bone spicule retinal pigmentary changes progressing from the periphery centripetally, a waxy optic disc and a cystoid macular edema (ME) ,10,11,12A further hallmark is the retinal vessel attenuation, affecting primarily the arteriolar, and later on the venular vessels ,23,24.One novel biomarker measuring the metabolic demand in RP is retinal vessel oxygen saturation . Using rMuch attention is given nowadays to the presence of intraretinal hyperreflective foci (HRF) in OCT images as an additional well-recognized sign in RP ,39,40,41Since the introduction of retinal gene therapy, it is questionable which patient may benefit and at which stage of degeneration the therapy should be implemented. In addition, apart from dose recommendation, the route of administration, as well as, evaluation of the local and systemic immune responses after ocular gene therapy, are mandatory .Several preclinical studies showed that gene therapy, for example, using rAAV (recombinant adeno-associated virus) might induce retinal toxicity. The latter is confirmed by means of loss of photoreceptors causing the thinning of the outer nuclear layer (ONL) or migration of retinal pigment epithelium (RPE) cells into the neuroretina ,43. ThusThus, in the present study, we aimed to evaluate to what extent the presence of HRF in RP may reflect the vascular, morphologic and metabolic findings by means of retinal vessel attenuation, the presence of ME and/or epiretinal fibrosis and retinal vessel oxygen saturation.The demographic data of our RP patients and the control group are presented in Patients and controls underwent all required standard evaluations by an experienced specialist for inherited retinal diseases (M.G.T).All RP patients presented with a clinical picture of the disease. The clinical phenotype of RP patients was characterized according to the typical funduscopic findings, diagnostic imaging , and electrophysiology (reduced and/or scotopic negative full-field electroretinogram).All participants underwent a detailed ophthalmic examination, which included, among others, refraction best corrected visual acuity , intraocular pressure examination by applanation, biomicroscopy, and funduscopy. Prior to diagnostic imaging, pupils were maximally dilated .Inclusion criteria for controls were Caucasian and best corrected Snellen visual acuity at distance >0.8, refractive error  of <6 diopters.Exclusion criteria for patients and controls were: the above-mentioned inclusion criteria not fulfilled, as well as the presence of ocular and/or systemic pathology other than RP , unstable fixation, and retinal vessel imaging and/or OCT images with inadequate quality.2  were obtained by simultaneously selecting the main vessels in all four quadrants. Their difference, known to be proportional to the oxygen saturation of the retina , was calculated, as well.Briefly, optic disc-centered fundus images, with a 50° field span, were taken for each eye, starting with the right eye, with the spectrophotometric oximetry unit for retinal vessel oximetry (Oxymap T1). Fundus images were recorded using a camera system, Digital Camera Topcon TRC 50DX. At least two images were taken and more images were added, if necessary. The image with optimal illumination set at least 8% the scale was further selected for analysis. An optic disc-centered image protocol was used where two concentric rings were created in the peripapillary area: one with a radius of 2 optic disc diameters, and the other with a radius of 3 optic disc diameters. The region between these two circles defined the area of interest where all measurements were obtained . The sofFor the evaluation of the retinal structure, a spectral domain optical coherence tomography  was achieved. All images were performed with the macular thickness protocol (OCT Protocol: 1024 × 496 (HR) 20° (6.3 mm); ART (23); Q: 41) and high-definition image protocol (HD 5 Line Raster), which allow high resolution image quality. In both, controls and RP patients the amount of hyperreflective foci along the horizontal whole 3rd single scan were calculated: within the central entire retina (HRF-ER) and the outer nuclear layer (HRF-ONL). In the present study, all HRFs within the central horizontal single HD OCT scan, were counted. The size of the HRF was not taken into account.The presence of HRF was defined based on hyperreflective tiny particles located within the retina. We evaluated their presence and counted them manually within the 2500 [μm] from the fovea and along the horizontal scans. A subgroup analysis was then performed taking the entire retina thickness into account (HRF-ER) and separately along the outer nuclear layer HRF-ONL; . The cou®  which allows for taking the dependency of the left and right eye in the same subject into account and is suitable for repeated measurements, in addition to both eyes being analyzed. A linear mixed-effects model was performed for each pair of the tested methods, where one parameter of the tested pair is a dependent variable.For statistical evaluation, ANOVA-based linear mixed-effect models were used with SPSS2, V-SO2 and A-V SO2), were taken as independent variables.The mean vessel diameter measurements (D-A and D-V) and retinal vessel oxygen saturation values , the eye and the group-effects were taken into account. In our ANOVA-based mixed-effects model, the eye and the groups were treated as fixed factors and the subject as a random factor. Results are presented as adjusted means and SD for controls with the respective In total, 132 eyes of 66 subjects were enrolled in the study: 84 eyes of 42 patients diagnosed with retinitis pigmentosa were compared to 48 eyes of 24 age-matched controls. Participants were enrolled based on the criteria described in In general, the RP group differed from the controls by its structural, functional and metabolic findings .p < 0.001; The RP group revealed clear differences compared to the controls concerning the presence of HRF-ER and HRF-ONL (p = 0.001).In order to link the amount of intraocular affection as part of the photoreceptor degeneration in RP we calculated the HRF-ONL/HRF-ER ratio. Again, RP patients could well be defined from controls by this sign  peripapillary retinal vessel diameters (D-A and D-V) were narrower in the RP group than in controls (2 and V-SO2 were higher in patients with RP  when compared to controls . The corresponding A-V SO2 difference, which tended to be lower in RP [35.64% (±7.12%)] compared to controls [37.61% (±2.87%)], did not reach statistically significant values (The mean (±SD) A-SOt values .p <0.001).The EZ line within the 5000 μm length of horizontal OCT scan was intact in the control group, whereas in the RP patients it showed interruptions and was significantly shorter at 2511.28 μm , structural  and metabolic  alterations a linear mixed-effects model, was performed.The interaction effect possibly indicates a different dependence of the number of HRF (HRF-ER and HRF-ONL) on the functional and structural variables between study groups. Therefore, we started with evaluation of the interaction HRF* group effect against the functional (BCVA), structural  and metabolic findings data , D-A (p = 0.018), EZ length (p < 0.001) and A-SO2 (p = 0.002) and A-V SO2 (p = 0.005).For the average values, the HRF-ER* group interactions were statistically significant only within RP  for the p < 0.011), D-A (p = 0.002), EZ length (p < 0.002) and A-SO2 (p = 0.008) and A-V SO2 (p = 0.014).For the average values, the HRF-ONL* group interactions were statistically significant only within RP  for the p < 0.001), but also with the A-V SO2 (p < 0.049). In addition, the higher HRF-ER count correlated with the presence of ME (p = 0.013), as well as with the simultaneous presentation of ERF*ME (p = 0.017).In the presence of macular edema in the RP group the higher amount of HRF-ER as well as of HRF-ONL correlated significantly with the BCVA, EZ-length (p < 0.001). This means, that when HRF were present in the ONL, they were found in the entire retina.For both, RP group and controls significant interactions were found between the number of HRF-ER and HRF-ONL . Their prevalence has been related to the progression of atrophy in age-related macular degeneration , to the Regarding RP patients, several studies have reported the HRFs to be available not only in the outer retinal layer but also in the choroid ,39,40,52There are several hypotheses explaining the origin of HRF in general. Some studies suppose that the HRF represents an inflammatory component like microglia or migrated RPE and/or the extravasation of lipoproteins following the breakdown of the blood-retinal barrier ,54. It hIn retinitis pigmentosa, based on the results of immunohistochemistry, other studies suppose the presence of HRF to reflect the degenerative process in response to apoptosis. In cadaver eyes with RP, a prominent neurite spouting of the rod, amacrine and horizontal cells, have been confirmed. These changes in the inner retina have been discussed to be a manifestation of regeneration on the part of the remaining inner retina even at the expense of normal visual function .Following that, and supported by other histopathology studies ,58, the Noticeably, the number of HRF in the outer nuclear layer  has beenp < 0.001). The same held true for the HRF-ONL* group interactions with the EZ length (p < 0.002). Correspondingly, we revealed significant interactions between the HRF-ER* group and BCVA (p < 0.011), but also HRF-ONL* group and BCVA (p = 0.011).In agreement, we also found a correlation between the HRF-ER* group and the EZ length  but also to the simultaneous presentation of ERF*ME (p = 0.017). These findings therefore suppose the presence of ME and/or ERF to be a part of remodeling in response to more advanced stage of photoreceptor degeneration.Furthermore, in our study, we found that the higher HRF-ER count in RP was linked to the presence of ME .Therefore, a relationship between RPE, photoreceptors, the choroidal morphology, and choroidal vasculature seems to be related to visual impairment and residual retinal function . Since RBased on the link found in the present study between the amount of HRF, retinal vessel attenuation and reduced oxygen use, the degeneration of photoreceptors seems to lead to structural and vascular remodeling. The measured metabolic demand in patients with retinitis pigmentosa probably serves as an additional hallmark allowing to evaluate the progression of the degeneration. This finding seems to reflect the decreased metabolic demand of the degenerating retina with the secondary remodeling of the inner retina and the choriocapillaris ,75,76,77p = 0.023). Furthermore, more attenuated D-V correlated well with the amount of HRF-ER and the HRF-ONL . All these findings reflect more altered vascular and structural remodeling with progression of photoreceptor degeneration.Several studies have shown that in progressive RP retinal damage morphologic and mictovascular changes are to be expected and found in the macular area. According to these studies the presence of an intact EZ line, the absence of ME and ERF on OCT translated into better BCVA ,80,81,82In conclusion, from the present study, we derive clear evidence that an increased number of HRF, attenuated peripapillary vessels, the presence of ME and ERF, and altered metabolic function in RP patients are linked to the more progressive and active stage of degeneration.This study concludes that by evaluating these parameters in RP a predictive model might be proposed. It would identify which individuals are at risk of developing a more aggressive photoreceptor degeneration of RP, and would suppose which ones may profit from future therapy. With arguably greater sensitivity, it would also point out those RP patients that may possibly develop therapy-induced retinal toxicity.Relatively small number of cases due to the rarity of the disease were included in the study.One further limitation is the absence of follow up, which could have helped to reevaluate the progression taking into account the number of hyperreflective foci and visual acuity.Compared to the study of Chu-Hsuan Huang  we dividEvaluation of the presence of HRF has been proposed in several studies to be done using the EDTRS grid of the macula. However, we decided to keep the data analysis using horizontal scans, so that to be able to take the presence of ME und ERF in RP into account.One remaining open question in the present study is whether the HRF is also present in healthy controls. The presence of HRF in normal eyes and with aging has not been discussed until now. Some possible explanations could be the decreased vessel dispensability, the ongoing vascular structural remodeling and also changes in viscoelastic properties occurring with ageing . All of There are some limitations of the present study, as for instance:Therefore, further prospective, randomized and controlled studies with a larger number of patients are required.


The medial prefrontal cortex (mPFC) and β-adrenoceptors (βARs) have been implicated in modulating anxiety-like behavior. However, the specific contributions of the β2-AR subtype in mPFC in anxiety are still unclear. To address this issue, we used optogenetic and microRNA-based (miRNA) silencing to dissect the role of β2-AR in mPFC in anxiety-like behavior. On the one hand, we use a chimeric rhodopsin/β2-AR (Opto-β2-AR) with in vivo optogenetic techniques to selectively activate β2-adrenergic signaling in excitatory neurons of the mPFC. We found that opto-activation of β2-AR is sufficient to induce anxiety-like behavior and reduce social interaction. On the other hand, we utilize the miRNA silencing technique to specifically knock down the β2-AR in mPFC excitatory neurons. We found that the β2-AR knock down induces anxiolytic-like behavior and promotes social interaction compared to the control group. These data suggest that β2-AR signaling in the mPFC has a critical role in anxiety-like states. These findings suggest that inhibiting of β2-AR signaling in the mPFC may be an effective treatment of anxiety disorders. Anxiety disorders constitute the most common psychiatric illness, with lifetime prevalence and steadily increasing occurrence ,2. NorepThe locus coeruleus (LC) is the major producer of NE in the brain and LC activation triggers NE release to the basolateral (BLA), the central nucleus of the amygdala (CeA), and the cortex ,11,12,13Apart from the hippocampus and the amygdala, the medial prefrontal cortex (mPFC) also plays a critical role in anxiety and fear ,19,20,21Chemogenetic activation of LC to mPFC neurons instead of LC to spinal cord neurons induced aversive and anxiety-like behavior , indicatTo dissect the roles of mPFC β2-AR in anxiety-like behavior, we recruit optogenetics to specifically manipulate β2-AR activity ,27 and mSystem blockage of β-adrenergic receptors can reverse the anxiogenic effects induced by optogenetic activation of LC NE neurons . In addiFirst, to test whether β2-AR are expressed abundantly in mPFC, which consists of anterior cingulate cortex (ACC), prelimbic cortex (PL), and infralimbic cortex (IL) , we use n = 9) spent significantly less time or entered less in the center of the OFT compared to EYFP controls (n = 10) during optogenetic activation , which is a measure of anxiety that is responsive to chronic administration of typical antidepressants ,31. Afteβ-adrenergic receptors have also been implicated in social anxiety disorders and β-blockers are commonly prescribed as anxiolytics in the clinic . Given tIn addition to acute anxiety, we test how optogenetic activation of mPFC β2-AR affect the social interaction in mice in the three-chamber social interaction test a. The miβ2-AR (ADRB2) gene is found to be strongly associated with GAD in the elderly . Inhibitn = 6 mice).To specifically knockdown the expression of β2-AR in mPFC pyramidal neurons, we inject the Cre dependent virus of β2-AR-miRNA-mcherry with AAV-CaMKIIα-cre into mPFC . After 3n = 12) mice spent significantly more time in or displayed a greater tendency to enter the center of the OFT than EYFP controls (n = 13) . Then, we utilize the EZM to further test the anxiolytic effect of miRNA silencing of mPFC β2-AR. We found that the miRNA-β2-AR/CaMKIIα group showed increased time and entries in the open zone compared to the control group , while the total distance showed no difference  d,e, suggfference i.p = 0.0049), indicating that the mice with β2-AR knock down in mPFC showed obvious anxiolytic-like behavior. In addition, the total food consumption in homecage after the NSFT showed no significant difference (p = 0.0354).To further confirm the anxiolytic effect by miRNA silencing of β2-AR within mPFC CaMKIIα neurons, we used NSFT, a classical test for anxiety assessment. After 24 h fasting, the same group of mice performed the NSFT. We found that the miRNA silencing β2-AR/CaMKIIα mice showed significantly decreased latency to eat compared to the control group . However, in the presence of a novel mouse, miRNA silencing of β2-AR mice showed a significant increase in the time spent in the social zone compared to the control . Interestingly, there were no differences in the number of entries and distance in the social zone, suggesting that miRNA silencing β2-AR/CaMKIIα-expressing animals entered the social zone and moved similarly to the control, but remained there for a longer period (p = 0.0521 and 0.051).As opto-activation of β2-AR in the mPFC pyramidal neurons induced social-anxiety behavior , we hypoTo investigate how β2-AR in mPFC modulates acute anxiety-like and depression-like behavior, we utilized optogenetics and miRNA silencing to specifically manipulate the β2-AR activity and expression in vivo. In this study, we found that optogenetic activation of β2-AR in mPFC excitatory neurons induced acute anxiety-like behavior in OFT, EZM, and NSFT, and depression-like behavior in TST, and reduced social interaction. Furthermore, miRNA knockdown of β2-AR in mPFC excitatory neurons induced anxiolytic and antidepression effects and promoted social interaction.Past studies have shown contradictory results regarding the effect of down regulation of β2-AR in anxiety. The Zhu group found that β2-AR knockout mice showed enhanced preference for the closed arm in the elevated plus maze and increased preference in the dark zone of the light dark box test, suggesting that β2-AR knockout increases anxiety level. Furthermore, β2-AR knockout mice exhibited less immobility in TST, indicating that β2-AR knockout induced anxiety- and depression-like behavior . This coThe mPFC is one of the key regions in regulating anxiety and mood ,38. HoweIn addition to excitatory neurons, β2-AR is also expressed in inhibitory neurons, including somatostatin, parvalbumin, calretinin, and calbindin interneurons in the mPFC . The rolOur data reveals that β2ARs in mPFC regulate anxiety-like behavior, but the cellular and molecular pathway contributing to β2-AR function in anxiety needs to be further investigated. Dong group reported that lactate metabolism regulated by β-arrestin-1 contributes to β2-AR functions in improved memory formation . Also, kSome early literature in humans showed that oral administration of β2-AR antagonists did not appear to be effective in acute anxiety neurosis or chronic anxiety ,53,54. TThough we can selectively activate β2-adrenergic signaling in excitatory neurons of the mPFC by using chimeric rhodopsin/β2-AR (Opto-β2-AR) with in vivo optogenetic techniques, this approach comes with some limitations. First, this non physiologic effect via optogenetics could not completely mimic β2-adrenergic signaling. Second, we cannot limit the expression of this recombinant receptor in excitatory neurons that intrinsically express b2-AR.In summary, using optogenetic β2-AR activation and the miRNA silencing technique, our data support the conclusion that β2-AR in mPFC excitatory neurons play a critical role in acute anxiety-like behavior. However, further studies of these receptors, circuits, and pathways are needed. Our finding provides new insights that β2-AR in the mPFC excitatory neurons regulates anxiety-like behavior and extends our understanding of the development of anxiolytic therapy.All the experimental work was carried out on adult male C57BL/6J mice (6–8 weeks old). They were kept in the cages with 24 h access to water and food chow. The animals were maintained in a holding room with a constant room temperature of 25 °C and a 12:12 h light and dark cycle. The experimental trials involving mice surgery were performed in accordance with the guidelines laid down by the Committee on the Use and Care of Animals, City University of Hong Kong and Department of Health, Govt. of Hong Kong SAR, Reference No. (20-17) in DH/HT&A/8/2/5 Pt. 1 and (20–112) in DH/HT&A/8/2/5 Pt. 1.AAV2/9-ef1a-Dio-opto-β2-AR-EYFP (Addgene plasmid No. 20948), AAV2/9-ef1a-Dio-EYFP, AAV2/9-CaMKIIα-cre, were packaged by Taitool Bioscience, Chine. To achieve the knockdown efficiency, the BLOCK-iT Pol II miRNAi expression vector kits were used . Six pre-miRNA sequences for β2AR receptors (β2AR receptors-miRNA) and a negative control sequence (NC-miRNA) were designed using Invitrogen’s RNAi Designer, created, and cloned into a pAAV-CMV-bGI-mCherry-miRNAi vector . The knockdown efficiency was then assessed by co-transfecting EGFP-tagged β2AR receptors with the β2AR receptors miRNA vectors in the human embryonic kidney (HEK293) cell line. The knockdown efficiency was confirmed by a decrease in the fluorescence signal or protein expressed by the EGFP-β2AR receptors vector. The sequence with the highest knockdown efficiency was chosen as follows: β2AR receptors -miRNA, TGCCTTCAATCCTCTTATCTA. The selected oligos were then either cloned into the linearized pAAV-ef1a-Dio-mCherry-miRNAi vector  using T4 DNA ligase. The plasmids were packaged into the AAV2/9 virus by calcium phosphate transfection with capsid and helper vectors on HEK293 cells. The titer of virus was determined by qPCR.Mice were anaesthetized using ketamine (100.0 mg/kg) and xylazine (8.0 mg/kg). Mice were placed on a stereotactic frame (RWD Instruments). Virus was injected into the mPFC bilaterally  using a modified microliter syringe (Hamilton) with a 32-gauge needle at a slow rate of 0.1 μL/min. After the injection was completed, the injection needle was left for an additional 9 min before it was slowly withdrawn totally. For the optogenetic experiment, optic fiber  was placed above mPFC with 10 degrees , and secured to the skull with dental cement . Then, mice were put to a heat pad to recover from anesthesia.12 v.g./mL, 0.10 μL, bilateral into mPFC, taitool Bioscience), AAV2/9-CaMKIIα-cre , AAV2/9-ef1a-Dio-miRNA-β2-AR-mcherry, AAV2/9-ef1a-Dio-NC-mcherry . All viral vectors were aliquoted and stored at −80 °C until use.The following viral vectors were used: AAV2/9-ef1a-Dio-opto-β2-AR-EYFP , AAV2/9-ef1a-Dio-EYFP  in PBS. The brains were extracted, postfixed overnight in 4% PFA at 4 °C, and cryoprotected in 30% sucrose. Brains were sectioned to a thickness of 30 mm using a sliding freezing microtome (Leica SM2010R) and preserved in a cryoprotectant 30% ethylene glycol, in PBS). Free-floating sections were washed in PBS, incubated for 1 hr in blocking solution  and 0.3% Triton X-100 in PBS), and incubated overnight at 4 °C with primary antibodies  in 0.1% Triton and 3% NGS in PBS. Sections were then washed with PBS 4 times and incubated for 2.5 h at room temperature with secondary antibodies  in PBS. Finally, sections were washed in PBS 4 times, mounted on slides and sealed with mounting medium . Mounted slides were imaged using an inverted laser scanning confocal microscope .For animal behavior study, all behavioral tests were performed in mice 9–12-week-old. We have 19 mice for the optogenetic experiment , and another 25 mice for microRNA silencing experiment . During optogenetic experiment, the optic fiber was connected to a laser source using an optic fiber sleeve. Opto-β2AR mice and control mice underwent the same procedure and received the same intensity of laser stimulation , 8–10 mW power; no seizure-like behavior observed for all the stimulation) continuously via optic fiber , during The test was conducted in a square open arena (500 × 500 × 500 mm) to quantify animals’ locomotor activity and anxiety-like behavior. The mice were positioned in the arena for 6 min under natural light condition (160 lux). For the optogenetic experiment, mice were kept in the OFT for extra 6 mice after optogenetic activation. The arena was cleaned with 30% ethanol between each test. The center area of OFT was defined as a square which occupied 50% the total area of the arena. The exploration distance, time in center area, and entries of the center zone were measured using Anymaze  tracking software.The EZM apparatus was made of grey plastic, 2000 mm in circumference, comprised of four 500 mm sections (two closed and two opened) to test anxiety-like behavior in mice. The apparatus was elevated 520 mm above the floor, and it had a path width of 45 mm with a 4 mm lip on the open section. The test was performed for 6 min under a condition of natural light (160 lux). For optogenetic experiment, mice will be kept in the EZM for an extra 6 mice after optogenetic activation. The exploration distance, time in the open zone, and entries to the open zone were measured using Anymaze  tracking software.The test was performed in a round open field arena . Food pellets were positioned in the center of the arena. The amount of food was about 5 g. For this experiment, mice were food restricted for a period of 24 h. After food restriction, the experiment was carried on by placing the animals in the corner of arena and measuring the time to approach and eat a pellet of food. The test duration was 6 min. Latency to approach and eat the food pellet were used to measure anxiety-like behavior using Anymaze  tracking software. Subsequently, animals were allowed to eat the food pellet for additional 5 min in their home cage. The total food pellet was weighed to calculate the amount of food intake.All mice were subjected to the tail-suspension test for a duration of 5 min. The mice were suspended by hanging their tails with adhesive plaster in an individual compartment of 400 mm height away from the floor. The immobility time were measured when mice were entirely motionless to test behavioral despair, which is typical anxiety-like behavior using Anymaze  tracking software.The social interaction behavior was performed in a rectangular arena  containing three chambers. Each chamber was 200 × 200 mm, divided by plastic walls with an open middle section to allow mice to freely move from between chambers. Two identical cage-like cylindrical containers were positioned in the middle of right and left chamber, one for each side. On the first day of this test, testing mice were put into the center of the arena for a 6 min duration of habituation, allowing them to freely move from each chamber; 24 h later, on the second testing day, a wildtype c57 mouse that had never approached the testing mice were put inside one of the containers  as a stranger. Testing mice were placed in the center of the middle chamber. Stranger mice were changed between each trial and the arena were cleaned by 30% ethanol. Each test duration was 6 min and the exploration distance, time in the social zone, and entries of social zone were measured using Anymaze  tracking software.p < 0.05, ** p < 0.01, *** p < 0.001, as determined by Student’s t-test (unpaired). Statistical analyses were performed in GraphPad Prism 9 .All data are expressed as mean ± SEM. Statistical significance was taken as *


Placemaking is a collective process that happens every day. However, little has been written about the ways older African Americans and Latinx immigrants create place in their everyday lives. Less has been said about the ways these efforts are examples of civic participation in the context of systemic inequality and oppression. Results from this phenomenological study that applies an intersectional life course perspective shows how older Black and Latinx immigrant adults maintain, preserve, and build their place in a society that continuously threatens their erasure. These results shine light on the ways Black and Latinx communities work to maintain the physical space , preserve their culture and history, and build structures to ensure access to resources that will empower their communities. Their efforts across the micro, meso, and macro social environments and across time, demonstrates a concerted effort towards creating a dignified and just place in our society to nourish and sustain future generations.


Ribosomes are complex macromolecules assembled from 4 rRNAs and 79 ribosomal proteins (RPs). Their assembly is organized in a highly hierarchical manner, which is thought to avoid dead-end pathways, thereby enabling efficient assembly of ribosomes in the large quantities needed for healthy cellular growth. Moreover, hierarchical assembly also can help ensure that each RP is included in the mature ribosome. Nonetheless, how this hierarchy is achieved remains unknown, beyond the examples that depend on direct RP-RP interactions, which account for only a fraction of the observed dependencies. Using assembly of the small subunit head and a disease-associated mutation in the assembly factor Ltv1 as a model system, we dissect here how the hierarchy in RP binding is constructed. Our data demonstrate that the LIPHAK-disease-associated Ltv1 mutation leads to global defects in head assembly, which are explained by direct binding of Ltv1 to 5 out of 15 RPs, and indirect effects that affect 4 additional RPs. These indirect effects are mediated by conformational transitions in the nascent subunit that are regulated by Ltv1. Mechanistically, Ltv1 aids the recruitment of some RPs via direct protein-protein interactions, but surprisingly also delays the recruitment of other RPs. Delayed binding of key RPs also delays the acquisition of RNA structure that is stabilized by these proteins. Finally, our data also indicate direct roles for Ltv1 in chaperoning the folding of a key rRNA structural element, the three-helix junction j34-35-38. Thus, Ltv1 plays critical roles in organizing the order of both RP binding to rRNA and rRNA folding, thereby enabling efficient 40S subunit assembly. In all forms of life, ribosomes are responsible not just for producing the right amount of the correct protein, but also the detection of damaged mRNAs. Both functions depend on the functional integrity of the ribosome pool, as well as efficient ribosome assembly, as reduced ribosome numbers can affect both mRNA selection , 2, as wIn eukaryotes, ribosomes are assembled from their constituent 4 ribosomal RNAs (rRNAs) and 79 ribosomal proteins (RPs) with the help of ~ 200 assembly factors (AFs), which are largely conserved from yeast to humans. These factors integrate rRNA processing with binding of RPs, chaperone rRNA folding, allow for regulation, prevent premature translation initiation, and facilitate quality control .after Rps19, The binding of ribosomal proteins (RPs) to ribosomal RNA (rRNA) is very hierarchical, as first characterized by Nomura and Nierhaus –9. A potin vivo [The small subunit head is constructed from the 18S rRNA 3’-domain and is the last of the subunit substructures to emerge. Because of its relatively late assembly, individual assembly steps can be identified using cryo-electron microscopy , as in vivo –24, 26. in vivo , and thein vivo . While tThe assembly factor Ltv1 has emerged as a critical player in 40S head assembly , 28, 29.Using a mutation in Ltv1 that causes a rare dermatological condition, LIPHAK syndrome , as wellRecently, the same mutation in human Ltv1, Ltv1_N168S was recovered in two unrelated families suffering from LIPHAK syndrome, a rare dermatological disorder characterized by altered skin pigmentation arising from subcutaneous oxygenation defects . We reasLtv1 is a late-binding and acting 40S ribosome assembly factor. Thus, we wanted to confirm that the growth defect from the Ltv1_L216S mutation reflected a ribosome assembly defect, as expected. Absence of Ltv1 leads to defects in rRNA folding and recruitment of late-binding proteins Rps10 and Asc1 and reduces 40S ribosome levels , 26. MutTo test if the mutation affected the formation of 80S-like ribosomes, we used a previously described assay that takes advantage of the observation that 80S-like ribosomes accumulate to high levels in yeast strains depleted of the essential ATPase Fap7 , 21, 33.We and others have previously shown that the formation of 80S-like ribosomes requires the phosphorylation of Ltv1 , 29, as Phosphomimetic (aspartate) mutations in the Ltv1 phosphorylation site  can rescue the depletion of the Hrr25 kinase , and proThe observation that Ltv1_L216S cannot be phosphorylated by Hrr25, and that a phosphomimetic mutation cannot rescue the release or growth defects from the L216S mutation, suggest that Ltv1_L216S is positioned differently on pre-40S ribosomes, so that it is no longer accessible to the Hrr25 kinase, and such that S336, S339 and S342 no longer weaken binding when mutated to phosphomimetic aspartate residues. In contrast, the interface with Enp1 appears to remain largely unchanged: an Enp1 mutation, Enp1_WKK , rescues the non-phosphorylatable alanine mutations in Ltv1, Ltv1_S/A , becauseTogether, the data in this section demonstrate that Ltv1_L216S impairs ribosome assembly, because it cannot be phosphorylated and released, and strongly suggest that these defects arise because Ltv1 is mispositioned on pre-40S ribosomes.https://alphafold.ebi.ac.uk/), and then utilized the matchmaker function in Chimera [To better understand how the Ltv1_L216S mutation alters its function in the context of the genetic and biochemical data that indicate mispositioning of the mutant protein, we wanted to visualize the location of the mutation in the context of the pre-40S structure. Unfortunately, only a small portion of Ltv1 has been visualized in current cryo-EM structures , 15. We  Chimera  to placeOverall, this structure for Ltv1 and its placement in pre-40S does not lead to significant steric clashes, but rather is compatible with the pre-40S structure and earlier pre-40S structures . In the To further validate this structure and the placement of Ltv1, we next designed point mutants in ribosomal proteins, which are expected to disrupt Ltv1/RP interactions, and then tested their genetic interaction with Ltv1_L216S.Inspection of the structure for Ltv1 on the nascent 40S revealed an interaction between Ltv1 and lysine residues K75 and R76 in Rps3 . We therIn addition to interactions with Rps3, Rps15 and Rps20, which have been previously validated , 29–31, Together, these mutational and genetic analyses, combined with the previous biochemical data validate the structure of Ltv1. Additional validation comes from the analysis of rRNA mutations in the next section.The structure reveals that L216 is part of a short alpha-helical segment adjacent to helix 34 h34, . H34 is However, premature folding of l31 leads to misfolding of j34-35-38, as it stabilizes misfolded structures of the junction . The strTo test whether Ltv1 chaperones the folding of j34-35-38 directly, or rather indirectly affects its folding by preventing the premature folding of l31, or both, we carried out a series of genetic experiments. Deletion of snR35, a snoRNA which directs pseudouridylation of U1191 in l31, and prevents the premature folding of l31, results in misfolding of j34-35-38 . This isTo further confirm this conclusion, we next tested whether rRNA mutations in the helices that surround the junction and are adjacent to Ltv1 would be sensitive to the Ltv1_L216S mutation. We therefore mutated U1285, U1286 and A1422, which are part of a bulge at the top of helix 34, as well as G1288 and C1327, the base pair which closes h35 , and tesFor this purpose, we deleted the Ltv1 gene from a strain where RNA polymerase I has a mutation that renders it temperature sensitive, thereby shutting off all transcription from the endogenous rDNA genes above 32 °C . When suGiven that the Ltv1_L216S mutant is only partially functional, we wanted to use its defects to gain additional insight into the functions of the wild type protein. Our previous work had shown that Asc1 and Rps10 were substoichiometric in ribosomes lacking Ltv1 , suggestTo better understand the molecular basis for the loss of many head RPs and determine whether additional rRNA regions are misfolded in the ribosomes from the L216S mutant yeast, we utilized DMS-MaPseq to probe the structure of mature 40S subunits from yeast expressing wild type Ltv1 or L216S Ltv1. As previously described , 38, ribThe Ltv1_L216S mutation impairs movement of Tsr1 to the beak).Six of the 18S rRNA residues with altered DMS accessibility are located in the 40S head. In addition, there is an additional cluster of 5 residues with altered DMS accessibility on the subunit interface . This clFour of the residues that are more DMS accessible in ribosomes from Ltv1_L216S cells, A1189, A1196, C1197 and A1515 are directly adjacent to residues in Ltv1 , furthernot depleted from the head of the 40S subunit in ribosomes from Ltv1_L216S cells , which mutates an interaction between Tsr1 and rRNA in the head , Rps15_S136F, which leads to defects in the resulting ribosome population . HoweverSaccharomyces cerevisiae strains  were grown at 30°C for >16h in YPD to deplete Fap7, and harvested in the presence of cycloheximide as described , 33, 43.5,000–7,500 OD of clarified lysate (~ 200 μl) were loaded onto a 10–50% sucrose-gradient and centrifuged at 40,000 rpm for 2 hours using a SW41Ti rotor before fractionation into 700 μl fractions. Northern blot samples were prepared by phenol chloroform extraction on 200 μl of each fraction. For Western blotting, fractions were mixed with SDS loading buffer, and ran on an SDS-PAGE gel, transferred and probed with the appropriate antibodies, as explained in , 33, 43.Northern blots were prepared from ΔLtv1 strain supplemented with either Ltv1, or Ltv1_L216S. The cells were grown overnight in minimal media, diluted into fresh YPD and harvested. Total RNA was isolated through phenol/chloroform RNA extraction, denatured in formaldehyde, and separated on an agarose gel. The following probes were used: 20S: GCTCTCATGCTCTTGCC; 18S: CATGGCTTAATCTTTGAGAC; 25S: GCCCGTTCCCTTGGCTGTG; and U2: CAGATACTACACTTG.600 for 48 hours while the plate was shaking at 30°C.Cells were grown in glucose minimal media or YPD overnight, diluted into fresh minimal media or YPD for 3–6 hours, before diluting them into a 96-well plate at a starting OD of 0.04–0.1 and placed in Synergy.2 plate reader (BioTek) to record ODrRNA mutants were grown in minimal media or YPD at 30°C overnight and diluted into fresh YPD to incubate for 3h at 30°C before changing the temperature to 37°C for 3h. Cells were then inoculated into YPD in a 96-well plate, where the growth was recorded for 48 hours at 37°C.2) supplemented with 1 mg/ml heparin, 1 mM benzamidine, 2 mM DTT, and protease inhibitor cocktail (Sigma-Aldrich) and lysed under liquid N2. The cell lysates were thawed and clarified before layering 200 μl onto 500 μl sucrose cushion and centrifuging at 70k rpm for 65 min in a Ti110 rotor. The supernatant was discarded, and the pellet containing the ribosomes was resuspended in high salt buffer  and layered onto another 500 μl sucrose cushion. The tubes were centrifuged at 100k rpm for 75 min, and the pelleted ribosomes were resuspended in subunit separation buffer , incubated for 1h at 37 °C, and loaded onto a 5–20% sucrose gradient  and centrifuged for 8 h at 30k rpm. To isolate the separated subunits the gradients were fractionated and 40S subunits collected, buffer exchanged into ribosome storage buffer (ribosome buffer with 250 mM sucrose and 2 mM DTT) and concentrated to be flash frozen and stored at −80°C for subsequent mass spectrometry or Western analysis.Cells were resuspended in ribosome lysis buffer Purified ribosomes were prepared as stated above and precipitated in acetone. Three biological replicates of WT Ltv1 and Ltv1_L216S were analyzed.2, and 0.2 μM RNaseP RNA at 30 °C for 5 min. DMS reactions were stopped by the addition of 0.4 volumes stop buffer , and purified using phenol chloroform precipitation.Prior to flash freezing the purified ribosomes were treated with or without 1% DMS (Sigma-Aldrich) in the presence of 80 mM Hepes pH 7.4, 50 mM NaCl, 5 mM Mg(OAc)2 at 94 °C for 10 min. Fragments were separated on a denaturing 15% gel, and fragments between 50–80 nt were cut out and RNA was eluted overnight in RNA gel extraction buffer , 0.25% vol/vol SDS (sodium dodecyl sulfate). Eluted RNA fragments were dephosphorylated using T4 PNK (NEB), ligated to an adaptor using T4 RNA ligase II truncated KQ(NEB), and then gel purified. TGIRT III (InGex) was used to reverse transcribe RNAs in 50 mM Tris HCl, pH = 8.3, 75 mM KCl, 3 mM MgCl2, 1 mM dNTPs, 5 mM DTT, and 10 U SUPERase·In (Invitrogen) for 1.5 h at 60 °C. The RNA was later hydrolyzed by 250 mM NaOH and the cDNA was circularized using CircLigase II (Lucigen). The circularized cDNA was shipped to the Scripps La Jolla Genomics Core for final library preparation with Illumina sequencing adaptors and sequenced using single-end sequencing on the Illumina NextSeq 500 platform.RNA library preparation was adapted from  and perfhttps://github.com/borisz264/mod_seq). The adaptor sequence (GATCGGAAGAGCACACG TCTGAACTCCAGTCA) was trimmed from raw sequence via Skewer [DMS-MaPseq data processing followed a GitHub pipeline . The HA-antibody was from Supplement 1