Case Presentation. This is a case of acute inflammatory pericarditis with a small pericardial effusion after receiving the first dose of AstraZeneca COVID-19 vaccine in a healthy adult patient who had no other symptoms suggestive of other viral illness in addition to the negative COVID-19 PCR. A 48-year-old healthy male presented nine days after receiving the first dose of COVID-19 AstraZeneca vaccine. The symptoms started three days after the vaccine, when he complained of progressive retrosternal chest pain with low-grade fever and generalized fatigue, followed by exertional dyspnea after a few days. The diagnosis of acute inflammatory pericarditis with small pericardial effusion was established, and the patient was accordingly treated. One week later, the patient showed significant clinical improvement with the resolution of his pericardial effusion. After 39 days, there was a significant radiological resolution of signs of acute pericarditis.  Clinical trials of the COVID-19 vaccine reported the safety and efficacy of mRNA vaccines (AstraZeneca) to help control the disease. Few previous reports have shown various side effects associated with COVID-19 vaccines that vary in severity. The possibility of pericarditis and myocarditis has been observed in people who have received an mRNA COVID-19 vaccine which we are reporting. Acute inflammatory pericarditis can be a rare presentation after receiving the first dose of this vaccine, and it is enriching to share such rare presentations in the era of COVID-19 for better management and outcomes after vaccination.  The ongoing COVID-19 outbreak is still under investigation, and guidelines are regularly modified since we are continuously learning about this novel disease, although multiple vaccines have been shown to be effective against COVID-19. However, we report a case of unique clinical manifestation that has not been reported widely in the literature, after receiving the first dose of AstraZeneca COVID-19 vaccine, and that it may help raise awareness of the possible diagnosis and the possibility of inflammatory pericarditis after COVID-19 vaccination. The viral respiratory infection associated with the novel coronavirus disease COVID-19) that firstly emerged in Wuhan in China in December 2019 has spread out rapidly among people worldwide 9 that fi, 4. The A 48-year-old male with an unremarkable medical history presented with retrosternal chest pain aggravated by lying supine and relieved in a sitting position. Also, he had a low-grade fever, fatigue, and exertional dyspnea. The low-grade fever was mildly improving when the patient took paracetamol. The chest pain started three days after he had received the first dose of the COVID-19 vaccine AstraZeneca. A written consent form was obtained from the patient before collecting any information about his medical condition.Vital signs and physical examination were within normal limits. Initial laboratory workup including complete blood count, cardiac enzymes, liver function test, and renal function test was within normal limits including a negative COVID-19 PCR. However, the C-reactive protein and the erythrocyte sedimentation rate were elevated reaching 75 mg/L and 77 mm/h, respectively. In addition, the D-dimer test showed a high result reaching 1.44 mg/L. The patient's chest X-ray  showed fThe patient was seen after one week in the cardiology clinic where he showed significant clinical improvement, with the resolution of the chest pain and fever, with minor exertional dyspnea. A timely follow-up with the patient was set after 39 days of the treatment, where he showed total resolution of his symptoms except for mild dyspnea. An enhanced CT scan of the chest  showed mAbout 58 vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed, and in clinical trials, one of them is the AstraZeneca vaccine. . Once thThe occurrence of pericarditis after immunization is extremely rare, and the number of cases of pericarditis reported after immunization to other vaccines such as influenza and hepatitis B virus is low . On the The most common cause of pericarditis is a viral infection which could be direct or secondary immune-mediated. It has been suggested that vaccination may trigger an autoimmune response due to antigenic mimicry as well as due activation of quiescent autoreactive T and B cells . Our casIt is difficult to confirm a definitive link between pericarditis and the COVID-19 vaccine; however, a link may exist which is supported by the fact that the patient is a previously healthy male without a previous history of the same symptoms. Additionally, several case reports suggest that COVID-19 infection, COVID-19 vaccine, and other vaccines may trigger an autoimmune reaction . The diaAlthough pericarditis is emerging as a potential rare adverse effect after COVID-19 vaccination (AstraZeneca), it is rare and the majority of cases of myocarditis/pericarditis reported after mRNA COVID-19 vaccines have occurred within 1-5 days (median 2 days) following the second dose. However, every effort should be made to diagnose early any crucial cardiac complication followed by the COVID-19 vaccination. Although the overall benefit-risk of the COVID-19 AstraZeneca vaccine remains positive, yet the healthcare provider needs to maintain a high index of cardiac symptoms to prevent further consequences.


Smart health applications have received significant attention in recent years. Novel applications hold significant promise to overcome many of the inconveniences faced by persons with disabilities throughout daily living. For people with blindness and low vision (BLV), environmental perception is compromised, creating myriad difficulties. Precise localization is still a gap in the field and is critical to safe navigation. Conventional GNSS positioning cannot provide satisfactory performance in urban canyons. 3D mapping-aided (3DMA) GNSS may serve as an urban GNSS solution, since the availability of 3D city models has widely increased. As a result, this study developed a real-time 3DMA GNSS-positioning system based on state-of-the-art 3DMA GNSS algorithms. Shadow matching was integrated with likelihood-based ranging 3DMA GNSS, generating positioning hypothesis candidates. To increase robustness, the 3DMA GNSS solution was then optimized with Doppler measurements using factor graph optimization (FGO) in a loosely-coupled fashion. This study also evaluated positioning performance using an advanced wearable system’s recorded data in New York City. The real-time forward-processed FGO can provide a root-mean-square error (RMSE) of about 21 m. The RMSE drops to 16 m when the data is post-processed with FGO in a combined direction. Overall results show that the proposed loosely-coupled 3DMA FGO algorithm can provide a better and more robust positioning performance for the multi-sensor integration approach used by this wearable for persons with BLV. Mobility and wayfinding are significant obstacles faced by people with BLV, specifically in urban areas. Degradation of the visual system can lead to a dramatic reduction in mobility. It has been shown that 80–90% of people with BLV spend the majority of their time inside buildings, and 30% rarely leave home alone ,2.Accurate positioning is essential for localization and navigation in urban canyons. Pedestrians with BLV who live in urban areas could benefit significantly from an integrated navigation solution for use during the activities of daily living. Much research has been performed to improve the autonomy of people with BLV, especially their ability to explore their environment. Rizzo et al. developed an advanced wearable in the form of an instrumented backpack equipped with microcomputers and sensors; this wearable incorporates cameras, inertial measurement units (IMUs), and GNSS positioning to provide a more comprehensive and full-featured navigation solution .This study develops a real-time 3DMA GNSS-positioning system based on state-of-the-art 3DMA GNSS algorithms to advance the approach used for localization by the wearable. The integration of shadow matching and likelihood-based ranging 3DMA GNSS methods were selected to evaluate positioning hypothesis candidates’ likelihood scores. The position was then integrated with the velocity estimated by Doppler measurements using the FGO as loosely coupled.Several experiments were designed in New York City (NYC) to acquire GNSS data during urban commuting . A commercial-grade receiver, u-blox ZED-F9P, was connected to an Nvidia microcomputer, Jetson TX2. The performance of the proposed system was evaluated with the data recorded in a series of trips that took place on the lower east side of NYC . The remainder of this paper is organized as follows: 4ION , is an advanced wearable consisting of a backpack with wide-angle high-resolution cameras on the support straps; these cameras, with integrated microphones and IMUs, are connected to a light-weight, portable computer for real-time analysis [The VISanalysis ,6,7,8. T4IONVIS system can process 720p video at 10 fps (dependent on the scene/task) and is robust without adding significant cognitive load to the end-user. This wearable runs off a laptop-battery with 66 Wh at 0.5 kg, yielding 3 h of run-time function.The platform provides real-time feedback using a binaural bone conduction headset and a haptic interface, allowing obstacle avoidance and situational awareness. More specifically, this mobile platform enables the users to understand their dynamically changing environment, giving them the agency to travel and wayfind independently. Our current Precise and reliable positioning is required to support safe navigation services for persons with BLV. GNSS provides continuous positioning in the global frame in absolute coordinates. However, the performance of GNSS navigation in the urban environment is unsatisfactory. GNSS signals can be blocked or reflected over buildings, resulting in non-line-of-sight (NLOS) reception and the multipath effect . These eOne of the most frequently used approaches to integrating the GNSS with IMU is pedestrian dead reckoning (PDR) . The intA camera is another popular aid for an integrated positioning system, which can provide the receiver’s orientation in the local frame. Visual odometry (VO)  can extrResearch also suggests using a sky-pointing fisheye camera to detect the NLOS signal via image recognition algorithms ,14. TherBesides the aid of external sensors, improving the positioning accuracy of GNSS itself can definitely benefit the whole positioning system. Therefore, researchers are trying to identify and mitigate the NLOS error to improve the GNSS positioning alone. The consistency-check method  can deteDual-frequencies measurements also demonstrate good performance in mitigating the multipath effects and isolating NLOS errors. Researchers used the nature of the higher resolution of L5-band measurements to design a new weighting scheme; the weighted least squares (WLS) method has been demonstrated to improve performance .One popular approach to improve GNSS positioning in urban canyons is using a 3D building model to identify and even correct the NLOS reception error. Different research has proposed to simulate the propagation path and model the error for code  and carrShadow matching ,25 matchAnother 3DMA GNSS is the ranging-based method. The receiver location is determined by comparing the modelled and received pseudorange. Pseudorange measurements are modelled at each candidate. For the NLOS-predicted satellite, the NLOS reflection delay is also modelled based on a geometrical or statistical approach. The absolute position of the reflecting point has to be determined for the geometrical approach. A popular approach is ray-tracing ,28. It tPerformance of shadow matching and ranging-based 3DMA GNSS are different due to the healthy satellite and building geometry distribution. Shadow matching usually outperforms in the across-street direction, while ranging-based 3DMA GNSS obtains higher accuracy in the along-street direction. The complementary nature of the two approaches inspired researchers to integrate them. The latest study shows that an integrated solution of 3DMA GNSS can provide positioning accuracy of around 10 m or less in urban canyons with both single-frequency  and multHowever, most of these approaches only concern positioning in a single epoch. Hence the performance is not robust for continuous positioning. As a result, a temporal connection is required to improve the reliability of urban GNSS positioning. There is research using the Kalman filter (KF) and extended Kalman filter (EKF) to recursively update the recent state through the prediction based on past estimation and error of current measurements ,37. ReseAnother approach is using factor graph optimization (FGO) , which oDifferent research demonstrates that FGO can provide an excellent positioning performance. This study integrates 3DMA GNSS with velocity estimated by Doppler measurements as a loosely-coupled solution, and states were optimized via FGO. The integrated solution can provide a more robust trajectory for pedestrian applications, such as this wearable.This section introduces the proposed loosely-coupled 3DMA GNSS- and velocity-positioning system via FGO. The flowchart is shown in 3D models of New York City (NYC) were obtained from open-sourced repositories released by the Department of Information Technology & Telecommunications’ (DoITT) 2014 aerial survey  with a lA skymask is a skyplot with building boundaries for a single location. It is an array with a total of 360 entries that represents the azimuth angle from 0° to 359°. Each entry stores the highest elevation angle of the building blockage in degrees in the corresponding entry (each azimuth angle). Skymasks are generated in an offline stage. The intentional coverage area for the 3DMA GNSS was first selected, and then the 3D city models were downloaded. The models were imported into the Rhino 7 3D-modelling software , and the3DMA GNSS evenly distributes the hypothesis positioning candidates during the online stage around the initial position. After that, the simulated measurements are generated to be compared with the received measurements for each candidate. Due to their computational efficiency, this study integrates shadow matching and likelihood-based ranging 3DMA GNSS. The implementation can be found in .An effective hypothesis positioning candidates sampling is important for 3DMA GNSS. The sampling area must cover the receiver location to achieve the theoretically best performance. Enlarging the sampling radius ensures the receiver location is being covered. However, this creates a massive computational load for the low-end microcomputer, which is not practical for a real-time application. Required computational time is proportional to the number of sampled candidates and received satellites. The computational time is within the necessary output rate. Thus, we proposed to use the surrounding skymasks and principal component analysis (PCA) to determine the street direction and distribute the sampling candidates effectively.Candidate distribution is based on the weighted least squares (WLS) for the first epoch and using the previous epoch FGO solution as the initial position after it is available. An initial circle with a sampling radius, The averaged skymask is then converted to vectors in the Earth-Centered-Earth-Fixed (ECEF) frame together with the transformation matrix, Therefore, we can obtain the eigenvalues, Finally, we can filter the initial circle with the ellipsoid based on the determined eigenvalues and eigenvectors. The length of the semi-major and semi-minor axes are The distributed candidates are an ellipsoid with a semi-major axis of 50 m. The separation for each candidate is 4 m. The above settings were determined empirically and suitable for real-time processing on the low-end microcomputer used in this study. A semi-major axis of 50 m can cover the position error of the initial position in most cases. In comparison, separation with 4 m can reduce the number of distributed candidates while maintaining an acceptable accuracy level.The proposed distribution can effectively distribute the hypothesis position candidates based on the surrounding environment. The prevention of candidate distribution at the next block can potentially mitigate the local minima issue caused by the high similarity of building geometry, as shown in To conclude, the skymask context-based candidates sampling can effectively distribute the position candidates. It has two main advantages. The first is to reduce the computational load by reducing the number of distributed candidates based on the surrounding environment. The second advantage is that candidates are most likely distributed on the same street. Therefore, the local minima on the next street can potentially be excluded.For each candidate found at .Shadow matching evaluates the visibility consistency between the measured carrier-to-noise ratio ( (AGNSS) ,48 and p (AGNSS) .With likelihood-based ranging 3DMA GNSS, we model the pseudorange at each candidate position and compare it with the received pseudorange measurements. The NLOS predicted satellite at a candidate, likelihood-based ranging 3DMA GNSS remaps NLOS pseudorange difference to a LOS one using the distribution model.The integrated solution of 3DMA GNSS, The receiver location is then optimized via FGO as a loosely-coupled solution.This study also optimized the 3DMA GNSS solution as a batch via forming the graphical optimization. It is associated with FGO, connecting two consecutive epochs’ solutions with velocity. The overall structure of the FGO process is shown in The error factor between the 3DMA GNSS solution, x-, y-, and z-axis, respectively. And constant Receiver velocity, ) method . The err dataset  that covA constant velocity motion model  is incluThe cost function for the position estimation of the proposed loosely-coupled 3DMA GNSS via FGO is formulated as,A commercial-grade receiver, u-blox ZED-F9P, was connected to a microcomputer, Nvidia Jetson TX2. A total of four satellite constellations with a single frequency were enabled during the experiments: GPS L1, GLONASS G1, Galileo E1, and Beidou B1. We modified the open-source library RTKLIB  for the The ground-truth reference trajectory was obtained via post-processing. The pedestrian subjects who collected the data walked straight lines and made their best attempts not to veer. Starting and ending locations and locations in-between were labelled manually. We also equipped a smartphone during the experiment and recorded the device location output. We used the smartphone output location to interpolate the longitudinal speed and project the vector between starting and ending location.NMEA: receiver output solution.WLS: weighted least squares method [s method ; uses ps3DMA GNSS: snapshot state-of-the-art 3DMA GNSS with positioning hypothesis candidates [ndidates .LC-FGO (proposed): real-time forward (instantaneous) processed loosely-coupled FGO solution with integrated 3DMA GNSS and velocity.LC-FGO-PP (proposed): combined (forward and backward) processed loosely-coupled FGO solution with integrated 3DMA GNSS and velocity.The evaluation was aimed at comparing the proposed algorithms in both a real-time and post-processing manner, also with several conventional solutions:The optimization frame was under the ECEF coordinate system. The comparison is divided into root-mean-squared error (RMSE) and standard deviation (STD) positioning error in meters. Note that both LC-FGO and LC-FGO-PP share the same graph structure. Only LC-FGO-PP uses historical and future factors and is optimized in a combined direction forward and backwards.A total of 11 experimental navigation trips were conducted in New York City. The positioning results of different trips are shown in We first present a trip with a good positioning performance (Trip 6). It starts from a relatively open-sky area and travels along straight to a deep urban canyon. The plots are shown in In this experiment, it can be observed that the 3DMA GNSS outperforms WLS by twofold, and the positioning RMSE are 18.27 m and 39.17 m, respectively. Many solutions for WLS were located on the opposite side or on the wrong street, as shown in Trip 6 is followed by a navigation trip with bad positioning results (Trip 2). Trip 2 begins in a deep urban canyon with a walk along the street to a relatively open area which is the opposite to Trip 6. The map and error plot of this experiment are shown in Similar to Trip 6, both 3DMA GNSS and two LC-FGO algorithms outperform WLS. The RMSE of WLS, 3DMA GNSS, LC-FGO, and LC-FGO-PP are 59.15 m, 29.14 m, 33.56 m, and 24.66 m, respectively. However, the LC-FGO is not outperforming the 3DMA GNSS. The overall positioning error is larger than that in Trip 6 because the environment is more complex. The average skymask elevation angle is higher in Trip 2, resulting in a more severe NLOS reception that mostly occupies a large portion of the total received satellites. The main error comes from the last 200 epochs. The performance of 3DMA GNSS keeps fluctuating during this period. The natural difference between 3DMA GNSS and FGO results in the average performance of this experiment. 3DMA GNSS is a snapshot estimation, and each epoch performance is independent of the others. However, the FGO is different, especially for the forward FGO. The fluctuation of the forward FGO will keep accumulating errors in batch optimization. Therefore, the future estimation is affected. However, the combined FGO, LC-FGO-PP, has a much stronger constraint that tries to optimize the solution in both directions. As a result, the positioning error can be suppressed. Therefore, if the performance of LC-FGO has to be improved, marginalization analysis must be done to find the acceptable error of this graphical problem. And we must adaptively select the existing trustworthy information in the sliding window.Near the end of the experiment, some 3DMA GNSS solutions wrongly estimated the position of the next block, as shown in Lastly, we also demonstrate a vehicle case in Hong Kong using the same receiver. The experiment covers different scenarios of an urban city, from open-sky areas to deep urban canyons. The data can be found in . The datBoth 3DMA GNSS and FGO outperformed WLS in this case. RMSE of WLS, 3DMA GNSS, LC-FGO, and LC-FGO-PP are 14.92 m, 7.94 m, 8.09 m, and 5.80 m, respectively. Overall, the positioning outperformed what was noted for New York. There are two main reasons for the excellent positioning performance. Firstly, the local environments in NY and HK were different; the testing areas in NY were more urbanized, i.e., the average skymasks’ elevation angle at ground truths in all navigation trips was 58.6° and 46.9° for New York and Hong Kong, respectively. Secondly, measurement noise was notably higher in NY, likely secondary to motion variation . The vehThe HK dataset reveals a better pseudorange quality, and it is reasonable to expect better positioning performance.A similar conclusion can be made in this vehicle-mounted experiment based on the results. 3DMA GNSS and LC-FGO obtain similar performance in this data, but comparing the positioning error shown in One of the main contributions of this study is to develop a real-time positioning system. Therefore, the processing time of a single epoch solution is important to guidance for a real-time operation that needs to maintain an output rate of 1 Hz. The computational load is directly proportional to the number of distributed candidates (sampling radius) and available satellites. From the result, the average number of total received satellites (including LOS and NLOS) and sampled candidates are 26 and 1143, respectively. The processing time for a single epoch solution is 0.91 s. In other words, the implemented system can provide a real-time operation at a 1 Hz output rate. If a higher output rate is required, using GPU has a huge potential to accelerate the process for real-time applications, such as presented work on using GPU for ray-tracing simulation  and corrAnother important point for 3DMA GNSS implementation is the format employed to store the information of 3D building models. It is impossible for a microcomputer to generate skymask online or in real-time. Therefore, the skymask is pre-generated offline and stored in CSV format, as in . If we wThis study developed a real-time loosely-coupled 3DMA GNSS with a Doppler measurements positioning system via FGO, and skymask context-based candidate sampling. Our approach distributes the candidates more effectively and mitigates local minima issues. Based on the experimental results, the positioning RMSE of loosely-coupled 3DMA GNSS with Doppler measurements via FGO is around 21 m with STD 15 m (on average). Performance can be further improved when optimizing in a combined direction with RMSE reduced to about 16 m with a STD of 13 m. The FGO can provide a lower standard deviation error than the candidate-based 3DMA GNSS, which means that it can provide a smoother and more robust solution.However, the performance of LC-FGO still has space to be improved. The results show that candidate-based 3DMA GNSS intermittently outperforms LC-FGO. The reason is the positioning error of 3DMA GNSS keeps contributing to the integration with Doppler measurements. Accumulated error results affect future batch optimization. An adaptive scheme should be developed to select the high confidence information in the sliding window. Moreover, tighter integration of the 3DMA GNSS with Doppler measurements should be effected to improve the performance.Furthermore, bad Doppler measurements result in wrongly estimated velocity. This will degrade the FGO performance. Doppler measurements error mitigation or correction is the key to improving the positioning. In future research, we will explore how 3DMA GNSS can more tightly integrate with Doppler measurements to provide a more robust positioning in the urban canyons for smart health applications and beyond.


The food supply system is facing important challenges and its sustainability has to be considered. Genome-editing technology, which accelerates the development of new variety, could be used to achieve sustainable development goals, thereby protecting the environment and ensuring the stable production of food for an increasing global population. The most widely used genome-editing tool, CRISPR/Cas9, is easy to use, affordable, and versatile. Foods produced by genome-editing technologies have been developed worldwide to create novel traits. In the first half of the review, the latest scientific findings on genome-editing technologies are summarized, and the technical challenge in genome sequence analysis are clarified. CRISPR/Cas9 has versatile alternative techniques, such as base editor and prime editor. Genome sequencing technology has developed rapidly in recent years. However, it is still difficult to detect large deletions and structural variations. Long-read sequencing technology would solve this challenge. In the second part, regulatory framework and approach for genome-edited foods is introduced. The four government ministries, including the Ministry of Environment, the Ministry of Agriculture, Forestry and Fisheries, and the Ministry of Health, Labour and Welfare (MHLW), started to discuss how the regulation should be implemented in 2019. The SDN-1 technique is excluded from the current genetically modified organism (GMO) regulation. The Japanese regulatory framework includes pre-submission consultation and submission of notification form. In the last part of this review, transparency of regulatory framework and consumer confidence were described. Since maintaining consumer trust is vital, transparency of regulatory framework is a key to consumers. The information of notification process on approved genome-edited foods is made public immediately. This review will help regulators build regulatory frameworks, and lead to harmonization of the framework between the countries. EcoRI specifically recognizes GAATTC and cleaves the sequence after the first G . Conventional restriction enzymes cleave only one predetermined sequence , whereas sequence-specific enzymes used in genome-editing technologies target any genome sequence. Genome-editing technologies have become popular because they are applicable for modifying most genomic regions. The technologies are now used in agricultural and medical fields1,2). The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) is useful for replacing mutated nucleotides causing genetic diseases with the original nucleotides3).Genome-editing technologies enable the modification of one or more nucleotides in the target region using an enzyme that recognizes a specific base sequence in the genome. Classic examples of enzymes used for editing genomes are restriction enzymes, which are often used in biochemical experiments, including analyses of PCR products by agarose gel electrophoresis and analyses of the genome of a microorganism by pulsed-field gel electrophoresis. For example, the restriction enzyme 4), and it has been used to study a variety of organisms from animals to plants5,6,7,8,9). The enzyme has also been used to elucidate specific gene functions by gene disruption and to create new plant varieties. Third, CRISPR/Cas9, developed in 2012, has been a game-changer. CRISPR/Cas9 is surprisingly easy to use and inexpensive, which has led to its rapid adoption in many research fields.There are three well-known sequence-specific enzymes in genome-editing technology. Zinc finger nucleases were first developed in 1996 and revolutionized genetic research, but the difficulty and cost of their construction prevented their general use in research. Second, the utility of the highly specific transcription activator-like effector nuclease (TALEN) was reportedAgrobacterium tumefaciens to insert target genes into the genome of plants. For example, herbicide-tolerant crops were created by introducing an herbicide resistance gene using the A. tumefaciens . The first commercialized papaya ring spot virus-resistant papaya was created, and commercialized in 1996. Mutation breeding has been performed since 1940s. After irradiating crops with γ-rays, the desired crop traits were selected through laborious selection steps and backcrossing. In addition, backcrossing has been used to restore the genetic background of the original crop. Mutation breeding using γ-rays or chemical mutagens is not regulated in any countries all over the world. On the other hand, EU has regulated GM plants in a technology-based manner and USA in a product-based manner. The regulatory approach for genome-edited products is discussed in many countries.Before the advent of genome-editing technology, it was necessary to use vectors derived from the soil bacterium 10). Since then, the Ministry of Environment and the Ministry of Health, Labour and Welfare (MHLW) initiated discussions on the handling of genome-edited organisms in Japan.At the 2018 OECD Conference on Genome Editing  in Paris, researchers and government officials discussed and shared information on recent technologies, applications and regulatory approaches in the member countriesThis review summarizes the latest information on genome-editing technologies, clarifies the technical issues, and describes how Japan regulates foods produced by genome-editing technologies (hereinafter referred to as “genome-edited products” or “genome-edited foods”).11,12,13,14). The first 8-12 PAM proximal base pairs form a seed region where mutations are less likely to be tolerated, and the PAM distal region is more mutation tolerant. To improve the sequence specificity, Cas9 orthologs were developed. Cas12a (Cpf1) was revealed to have higher specific cleavage activity15,16,17). The protospacer motif of Cas12a (21–24 bp) is longer than that of Cas9 (20 bp). Cas9 and Cas12a create a blunt end proximal to PAM and a staggered end distal to PAM, respectively (18).Genome-editing technologies have been used to add, remove, or substitute DNA sequences in specific genomic regions. CRISPR/Cas9 system is currently the most commonly used technique because it is easier to use and more accurate. An overview of CRISPR/Cas9 system is shown in 19). This method is expected to minimize adverse effects due to the shorter half-life of Cas9.-Genome-editing technologies can also enhance or suppress gene expression by inducing base substitutions or cytosine methylation without double-stranded DNA breaks. BE can substitute a single base by breaking one strand of DNA (left). This technique was expected to suppress off-target mutations. The cytosine-based editor (CBE), which converts cytosine (C) to thymine (T), and the adenine-based editor (ABE), which converts adenine (A) to guanine (G), were developed20,21,22,23,24,25). It was, however, later reported that unexpected genome-wide and transcriptome-wide mutations frequently occurred26,27,28). The base editing technologies were found to induce unintended base substitutions in mouse and rice DNA27,28). Therefore, ABE and CBE are still undergoing efforts to control unintended mutations. A new BE technique converting C:G to G:C was recently reported29). Prime Editing (PE), which induces base substitutions using reverse transcriptase, was developed30) (right). Although PE is a more flexible technique than ABE and CBE, it requires a complex vector construct. PE was used to edit bases in rice and mice with up to 22% of editing efficiency, however, gRNA scaffold insertion and substitution were detected20,31,32,33,34). PE3  is now reported to be more efficient35).Vectors encoding Cas9 protein and gRNA sequence have been used to introduce gRNA/Cas9. Recently, the Cas9 protein/gRNA complex can be introduced directly into cellsd of DNA , left. Tloped30) , right. CRISPR/Cas9 system has many tools, protocols, and experimental conditions. Therefore, a variety of results can be obtained with different genome-editing techniques. The frequency and outcome of genome editing at target and off-target sites vary widely depending on the experimental conditions.36), whereas base substitutions are rare. Insertions and deletions (indels) may result in frame-shift mutations that alter the amino acid sequence. Therefore, the technical issues to be evaluated after the absence of foreign DNA in the genome has been confirmed include the effects of new open reading frames (ORFs) around the target site. The potential toxicity and allergenicity of candidate proteins encoded by the new ORFs must be evaluated. SDN-2 is used to introduce desired base substitutions. Because base substitutions do not lead to altered ORFs, the issues to be addressed, if any, may include whether the desired phenotype is due to the base substitution. If foreign DNA is integrated into the genome, the products are considered to be GM foods . Unlike SDN-1, SDN-2 uses a DNA template for introducing base substitutions. Although the donor vector can be integrated into the genome, the integration can be detected by next-generation sequencing (NGS) and conventional PCR analyses. However, the template DNA may induce base substitutions outside the target site (off-target effect) regardless of CRISPR/Cas926,27,28). The off-target base substitutions should be considered in SDN-2. The repair outcomes are sometimes the same between SDN-1 and SDN-2. Since SDN-3 products contain foreign genes or short sequences, it is necessary to investigate the impact in terms of what the inserted genes are.Genome-editing technologies used to produce genome-edited foods are classified into the three categories of technique (site-directed nuclease (SDN)-1, SDN-2, and SDN-3) according to whether a DNA template is used or whether foreign DNA remains in the genome (left), thereby increasing the chances of translocations . The resulting product technically belongs to the SDN-3 category, but it may be more appropriate to consider it as an SDN-2 product . This type of product will not be regulated as a genetically modified organisms (GMO) if the process  is not disclosed. An appropriate NGS analysis will reveal the random integration of the vector used into the genome, regardless of the technique . Common technical issues for SDN-1, -2, and -3 include the formation of structural variations, including translocations . A report providing an overview of these genomic modifications was recently published38). The GMO Panel suggested that compared with the effects of conventional breeding, genomic modifications induced by SDN-1, SDN-2, or ODM are not associated with additional risks. The GMO Panel also indicated that analyses of potential off-target sites are not useful for risk assessments because of the limited availability of fully sequenced plant genomes39). The genome sequences of many organisms, including major crop species, are available in public databases, including OryzaGenome and GenBank (accession numbers start with GCA).In 2012, European Food Safety Authority (EFSA) released the report, “Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with a similar function”, which indicated that the main difference between the SDN-3 technique and transgenesis is whether a predefined genomic region is targeted. The SDN-3 technique can minimize the damage associated with the disruption of genes and/or regulatory elements in the recipient genome40,41,42). Therefore, the mutations detected are specific to the genome-edited products. It is difficult to assess the impact of a mutation occurring at a particular position in a gene . There are issues associated with SDN-1 because more than 99% of the off-target mutations can be removed during the backcrossing over several generations39,43). However, if backcrossing is performed for only two generations, 25% of the unintended mutations would theoretically remain in the genome. Is genome editing followed by two-generation backcrossing always enough to evaluate the safety? Genome-editing technologies are now widely used to modify diverse organisms 44,45,46). Some fruit trees are difficult to backcross.Draft genome sequences may be also useful for detecting mutations specific to genome-edited products because mutation analyses are generally performed to find mutations present only in genome-edited products, not in the original products. All mutations in the original products are subtracted from the corresponding genome-edited products using an appropriate pipeline, including GATK, BWA, and bcftools47). Moreover, CRISPR/Cas9 system induces complex rearrangements48,49). Thus, elucidating the genome-editing outcomes and the subsequent breeding conditions is important.There are generally no specific risks associated with the long-term presence of gRNA alone, whereas the presence of both gRNA and Cas9 may result in many unintended modifications. Recent research demonstrated that Cas9 alone can activate p53-inactivating mutations, leading to long-term irreversible changes in cultured cells50,51,52). The parallel use of Nanopore and Illumina sequencing technologies is becoming a powerful option for investigating genome sequences. In addition, DNA or RNA that encode Cas9 and gRNA, as well as the Cas9 protein/gRNA complex, is inserted into plant cells by methods using Agrobacterium species, biolistic bombardment, and nanoparticles. Plant viruses, polyethylene glycol, and de novo meristem induction may be applicable in the future53).The recent rapid advances in sequencing technologies, especially the Nanopore sequencing technology that produces a single read comprising up to 4 Mb, enable the assembly of full genome sequences−7. The deletions of 1–16 bp were the most common mutations (63%) induced by γ-irradiation (80–300 Gy), followed by large deletions  and single-base substitutions (13%). Medium-sized deletions (100 bp to 8 kb) were rarely induced by γ-irradiation54). The rate of naturally occurring mutations was estimated to be approximately 7 × 10−9 base substitutions per site per generation55,56), most of which are C:G to T:A transitions. The deletions exceeding 3 bp occurred at a rate of 0.5 × 10−9. The intergenic mutations (55%) were the most common, whereas non-synonymous mutations occurred less frequently (11%). Natural mutations and mutations induced by γ-irradiation mainly occur in introns and intergenic regions, slightly in exons.Before GM plants were developed, mutation breeding via γ-irradiation and the use of chemical mutagens, such as ethyl methanesulfonate, was used to generate plants with novel traits. Specifically, γ-irradiation initially triggers DNA double-strand breaks at different sites depending on the radiation dose, which results in the induction of various mutations in different genomic regions . Backcrossing and selection according to the phenotype are essential steps to establish the desired agricultural products. A recent study revealed that the final products have deletions shorter than 10 bp at a rate of nearly 2 × 10−9). The frequency of double-strand breaks at off-target sites depends on the cell type. The use of ribonucleoproteins comprising gRNA and Cas9 can reduce the chances of off-target mutations compared to using plasmid DNA57).Genome-editing technologies precisely induce DNA double-strand breaks at the target sequence at a much higher rate (approximately 0.2 or higher) than the spontaneous mutations described above (0.5 to 7 × 1058), and thus a γ-irradiation at 300 Gy can initiate 6,000–12,000 DNA double-strand breaks per cell. Spontaneous DNA damage occurs in the order of 10,000–100,000 events per cell per day59,60). These results suggest that many of the mutations induced by irradiation could be repaired inside cells.Mutation breeding by γ-irradiation can induce many random mutations. A γ-irradiation at a dose as low as 1 Gy can lead to 20–40 DNA double-strand breaks per cell61,62). ML and deep learning techniques will be soon used to establish in silico allergenicity prediction methods.To date, the safety of GM plants has been evaluated by examining the proteins encoded by the integrated genes and the potential proteins encoded by new ORFs in terms of their toxicity and allergenicity. Regarding SDN-1, foreign genes and sequences are not present in the final products (null segregant). However, new ORFs can emerge because of the introduction of indels in the coding region of an endogenous gene  is a neurotoxin present in scorpion venom. Pompilidotoxin in solitary wasp venom is only 13 amino acids long (RIKIGLFDQLSRL). Phalloidin and amanitin are cyclic peptides consisting of seven and eight amino acids, including D-amino acids. Among allergens, Api m 3 is considered to be the smallest allergenic protein (26 amino acids).63) and Oxford Nanopore64) is that they can be read much longer at a time. Using PacBio and Oxford Nanopore sequencing, long repeated regions can be analyzed, such as telomeres, centromeres, and low-complexity regions. Software for mapping, variant detection, and annotation can be used to study the alterations in genomes at single-nucleotide resolutions. An in-depth NGS analysis can decipher structural variants, including complex DNA rearrangements and chromothripsis65,66,67). However, the results vary among analysts. Standard guidelines for NGS analyses are urgently needed for risk assessment. Otherwise, the results of genome analysis may differ for each applicant.Many bioinformatics tools have been developed. For example, NGS is a powerful tool facilitating whole-genome analyses. To date, there are two technologies, short- and long-read sequencing technologies. The short paired-end sequencing technology in the Illumina platform is one of the most frequently used methods for analyzing genomes. The advantage of the long-reads in PacBioIn what fields have genome-editing technologies been studied? Recent trends were investigated through cluster analysis of published manuscripts describing research involving genome-editing technologies, especially CRISPR/Cas9. Research articles published between January 1, 2015 and October 20, 2021 were collected  and then separated into clusters based on t-distributed stochastic neighbor embedding (t-SNE), which is a dimensionality reduction technique to visualize high-dimensional data in a two-dimensional figure , followed by TALEN (3%). Rice (13%), tomato (7%), and wheat/barley (3%) were the most commonly studied plants, whereas pig (7%), chicken (2%), and cow (2%) were the main animal species that were investigated . The stuin vivo and ex vivo somatic cell genome-editing techniques developed to correct mutated sequences responsible for human genetic diseases have been widely studied and may be useful if the benefits overweigh the risks.Genome-editing technologies apply to the characterization of genomes of diverse organisms . In clinical applications, 68).Agriculturally important plants and animals are also being modified by genome-editing technologies. A variety of genome-edited plants would be commercially produced to increase yields and optimize the use of agricultural land . Genome-editing technologies could be used to achieve sustainable development goals, thereby protecting the environment and ensuring the stable production of food for an increasing global population. In recent years, there has been growing interest in genome-editing fish and other animals. However, animal welfare protocols and standards must be considered. In addition, several issues will need to be addressed, including whether the mass production of GM or genome-edited animals for food will be accepted by consumers. The increased use of genome-editing technologies in the agricultural field will depend on the understanding and acceptance of the general public. Genome-editing technologies may facilitate or enhance already controversial commercial practices in livestock breedingThe regulatory approaches for genome-edited products were studied in four different agencies, the Ministry of Environment, the Ministry of Economy, Trade and Industry (METI), the Ministry of Agriculture, Forestry and Fisheries (MAFF), and the MHLW. This chapter describes the results of the studies conducted by each ministry in chronological order.On February 8, 2019, the Ministry of Environment released a notification regarding organisms obtained through genome-editing technologies based on the Cartagena Act . The Cartagena Act does not regulate genome-edited organisms generated using RNA and recombinant proteins . Organisms generated by the transient expression of DNA construct encoding TALEN or CRISPR/Cas are also not covered by the Cartagena Act if they lack foreign genes or their fragments. Applicants are requested to provide the following information regarding genome-edited organisms:1) Proof that the organism does not contain the remnants of extracellularly processed nucleic acids or their replicated products, as stipulated in the Cartagena Act2) Taxonomical details regarding the modified organism3) Method describing the use of genome-editing technologies4) Modified gene and its functions5) Changes in the target traits induced by the modification6) Changes in non-target traits 7) Purpose of use of the organism8) Discussion regarding the possible effects on biological diversity when the organism is releasedThe METI requested that information be provided for use in open-field cultivation where containment measures have not been applied (https://www.meti.go.jp/policy/mono_info_service/mono/bio/cartagena/genome_yoryo.pdf).On October 9, 2019, the MAFF released information regarding the regulatory procedures for organisms generated by genome-editing technologies. The procedures are applied to plants and animals in the agriculture and fisheries industries. Self-cloning organisms and naturally occurring organisms  were excluded. Applicants were required to consult with the appropriate agencies to determine whether the generated organisms were fallen into self-cloning or naturally occurring species based on scientific evidence.This review focuses on genome-edited foods, and therefore the results of scientific discussion in the MHLW are described in detail. The MHLW also released the Food Hygiene Handling Procedure (https://www.mhlw.go.jp/content/000550824.pdf) and started the pre-submission consultation and notification system on October 1, 2019. Applicants were requested to provide the following information for the pre-submission consultation:1) Names of the item and breed as well as a summary of the current use and intended use of the food2) Method describing the use of genome-editing technologies and details regarding the genomic modifications3) Proof that there are no remaining foreign genes or their fragments4) Proof that the confirmed changes in DNA do not result in the production of new allergens that adversely affect human health or increase the abundance of known toxic compounds5) Whether the modification to increase or decrease specific components affects the metabolic system6) Year and month of marketing (if decided)Discussion of genome-edited foods on the SubcommitteeThe MHLW Subcommittee on GM Foods, Newly Developed Food Committee of the Food Sanitation Council under the Pharmaceutical Affairs and Food Sanitation Council discussed the regulatory procedures for genome-edited foods as follows. First, the term “genome-editing technologies” was defined. As mentioned in “1. Introduction”, genome-editing technologies use sequence-specific enzymes to create new functions. The editing outcomes of genome-edited products are equivalent to those induced by conventional breeding and radiation breeding, which are exempted from the current GMO regulation. To date, the products have been classified into GM foods if foreign genes and their fragments exist in the final products based on the rule of recombinant DNA technology . Therefore, the proof of the absence of foreign genes is a necessary condition for foods to be judged as genome-edited foods. Applicants should provide solid data confirming the absence of foreign genes and their fragments in the genome. Southern blot and PCR analyses have been used to produce the required proof, however, NGS is becoming a popular technique for that purpose. There was no discussion during the Committee members’ review process as to how short a gene fragment must be examined. In general, fragments shorter than 20-30 bases can be ignored unless they have any function.Next, it was discussed what molecular characterization of genome-edited foods applicants are required to declare. In most cases, genome-edited foods are produced by introducing indels (SDN-1). As the resulting frameshift may generate a novel protein, toxicity or allergenicity of the protein must be appropriately examined. BLAST, UniProt, and COMPARE databases are commonly used. The same approach as the current GMO regulation was considered appropriate for evaluating the safety of genome-edited foods. The genetic stability of genome-edited organisms must be studied over a few generations at least.in silico tools, such as CRISPRdirect (https://crispr.dbcls.jp) and Cas-OFFinder (http://www.rgenome.net/cas-offinder/). Applicants can design the specific target site. If in silico prediction tools detect off-target sites, PCR analysis can provide experimental evidence that the off-target mutation does not happen.Off-target effects were also discussed in the Committee. Genome-editing technologies work in a sequence-specific manner. Applicants can search for similar sequences to the target sequence in advance by using web-based Finally, the regulatory approach of genome-edited products in which metabolic pathways have been altered was discussed. When one of the genes involved in a complex metabolic pathway that can give an impact on many surrounding genes was changed, applicants are required to submit a diagram showing which gene was altered on the metabolic map and its relationship to other related genes. Based on this information, the effect on the metabolic system needs to be considered. For example, fatty acid profiles would change and influence on human health when genes related to the formation of unsaturated fatty acid are genome edited. Therefore, the changes in gene products that exist in the metabolic pathway need to be investigated. After the scientific discussion, the regulatory procedures for genome-edited foods were released on September 19, 2019. The pre-submission consultation and notification system was started on October 1, 2019.Applicants (developers) first submit a dossier containing the required information to the MHLW according to the regulatory procedures for foods produced by genome-editing technologies. The MHLW Subcommittee on GM Foods confirms the required information is included in the submitted documents. The most important criterion is the absence of any foreign genes or their fragments and backbone sequences of the vector used (null segregant) in the genome. If the Subcommittee members agree to judge the submitted product as a genome-edited product, the applicants can sell the product in the market after the proper notification form has been submitted. In addition, the MHLW will publish information explaining how the Subcommittee confirmed the product is safe. If the submitted product is considered to be a GMO, the MHLW will ask the Food Safety Commission of Japan (FSCJ) to assess its safety, similar to other GMOs. If the MHLW is unable to determine the safety of the submitted product at the Subcommittee level, even though the use of genome-editing technologies has been described and proof of the null segregant has been provided, the MHLW will consult with FSCJ to obtain a scientific evidence-based opinion. These steps in the regulatory framework are illustrated in Once a crop is designated as a genome-edited crop, it is considered the same as a crop obtained through conventional breeding. Therefore, backcrosses between genome-edited plants and conventional plants no longer need to be reviewed by the MHLW.The regulatory approach in the framework described above is guided by the following principles.The Subcommittee members discuss the contents submitted by applicants from a scientific standpoint while also taking into consideration the effectiveness.The process that led to the decision to be considered notification for genome-edited foods is made public. If new findings on genome editing technologies are obtained, the framework will be reviewed as necessary. The MHLW will facilitate risk communication to promote public understanding and acceptance.SIGAD3, which negatively regulates the GABA level, was knocked out by the insertion of one base using vectors encoding Cas9 and gRNA. Southern blot and extensive PCR analyses covering the entire region of the vector used proved the absence of foreign sequences. The toxic glycoalkaloid tomatine was undetectable in the final products. The off-target analysis was performed using CRISPRdirect and Cas-OFFinder as well as PCR amplifications followed by Sanger-based sequencing. The mutations were detected at the target and the predicted off-target sites (55 sites) by the analyses. Whether new ORFs encode allergenic proteins was examined using COMPARE and FARRP (https://farrp.unl.edu) databases. Genetic stability was confirmed through three generations . Tomatoes with increased GABA content were approved in 2020. In 2020, two genome-edited fish were submitted to the MHLW. The MHLW Subcommittee has initiated discussions on the regulatory procedures for genome-edited fish. The current safety assessments of GM foods have been conducted only for plants. The discussion centered on how to consider an event. In GM plants, a plant of a single genomic pattern  can be evaluated. However, fish are bred in groups. After five-time discussions with the Committee members and external experts, the Subcommittee decided that a population with identical genetic changes at the target site on both alleles  may be considered a single event for SDN-1 mutations. Two genome-edited fish species (sea bream and tiger pufferfish) were approved in 2021. Using Cas9 mRNA and gRNA, the myostatin gene mstn and the leptin receptor gene lepr were knocked out by 14- and 4-base deletions in sea bream and tiger pufferfish, respectively. Whole-genome sequencing and PCR analysis were conducted to reveal the absence of foreign sequences and off-target mutations. Protein allergenicity was investigated for the 10 and 61 newly emerged ORFs in sea bream and tiger pufferfish using FARRP and ADFS  databases, showing no potential allergens. Red Seabream and Tiger Pufferfish with increased muscle mass were approved on September 17, 2021 and October 29, 2021. Thus, three genome-edited foods have been approved to date in Japan.To date, three genome-edited foods have been notified. Genome-edited tomato containing 4- to 5-fold more γ-aminobutyric acid (GABA) than wild-type tomato was first approved on December 11, 2020 and released in the market. The tomato glutamate decarboxylase gene 69). However, the Food and Drug Administration (FDA) subsequently found the presence of plasmid sequences in the genome-edited cattle70). The developers reanalyzed the genomic sequence and confirmed the presence of the plasmid sequence71). Finally, the developed cattle were incinerated. To accelerate the development of genome-edited plants, new techniques such as de novo meristem induction and the use of RNA viruses as vectors72), will be applied, and large-scale genome-editing experiments will be conducted, which will allow for increasingly efficient genomic modifications.The number of plants and animals produced by genome-editing technologies is expected to increase. In assessing the safety of genome-edited foods, an appropriate analysis should be conducted to ensure that residual DNA sequences or changes in composition are not missed. In one case, the insertion of a plasmid DNA sequence was overlooked. In 2016, a hornless cow was developed using TALEN, and analysis immediately after its development confirmed the absence of foreign DNA73) and the “Executive Order (EO) on Modernizing the Regulatory Framework for Agricultural Biotechnology Products” in 201974). The Animal and Plant Health Inspection Service of the USDA issued the SECURE rule75), which applies to genetically engineered organisms modified by genome-editing technologies and recombinant DNA technology. Notably, the SECURE rule does not apply to SDN-1 products, which means that SDN-1 is no longer genetically engineered plants. Genetically engineered animals are under the jurisdiction of the FDA. Whether the FDA continues to be involved in regulating GM plants though the consultation is unclear because the oversight of genetically engineered crops is now the responsibility of the USDA. However, the problems associated with genome-edited hornless cattle have reinforced the need for input from the FDA to ensure food safety.The USA updated the regulatory framework according to the “Memorandum on Modernizing the Regulatory System for Biotechnology Products” in 201537). In addition, the scientific opinion on SDN-1, SDN-2, and ODM was released. In the opinion, EFSA GMO panel did not identify new risks specifically linked to SDN-1 and SDN-2 techniques38,39). In April 2021, the European Commission published the Commission Staff Working Document, “Study on the status of new genomic techniques under Union law and in light of the Court of Justice ruling in Case C-528/16”76). This document states that products generated by genome-editing technologies can also be obtained through conventional breeding and cisgenesis. Thus, applying different levels of regulatory rules to products of similar risk levels may not be justified. Strict risk assessment guidelines are not suitable for case-by-case assessments and make it difficult to revise risk assessment requirements based on the basis of scientific progress. The final decision has not been made on regulatory procedures for genome-edited foods.In the EU, EFSA published a report on the assessment of the safety of plants developed through cisgenesis/intragenesis and zinc-finger nuclease-3 (corresponding to the current SDN-3) in 2012In the United Kingdom (UK), Food Standards Agency (FSA) plays in regulating food and feed and how FSA intend to develop a new regulatory framework to support the objectives of the Genetic Technologies  Bill, which is currently progressing through the various stages of Parliamentary scrutiny. The regulatory framework would contain an authorization process for food and feed products produced using PB technology. This process includes safety, transparency, proportionality, traceability, and building consumer confidence.77), although the definition of “novel” is unclear. Health Canada announced that a new guideline will be issued soon.Canada has a product-based risk assessment framework for plants with novel traits. This framework, developed by the Canadian Food Inspection Agency, applies to products obtained using genome-editing technologies77,78,79).In 2015, Argentina established a case-by-case consultation process to determine whether genome-edited products comply with the GMO regulations set by the Argentine Biosafety Commission (CONABIA). In Brazil, the National Biosafety Commission (CTNBio) approved Normative Resolution No. 16, which includes updated regulations for novel plant breeding technologies. Under these regulations, SDN-1 and SDN-2 products are evaluated as non-GMOs on a case-by-case basis80). The situation is the same in other countries. Consumer acceptability of GMO has gradually increased. However, the ratio of acceptability is still low in Japan. The results of the web survey showed that consumer perceptions of “genome editing” was about 54% . Twenty percent of the survey respondents were positive about promoting genome-edited high GABA tomatoes.Maintaining consumer trust is vital. For example, FSA in the UK ensures consumer trust and interests by developing policies that put the consumer first (https://bills.parliament.uk/bills/3167). FSA commissioned research into consumer perceptions of genome-edited foods. The key findings suggested that there was very low knowledge of genome-edited foods80). Most consumers feel that labeling should always inform the consumers of the presence of genome-edited ingredients using the full term “genome-edited”. A transparent regulatory framework, labeling system, and safety evaluation will provide consumers with confidence in genome-edited foods.Transparency of regulatory framework is a key to consumer confidence. FSA reported that the regulatory framework must be clearly communicated and accessible to consumers and other stakeholders, and stakeholders must be involved in the development and operation of the framework to maximize open access to informationFoods produced by genome-editing technologies have been developed worldwide to create novel traits. Compared with the products of conventional mutation breeding, SDN-1 products do not have additional risks if genome-editing technologies are properly used, and the products are appropriately analyzed. SDN-2 products are similar to SDN-1 products, except for the use of a DNA template. The difference between SDN-1 and SDN-2 products cannot be distinguished by examining the final product alone. Genome-editing technologies currently include a variety of techniques, protocols, and experimental conditions. The outcomes also depend on cell types and organisms. The techniques used and the genome-edited outcomes should be considered carefully.The consultation and notification system for genome-edited products in Japan requires information on molecular characteristics, proof of the absence of foreign DNA sequences, the results of off-target analysis, and the data for the quantitative analyses of toxicants and other chemical compounds. The submitted dossier is reviewed by the MHLW Subcommittee on a case-by-case basis. Once the Subcommittee judges genome-edited products, applicants can commercially produce and sell their products after submitting the required notification form. The content of the notification form is then published on the MHLW website.


The Maros-Pangkep karst in southwest Sulawesi, Indonesia, contains some of the world’s oldest rock art. However, the Pleistocene images survive only as weathered patches of pigment on exfoliated limestone surfaces. Salt efflorescence underneath the case-hardened limestone substrate causes spall-flaking, and it has been proposed that the loss of artwork has accelerated over recent decades. Here, we utilise historical photographs and superposition constraints to show that the bulk of the damage was present before 1950 CE, and describe the role of anthropogenic sulphur emissions in promoting gypsum-salt efflorescence and rock art decay. The rock art shelters have been exposed to domestic fire-use and intensive rice cultivation with post-harvest burning of straw for hundreds (if not thousands) of years, both of which release chemically reactive sulphur oxides for gypsum formation, with cumulative effects. Analysis of time-lapse photography indicates that the rate of rock art loss may be on the decline, consistent with the history of fire-use in southwest Sulawesi. At present, vandalism and sulphur emissions from diesel-powered traffic and cement-based infrastructure development constitute localised threats. Our findings indicate that there are grounds for being cautiously optimistic that targeted conservation measures will ensure the longevity of some of our oldest artistic treasures. More th15. Case-hardening is produced by the precipitation of solutes at the limestone’s surface to form a durable crust. However, geological salts, notably the sulphate mineral gypsum (CaSO4.2H2O), can easily accumulate in the void spaces created below the case-hardened surface by the dissolution of cement matrices15. Changes in temperature and relative humidity cause the gypsum crystals to expand and contract and, over time, spall-flakes are dislodged from the limestone surface. As a result, much of the older Maros-Pangkep artwork is patchy and poorly preserved  do not support climate change on its own as the driver of gypsum efflorescence. Instead, our analysis of historical photographs of rock art panels shows that most of the limestone exfoliation has been inactive since 1950 CE. Without a clear connection to climate change, the degradation of the Maros-Pangkep rock art requires more proximal causes. The triggering of gypsum crystal growth and weathering of carbonate building stone by atmospheric SO23. SSTs increased during the deglaciation and after ~10 ka they were generally equal to or up to 0.5 °C warmer than pre-industrial times.The available marine and terrestrial palaeoclimate records for southwest Sulawesi provide histories of local sea-surface temperature (SST) and summer monsoon rainfall over the last 40 kyr  in speleothems from the Maros karst show a substantially drier austral summer monsoon between ~40 and 12 ka  modern values by ~8 ka.Oxygen-isotope ratios . Quality-controlled instrumental rain-gauge station data for southwest Sulawesi extend back to 1950, but the SOI covers 145 years from 1876 to 2021 , with 6-7 El Niño years each, are essentially the same, consistent with the findings of other studies27.Figure 26  and algal growth (31%) dominant3. Importantly, the available assessment data on rock art degradation in Maros-Pangkep, based on broad criteria, show significant differences in the preservation of artwork among shelters11  during the industrial revolution and gypsum formation on carbonate building materials (e.g. limestone) is well documented20. About 5-10% of the SO2 produced by the combustion of fossil fuels  is converted to SO3 in the atmosphere and on carbonate surfaces16. When dissolved in water, SO3 produces sulphuric acid (H2SO4) that reacts with carbonate materials to produce gypsum. Before the industrial revolution, urban air pollution originated primarily from the combustion of wood for cooking and heating. The sulphur content of wood (0.05-0.2%) is less than coal (2-3%)36, but it has been shown that pre-industrial concentrations of wood-fired air-borne SO2 were sufficient to trigger the formation of gypsum on ancient carbonate building facades in Europe37.Here, we explore the possibility that the early onset of gypsum efflorescence in the Maros-Pangkep rock art shelters is due to human agency. An important clue to the underlying cause was provided by Suhartono38 would have introduced an influential new source of air-borne sulphur to the Maros-Pangkep rock art. Once established, open-field burning of rice straw would have been inextricably linked to the maintenance of rice agriculture40. Rice straw residues generally contain 0.1-0.2% sulphur and about 40-60% of it is lost during burning42. Low temperature rice straw burning (<500°C) can lead to the direct formation of SO3 (ref.43) and trace elements, such as iron in soot and particulate matter, catalyse the oxidation of SO2 to SO3 (ref.16). Soot is particularly harmful because it serves as a medium for SO2 absorption, which reinforces gypsum formation20. Recent work at Alero Cachaco rock shelter (Chile) identified a close link between layers of soot and gypsum deposited on the walls and ceiling from repeated domestic fire-use over the last ~3,000 years44.Along these lines, biomass burning associated with the rise of Austronesian rice agriculture ~3,500 years agoHomo sapiens) and fragments of burnt animal bones dating to 25-16 ka (ref.45). Also, the negative hand stencils in the Maros-Pangkep rock shelters are thought to indicate collective ‘ownership’ of an ancient dwelling place by an extended family group46. Thus the more than 1,000 hand stencils identified so far7 probably mark recurrent occupations and fire-use by many generations of family groups. A related point is that, in addition to releasing sulphur, repeated heating of cave walls and ceilings by fire (followed by cooling) can create micro-fractures in limestone48. This pre-conditioning would facilitate gypsum growth beneath case-hardened surfaces that have been breached. If this is the case, the link between human fire-use, air-borne sulphur and gypsum efflorescence could extend back to ~40 ka, the age of the earliest dated human hand stencil in the Maros karst1.Under this scenario, artwork in rock shelters that are well-suited for domestic fire-use would tend be the most vulnerable. The earliest evidence for domestic fire-use in the Maros-Pangkep karst comes from Leang Bulu Bettue, where excavations revealed a close association of human skeletal remains  and relative humidity at Maros-Pangkep is ideal for gypsum crystals to shrink and swell and damage rock art.It is important to note that most of the Maros-Pangkep rock shelters are adjacent to open-field burning around the periphery of the karst, and are thus prime locations for gypsum formation .Taken together, the long and varied histories for the exposure of Maros-Pangkep rock art to agricultural and domestic fire-use may explain the differences in surface exfoliation among shelters. The cumulative degradation of artwork should reflect the concentration of anthropogenic sulphur, exposure history, and extent to which the limestone has been pre-conditioned for gypsum growth by domestic fire-use. The legacy of this millennial-scale history of human fire-use was obvious in the mid-1800s. During his journeys from Makassar to the Maros karst in the dry-seasons of 1856 and 1857, Alfred Russel Wallace53). Damaged artwork was documented in three limestone shelters in February-April 1950 during the course of shallow excavations in Leang Pettae that encountered evidence for domestic fire-use . The condition of negative hand stencils on red background pigment discovered in Leang Pettae on 26 February 1950 was as follows: “The red mineral has peeled off in several places and has caused the loss of many details” . On 5 March 1950, poorly preserved hand stencils were noted at Leang Burung about 6 km southwest of Leang Pettae: “On the ceiling near the entrance we discovered traces of several hand-stencils, but the red paint appeared in such a state of weathering and was so blistered, that the stencils were hardly recognizable …..” (p. 96). “Blistering” of hand stencils also was evident in 1950 in the Leang Jarie rock shelter, ~12 km south of Leang Pettae.There are no systematic observations to show that rock art panel loss in the Maros-Pangkep occurred primarily in recent decades, or that the rate of loss is accelerating. However, historical photographs by the early archaeologist H. R. van Heekeren provide definitive evidence for advanced exfoliation of the rock art by 1950 . Our photograph in 2022 shows more of the extent of the pre-1950 exfoliation beyond the boar , and again in 2003-05 (ref.7). These historical reference points allow us to estimate the relative rates of rock art panel loss within two shorter and more recent time-intervals; 1985 to 2022 (~37 years) and 2004 to 2022 (~18 years).It is critical to determine if the rate of rock art damage has accelerated over the last ~70 years. Fortunately, the state of the limestone exfoliation in some of the rock art sites was documented in 1985-86  and Leang Sumpang Bita (Pangkep) sheltersere Fig. . The pre7. Our up-to-date photographs of artwork panels in nine different shelters, six in Maros and three in Pangkep, show that only minor exfoliation has occurred since 2004 across a total surface area of ~5.8 m2  show no perceptible exfoliation. However, the occasional appearance of damage specific to the rock art images, in the absence of change elsewhere on the panel, is a prime indicator of vandalism. For example, the time-lapse photography confirms that panel loss around the Austronesian-style anthropomorph image at Leang Sampeang has been inactive, to the finest detail, over the last 18 years. And, remarkably, there has been no discernible loss specific to the ancient motif itself between the time it was painted and 2004. Then, two patches of charcoal pigment were suddenly lost from the chest area of the figure between 2004 and 2017  increased substantially after 1950 Fig. . The acc55. A notable turning point occurred in 2009 with the issuance of Act No 32/2009 on Environmental Protection and Management, which curtailed agricultural burning. Biomass burning during the dry season (July–September) has been a major factor contributing to high SO2 and particulate concentrations in air in the city of Makassar on the Maros coastal plain57. However, SO2 and particulate air pollution in Makassar both show distinct downward trends starting around 2005 suggesting that the Maros-Pangkep rock art may already be benefitting from waning fire-use.It is important to note that the upward trajectory of rice straw burning in Indonesia has plateaued, and even started to reverse, over the last two decades Fig. . The plaThe results at hand highlight a genuine need to determine which of the Maros-Pangkep art galleries are actively under threat. A reasonably attractive scenario for conservation management would be one where only relatively minor ‘hot-spots’ of panel loss within particular shelters are currently active. In that case, targeted conservation measures could be deployed precisely where required. In this regard, the potential for localised exposure of artwork to vandalism, sulphur emissions from diesel-powered traffic and reactive sulphur-rich cement dust needs to be considered.The extent to which direct human interference has disfigured artwork in the Maros-Pangkep karst may have been under-estimated. Population growth, commercial development and burgeoning tourism in the Maros-Pangkep districts all raise the risk of vandalism to readily accessible rock art. Figure Time-lapse photography of rock art images set within the broader context of panel loss will be important to distinguish vandalism from chemical exfoliation. A key indicator of human interference is the sudden appearance of pigment loss specific to the artwork in the absence of change elsewhere on the panel. Ragged edges on the damaged area and associated scratch-marks may provide confirmation of human agency. Rock art galleries with vandalism hot-spots that emerge in time-lapse photography could be prioritised for protection by targeted deterrents.2 and acidic soot.Gypsum efflorescence caused by sulphur emissions from diesel-powered traffic should be considered where hot-spot panel loss, either within or outside of artworks, is detected in proximity to roads Fig. . Develop3. The width of the most active spall-patch under investigation has doubled from ~2.5 cm to ~5 cm between October 2018 and June 2021.A case in point is the Leang Jarie shelter, which is sign-posted on a main road between Makassar and central Sulawesi that passes within ~200 m of the site Fig. . Here, t58. Aside from the potential for physical damage, cement manufacturing facilities release SO2 and cement dust to the surrounding atmosphere. Sulphur dioxide emissions from cement factories are primarily related to the coal-fired kiln system that heats raw materials (limestone and clay) to form clinker. Perhaps of more concern, however, is that the production of Portland cement, the basic ingredient of concrete, requires the addition of 3-5% gypsum to regulate its setting time59. The main source of sulphur-rich dust is the stacks of the kiln system, but emissions can occur during grinding processes and may arise from storage and handling of finely ground materials60. Therefore, it is possible that powdery cement dust, with high SO3 content, could be adsorbed onto damp limestone surfaces in nearby rock shelters and react to form gypsum61.The expansion of limestone mining for cement production facilities along the periphery of the Maros-Pangkep karst poses additional threats to rock artA key point here, however, is that cement dust released during the construction of domestic and commercial infrastructure in recent decades may have posed a more insidious risk to the Maros-Pangkep artwork. Portland cement is used in virtually all concrete structures and, in the Maros-Pangkep districts, is generally mixed on-site for the construction of buildings, walls, culverts and, in some cases, secondary roads. Chemically reactive sulphur-rich dust released during the concreting process could act as a catalyst for gypsum formation. Again, a potential target is the Leang Jarie shelter, which is adjacent to a ~200 m wide strip of land with cement-based infrastructure between a main road and the edge of the karst Fig. . In thisOur findings show that there are grounds for being cautiously optimistic that most of the limestone exfoliation in the Maros-Pangkep rock art shelters is currently inactive. The available time-lapse photography of rock art panel loss from 1950 to 2022 raises the possibility that the rate of rock art exfoliation might even be on the decline. The pattern is consistent with the history of fire-use in southwest Sulawesi and air-borne sulphur as the primary agent driving gypsum efflorescence and rock art degradation over hundreds and perhaps thousands of years.There is an urgent need to determine which artworks are actively under threat. Time-lapse photography will be pivotal for assessing the status of individual rock shelters. The results presented here show that evidence for limestone exfoliation hot-spots, where present, should emerge within the broader context of pre-existing rock art panel loss. Locating historical photographs of the Maros-Pangkep artwork will be essential to evaluate the risk posed by hot-spot activity revealed through systematic observations. Active areas that do arise could be prioritised for targeted monitoring, mitigation and conservation measures to ensure the longevity of these artistic treasures for future generations.21) and core 70GGC  at 200-400 year resolution. Briefly, Mg/Ca was measured on tests of the planktonic foraminifer Globigerinoides ruber, which lives in the surface mixed layer, and converted to SST using calibration relationships appropriate for Makassar Strait. Age models for the records are based on AMS 14C dates on planktonic foraminifera with 14C reservoir corrections of 400-500 years subtracted from the dates before they were converted to kyrBP . A broad network of G. ruber Mg/Ca-based SST reconstructions defines a consistent ~3-4 °C increase in glacial-to-Holocene SST across the western sector of the Indo-Pacific Warm Pool23.The glacial-to-Holocene SST reconstructions for Makassar Strait in Fig. 18O data at ~50-year resolution for stalagmites GB09-3 and GB11-9 from Gempa Bumi Cave  in the Maros karst24. The age models for the records are based on 44 U-Th dates (converted to kyrBP). An ice volume correction has been applied to the stalagmite δ18O values to account for the effect of the 1‰ decrease in seawater δ18O across the glacial-Holocene transition24. The adjusted δ18O record is interpreted in terms of relative changes in monsoon rainfall on the premise that the δ18O of tropical rainfall is inversely proportional to rainfall amount. Measurements of Mg/Ca in stalagmites from the Maros karst have validated the large change in δ18O between drier glacials and wetter interglacials in southwest Sulawesi62.The ~40-kyr record of austral summer monsoon rainfall in Fig. 63. The record in Fig. Figure 63. Therefore, our presentation of the GPCC record in Fig. 64. Supplementary Figure The GPCC rainfall time-series are not bias corrected for systematic gauge measuring errors11. Permana7 assessed 727 hand stencil artworks in 36 cave sites in the Maros-Pangkep karst in July 2003, September 2004 and February 2005. The condition of each hand stencil was assessed as “clear” or “not clear”, based primarily on whether the outline of the stencil was easy to discern. Therefore, the assessments account for any type of damage to the hand stencils, not just exfoliation, and do not extend to the cave site as a whole. We calculated the percentage of hand stencils damaged at each site, with outcomes ranging from 0% (none damaged) to 100%  , biological weathering  and chemical weathering 11. There is a notable difference in the frequency of severe damage based on the two classification systems: Permana7 noted only one site in 2003-2005 where all hand stencils were damaged whereas in 2013 Mulyadi11 noted 12 severely damaged sites.MulyadiSupplementary Information.


Omicron variant of COVID-19 is characterized by exceptional transmissibility and by immune evasion with the ability infect people with naturally acquired or vaccine-induced immunity. However, lung involvement is poorly reported in patients who resulted positive by this new COVID-19 variant. COVID-19 breakthrough infections are defined as COVID-19 infection in fully vaccinated patients. Herein, we present a case of breakthrough infection in an elderly woman who came in emergency with dyspnea and with findings of COVID-19 pneumonia on chest computed tomography. The patient was vaccinated with a booster dose of an mRNA vaccine some months earlier and the Omicron variant was detected on real-time reverse-transcription polymerase chain reaction. However, the patient's condition remained stable. For our knowledge we report the first case with lung involvement due to Omicron variant in an elderly after the booster dose of mRNA vaccine. This case highlights as COVID-19 breakthrough infections may represent some concerns in the elderly patients in presence of virus variants. The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused millions of deaths worldwide. Poor outcomes are especially observed in elderly people and in those with comorbidities COVID-19 presentation may vary from asymptomatic forms to a multisystem disease with the involvement of the lungs, gastrointestinal system, and central nervous system. COVID-19 can have later manifest as long COVID COVID-19 vaccines represent an efficient strategy to control the spread and severity pandemic. COVID-19 vaccines have shown high efficacy in preventing severe disease in clinical trials Recent data have indicated that B.1.1.529 is more transmissible than previous variants due to its large number of additional mutations in the spike protein The efficacy of the COVID-19 vaccines should be enhanced given the decline in the immune response over the time, mainly in the elderly, in patients with comorbidities, and in those who are immunocompromised as such patients are also at major risk of developing severe disease [15]We report a case of COVID-19 pneumonia due to the Omicron variant in an elderly woman who had previously received a third dose of an mRNA vaccine.A 75-year-old woman with a history of arterial hypertension and chronic renal failure was vaccinated with 3 doses of COVID-19 mRNA .3/µL), elevated lactated dehydrogenase enzyme (465 U/L), elevation of C-reactive protein (16.6 mg/dL), and a mild elevation in the D-dimer level (367 ng/mL). A chest computed tomography (CT) was also conducted in the emergency room. The chest CT showed interstitial thickness with ground glass opacities and initial consolidations suggesting COVID-19 pneumonia, with a score of 13/20  with cough and dyspnea with positive results on a real-time reverse-transcription polymerase chain reaction test for SARS-CoV-2 on nasopharyngeal/oropharyngeal (NP/OP) swabs, which detected the B.1.1.529 (Omicron) variant. One week later, her condition worsened with a blood oxygen saturation level (SO2) of 92%. Upon laboratory examination, she had a high white blood count  along with conventional oxygen therapy, which was started together with LMWE 4000 IU (twice daily with subcutaneous administration for 10 days). The serology, performed with an immunoassay (Liaison XL), showed only the presence of SARS‐CoV‐2 anti‐spike IgG (>2080 BAU/mL) related to the previous vaccination .One week later, the patient showed a clinical exacerbation of the dyspnea with SO2 of 89%. On the chest CT was reported a mild worsening of the previous pneumonia with a CT-SS of 16/20 . A mild However, after other 10 days the patient showed a mild improvement of the dyspnea with SO2 of 95%. Another chest CT was repeated with improvement of the consolidation areas and with bilateral pleural effusion that was found increased on the right side .Fig. 3ChThe patient's clinical conditions remained stable and she reported the first negative results for SARS-Cov-2 on NP/OP swabs after 30 days from the hospital admission. Therefore, the patient was transferred to another Hospital where she was followed for the lung rehabilitation for other 20 days.COVID-19 pneumonia was usually reported on imaging during the first and second waves of the pandemic when COVID-19 vaccines were not available.A COVID-19 vaccine breakthrough infection is defined as the detection of SARS-CoV-2 in a respiratory specimen collected from an individual who is fully vaccinated and in which the SARS-CoC-2 infection usually manifests with mild symptoms. However, severe presentations are also possible mainly in the elderly, in patients with comorbidities, and in those who are immunocompromised The Omicron variant is characterized by immune evasion and increased incidence of reinfections and breakthrough infections On the other hand, the study of Hui et al However, in our case, the patient developed COVID-19 pneumonia 3 months after the third dose and was infected by the Omicron variant. In our patient the breakthrough infection can be also explained by the decline in the immune response over time after a booster dose of COVID-19 vaccines For our knowledge, we report the first case with lung involvement due to Omicron variant in an elderly after the booster dose of mRNA vaccine.Imaging continues to play an important role and should be performed for patients at high risk of severe breakthrough infections This case highlights that particular attention should be paid to the elderly and patients with comorbidities, even if they were fully vaccinated especially in the presence of virus variants. We should also consider that vaccines are based only on the spike protein; therefore, virus variants may continue to pose some risk New vaccines that are not only based on spike proteins and produce a protective immune response that declines more slowly over time should be taken in account Data regarding nasal spray vaccines that can block SARS-CoV-2 transmission are also promising On the other hand, early therapy in vaccinated and fragile individuals should be not overlooked after the third vaccine dose. The use of masks in all closed environments should be required in these individuals In conclusion, COVID-19 knowledge is continuously evolving and virus variants can represent some challenges in the elderly people. Therefore, a multilevel and integrated approach is necessary in order to manage COVID-19 patients. Vaccines updating and improvement are required.The authors comply with international and national ethical standards. The study was conducted in accordance with the Declaration of Helsinki.Barbara Brogna: Conceptualization, Methodology, Software, Validation, Formal analysis, Writing – original draft, Project administration. Chiara Capasso: Validation, Formal analysis. Giovanni Fontanella: Software, Formal analysis. Elio Bignardi: Conceptualization, Methodology, Software, Validation, Formal analysis, Writing – review & editing, Project administration.