How the FaRLiP response arose remains poorly studied. Here, we report ancestral sequence reconstruction and structure-based molecular evolutionary studies of the FRL-specific subunits of FRL-PSII. We show that the duplications leading to the origin of two PsbA (D1) paralogs required to make chlorophyll f and to bind chlorophyll d in water-splitting FRL-PSII are likely the first to have occurred prior to the diversification of extant cyanobacteria. These duplications were followed by those leading to alternative PsbC (CP43) and PsbD (D2) subunits, occurring early during the diversification of cyanobacteria, and culminating with those leading to PsbB (CP47) and PsbH paralogs coincident with the radiation of the major groups. We show that the origin of FRL-PSII required the accumulation of a relatively small number of amino acid changes and that the ancestral FRL-PSII likely contained a chlorophyll d molecule in the electron transfer chain, two chlorophyll f molecules in the antenna subunits at equivalent positions, and three chlorophyll a molecules whose site energies were altered. The results suggest a minimal model for engineering far-red light absorbance into plant PSII for biotechnological applications.Cyanobacteria are major contributors to global carbon fixation and primarily use visible light (400−700 nm) to drive oxygenic photosynthesis. When shifted into environments where visible light is attenuated, a small, but highly diverse and widespread number of cyanobacteria can express modified pigments and paralogous versions of photosystem subunits and phycobiliproteins that confer far-red light (FRL) absorbance (700−800 nm), a process termed far-red light photoacclimation, or FaRLiP. During FaRLiP, alternate photosystem II (PSII) subunits enable the complex to bind chlorophylls  By coupo 700 nm , as the  800 nm) ,4,5,6. Bof Chl a ,5,9,10. to crops ,12.d and f in FRL-PSII by some cyanobacteria is part of a wider acclimation process called far-red light photoacclimation, or FaRLiP [f, as well as FRL-specific subunits found in peripheral phycobiliprotein antenna complexes and the two FRL-acclimated photosystems [The implementation of Chls r FaRLiP ,9. FaRLir FaRLiP ,17,18,19r FaRLiP ; thus, tosystems ,13. In Posystems . For claSynechococcus sp. PCC 7335  was recently obtained by cryo-electron microscopy (cryo-EM) [f molecules in the antenna subunits and one site, ChlD1, that binds Chl d among the cofactors that comprise the electron transfer chain. It was found that the protein confers binding specificity due to FRL-specific residues that preferentially accommodate the structures of Chls d or f; these Chls exhibit polar formyl moieties at either the C2 position (Chl f) or the C3 position (Chl d) on the tetrapyrrole ring, whereas Chl a exhibits hydrophobic methyl or vinyl moieties at those locations, respectively. Additionally, the FRL-PsbD and FRL-PsbC subunits contained conserved FRL-specific residues on the stromal surface that are probably complementary to residues of FRL-specific allophycocyanin subunits of the peripheral phycobiliprotein complex [To uncover the architectural details of FRL-PSII, the structure of a monomeric FRL-PSII core complex from cryo-EM) . Althoug complex ,22.Synechococcus 7335 FRL-PSII structure with homology models of the ancestral sequences and PSII structures from cyanobacteria incapable of performing FaRLiP. These comparisons reveal insights into how the characteristics of modern FRL-PSII evolved over time and provide new insights into the diversity of FRL-PSII among modern cyanobacteria. The findings also provide a minimal model for FRL-specific characteristics that can be used as a starting point to engineer FRL absorbance into crops.Because FRL-PSII is such an important bioenergetic system in the contribution of cyanobacteria to global carbon fixation, understanding its evolution and diversity among extant organisms is also of great interest. Recent evolutionary studies have suggested that FaRLiP has been inherited in cyanobacteria mostly vertically from a common ancestor that likely existed during the early to mid-Proterozoic . Buildinhttps://web.expasy.org/decrease_redundancy/ (accessed on the same dates the sequence datasets were collected) from the Swiss Bioinformatics Resource Portal [All non-redundant and complete amino acid sequences were downloaded from the National Center for Biotechnology Information (NCBI) RefSeq database using PSI-BLAST with default settings and restricted to the phylum Cyanobacteria. PsbA and PsbD sequences were collected on the 17 August 2021, PsbB and PsbC sequences on the 6 January 2022, and PsbH sequences on the 14 February 2022. Redundancy was decreased to 98% sequence identity using the following tool e Portal ,24. In te Portal  using fie Portal .-asr. The ancestral sequence reconstruction function of IQ-TREE does not estimate ancestral sequence insertions or deletions; therefore, prior to homology modeling, all insertions were manually removed using the respective sequence from the Synechococcus 7335 FRL-PSII as the template. Trees were visualized using the software Dendroscope version 3.8.1 [Maximum likelihood unrooted phylogenies were inferred using IQ-TREE multicore version 2.0.3 . Best fion 3.8.1 . All seqSynechococcus 7335 deposited under PDB accession code 7SA3 using PyMOL [Synechocystis sp. PCC 6803  [To create homology models of ancestral FRL-PsbA, FRL-PsbB, FRL-PsbC, and FRL-PsbD, the subunit of interest was isolated from the cryo-EM structure of monomeric apo-FRL-PSII from ng PyMOL . For theis 6803) , PDB accf synthase, also known as ChlF [INT [To provide insight into the evolution of the FRL-PSII subunits, we generated phylogenetic trees for each of the core subunits: PsbA, PsbD, PsbC, PsbB, and PsbH . The evo as ChlF ,40,41 or as ChlF ,37, whic as ChlF . The ear as ChlF  shows thhlF [INT . The other subunits of PSII have not left a record of gene duplications antedating the last common ancestor of cyanobacteria, with the potential exception of the duplication leading to the family of Chl-binding proteins derived from PsbC , which are unable to support water oxidation or replace PsbC in active PSII complexes . Therefod-producing Acaryochloris sp. strains. This observation supports the hypothesis that the facultative FaRLiP response is unrelated to the constitutive expression of Chl d exhibited by Acaryochloris strains, and that the two strategies probably evolved convergently.The trees in Aphanocapsa sp. GSE-SYN-MK-11-07L (Aphanocapsa GSE), Pleurocapsa sp. PCC 7327 (Pleurocapsa 7327), Synechococcus 7335, and Fischerella thermalis PCC 7521 (Fischerella 7521). These were realigned using Clustal Omega [To explore the predicted sequence for each ancestor of the FRL-specific subunits, and to estimate the similarity of these subunits among species, we reconstructed the ancestral sequence for each . Specifically, the nitrogen atom of the sidechain for FRL-PsbC-Gln277 donates an H-bond to the 131-keto oxygen atom of Chl a 505 , the FRL-specific residues near Chl f 605 and Chl f 608 are not, including those near their C2 formyl moieties that confer binding specificity for Chl f over Chl a. This suggests that the FRL-PSII ancestor did not bind Chl f molecules at sites 605 and 608, and some extant representatives also might not. For example, the 18 FRL sequences in our dataset cluster in three distinct clades in the maximum likelihood phylogeny . The Fischerella spp. group is separate from other heterocyst-forming cyanobacteria , which instead maintain Phe in this position as found in the ancestral FRL-PsbB sequence as well as VL-PsbB sequences and the PsbB sequences from the FaRLiP-incapable T. vulcanus and Synechocystis 6803. The same is true for positions Phe33 and Leu62 near Chl f 605. It is plausible that this may represent a case of gene replacement via horizontal gene transfer, although convergent evolution cannot be entirely ruled out. It suggests that Chls f 605 and 608 may be unique to the subgroup containing Synechococcus 7335, which highlights the need for more structural data on FRL-PSII from other cyanobacterial species.Of the core subunits, FRL-PsbB has the largest percentage of FRL-specific residues conserved among the extant cyanobacteria in the subset, but not necessarily in the ancestral sequence . This suignments . Whereashylogeny . Out of y . The two sequences in the FRL dataset without Thr244 are that of Synechococcus 7335, from which the apo-FRL-PSII cryo-EM structure was solved, and one from a strain of Nodosilinia also with Val at the same position. Based on homology modeling, the sidechain of PsbB-Thr244 was previously suggested to confer Chl f-binding in site 611 of FRL-PSII from Fischerella 7521 by donating an H-bond to a C2 formyl moiety [f-containing sites than it is for Chl a-containing sites [f occupies this site in many extant FRL-PSII complexes, it is also likely to have been found in the FRL-PSII ancestor. To investigate this further, we modeled a Chl f into the Chl 611 site from the Synechococcus 7335 apo-FRL-PSII structure and modeled a Thr in position 244 instead of Val, occupying the identical rotamer position. Indeed, this approximation places the hydroxyl moiety of Thr244 within H-bonding distance of the C2 formyl moiety of Chl f 611 , distant from the conserved FRL-specific FRL-PsbH residues. We note that the single Chl f site found in PsbB of the Synechococcus 7335 apo-FRL-PSII structure, which is conserved in the FRL-PSII ancestor (site 614), and the site that probably contains Chl f in many extant species and may have been present in the FRL-PSII ancestor (site 611) are also located close to the stromal side of the complex  and some strains of Nodosilinea spp (e.g. WP_194026004.1), Chroococcidiopsis spp. (WP_199197231.1) and a few more. This is a 48-residue small subunit annotated as hypothetical, but it is also found within the FaRLiP gene cluster downstream of the psbH2 gene, suggesting that this could be a novel small subunit of FRL-PSII originating from fission of the ancestral gene for the double helical form of FRL-PsbH.Another unique observation of FRL-PsbH is that out of the 31 sequences in our dataset, 6 contain a second transmembrane helix at the C-terminus not found in the other sequences . These if. Previous work had suggested that ChlF evolved due to a psbA gene duplication that occurred before extant cyanobacteria began to diversify [f synthesis before the MRCA of the FaRLiP-capable cyanobacteria. It is reasonable to assume that the ability to produce and bind, even with low specificity, a few red-shifted pigments alone may have been sufficient to allow survival in FRL-enriched environments prior to the duplications leading to the FRL-specific subunits that could use these pigments more optimally. The second oldest FRL-PSII subunit, as seen in the phylogeny, is FRL-PsbA, which, like ChlF, may have originated from a duplication occurring before the radiation of cyanobacteria. Based on these observations it could be hypothesized that the availability of Chl f pigments and the capacity to bind Chl d at position ChlD1 were some of the earliest innovations towards adapting oxygenic photosynthesis to lower-energy photons, beyond the visible range. It can also be hypothesized that some of the earliest cyanobacteria could have had some ability to use FRL prior to the emergence of the gene cluster as found today. Therefore, it could be predicted that within the more basal clades, which remain poorly sampled, novel FRL use strategies may still be found. It is worth noting that the Chl d synthase has not yet been identified [Acaryochloris spp., there is still a possibility that the adaptations could be still related through the synthesis of Chl d. Once the protein(s) responsible for Chl d synthesis is(are) found in FaRLiP strains and Acaryochloris spp. it may be possible to resolve whether the two adaptations represent separate but convergent evolutionary processes for the use of FRL, or whether the Acaryochloris strategy and FaRLiP have a common root that followed diverging paths towards a similar adaptation.The phylogenetic analysis presented here suggests several possible transitions prior to the emergence of the MRCA of FaRLiP-capable cyanobacteria and the FaRLiP gene cluster . Taken aiversify ,38. Howeentified ,58. WhilpsbC and psbD duplicated at the same time since these two genes overlap in most cyanobacteria and in all plastids, including the basal branches, although the overlap is not conserved in the paralogous copies found in the FaRLiP cluster and there is also a stand-alone version of VL-psbD outside the FaRLiP cluster [2 methoxycarbonyl moiety of PD2 analogous to PsbD-Tyr191 observed in the Synechococcus 7335 apo-FRL-PSII cryo-EM structure [f molecule in site 507 and exhibited a red-shifted site energy for the Chl a in site 505. The last stage in the evolution of FRL-PSII was the addition of FRL-PsbB and FRL-PsbH, which may have been added at a later stage to extend FRL-PSII absorption further into the red and to enhance assembly. The FRL-PsbB ancestor contained two Chl f-binding sites, 614 and 611, the latter of which is related to the Chl f molecule in FRL-PsbC by symmetry with the core. The ancestor of FRL-PsbH appears to have played a role in altering the site energy of a Chl a molecule in PsbB, that in site 617, and may have influenced assembly dynamics of the ancestral FRL-PSII complex. We note that every antenna-related characteristic of the FRL-PSII ancestor we have described is present on the stromal side of the FRL-PSII complex , one Chl f in each antenna subunit, and possibly three Chl a sites (one in each antenna subunit and one at the PD2 site in the electron transfer chain) that are altered in their site energy. These modifications are achieved by altering a relatively small percentage of the total residues in the entire complex. These observations will be useful for engineering FRL absorption into crops with the aim of enhancing biomass production in the future.Most of this description of ancestral FRL-PSII characteristics is maintained in FRL-PSII complexes from extant cyanobacteria, with only the exception of site 611 probably binding Chl


An optimization-based decentralized motion planning algorithm is implemented to demonstrate the effectiveness of this motion capture system. Extensive experimental evaluations were performed to (1) compare the performance of four deep-learning algorithms for high-speed multi-quadrotor detection on event-based data, (2) study precision, recall, and F1 scores as functions of lighting conditions and camera motion, and (3) investigate the scalability of this system as a function of the number of quadrotors flying in the arena. Comparative analysis of the deep learning algorithms on a consumer-grade GPU demonstrates a 4.8× to 12× sampling/inference rate advantage that YOLOv5 provides over representative one- and two-stage detectors and a 1.14× advantage over YOLOv4. In terms of precision and recall, YOLOv5 performed 15% to 18% and 27% to 41% better than representative state-of-the-art deep learning networks. Graceful detection and tracking performance degradation was observed in the face of progressively darker ambient light conditions. Despite severe camera motion, YOLOv5 precision and recall values of 94% and 98% were achieved, respectively. Finally, experiments involving up to six indoor quadrotors demonstrated the scalability of this approach. This paper also presents the first open-source event camera dataset in the literature, featuring over 10,000 fully annotated images of multiple quadrotors operating in indoor and outdoor environments.Motion capture systems are crucial in developing multi-quadrotor systems due to their ability to provide fast and accurate ground truth measurements for tracking and control. This paper presents the implementation details and experimental validation of a relatively low-cost motion-capture system for multi-quadrotor motion planning using an event camera. The real-time, multi-quadrotor detection and tracking tasks are performed using a deep learning network You-Only-Look-Once (YOLOv5) and a  SeveralThe study presented in this paper investigates event-based cameras as an alternative to infrared motion capture systems. Specifically, this motion capture system uses an event camera and a state-of-the-art YOLOv5 deep learning-based, Convolutional Neural Network (CNN) to provide real-time estimates of the position and tracks of multiple quadrotors. Event-based cameras, also known as dynamic vision sensors (DVS), are neuromorphic sensors that record changes in light intensity asynchronously. These sensors are attractive to computer vision researchers and practitioners due to high-speed vision, low perception latency, and relatively low power requirements when compared to frame-based cameras ,10,11,12Need for Event Datasets: The growing interest in event-camera-based vision tasks has created a need for labeled event-camera datasets that are suitable for evaluating event-vision tasks [on tasks ,14,15,16on tasks .An event-based motion capture system for online multi-quadrotor motion planning. The implementation details and hardware–software architecture have been detailed in this manuscript. Comprehensive experimental validation results are provided to showcase demanding use-cases beyond a standard indoor laboratory setup. Additionally, key software functionality has been open-sourced.A novel open-source event camera dataset of multiple quadrotors flying simultaneously at various speeds and heights. To the best of our knowledge, this dataset is the first to involve event cameras in the capture of quadrotors in flight. Over 10,000 synthetic event images were captured over a series of flight test sessions taking place in both indoor and outdoor arenas. Challenging scenarios, including low-light conditions and unstable camera motions, are represented in the dataset. Bounding box annotations for each image are provided.The key contributions of this study are as follows:Several studies in the literature have used Vicon and OptiTrack infrared-based motion capture systems for multi-quadrotor experiments ,18,19,20The event-based motion capture system presented here draws its working principles from the computer vision tasks of event-based multi-object detection (MOD) and multi-object tracking (MOT). A relatively small yet growing body of work underscores the value of event-based cameras for multi-object detection and tracking ,24,25,26In particular, leveraging deep learning methods for event-based work combines the power of cutting-edge vision research and high-frequency event sensing . For exathors in . Here, tSimilarly, in , the autWe note that there is a growing body of literature on spiking neural networks (SNNs), which have shown potential to take advantage of the asynchronous nature of event data and low latency of event cameras . HoweverMost of the works mentioned above make use of open-source datasets for training the neural networks ,35,36. IThe remainder of this paper is organized as follows. In This section elucidates the hardware and software setup for capturing real-time event information from the camera and setting up the motion-capture process.The hardware consisted of the event camera, the quadrotors, a host computer, and a Google Colaboratory cloud computing environment.The event camera used in the experiments was a VGA-resolution contrast-detection vision sensor from Prophesee, shown in The quadrotor used in this study was an off-the-shelf, Python programmable, small unmanned aerial vehicle (sUAV). The quadrotor’s inertial measurement unit (IMU) featured an accelerometer, gyroscope, barometer, and magnetometer for flight control. The quadrotor flew at a maximum speed of 8 m/s . CommuniThe host machine was responsible for interfacing with the event camera, performing quadrotor detection and tracking, and executing the optimization-based motion planning algorithm. The host computer was equipped with the Windows 10 Operating System, an NVIDIA GTX 1080 GPU, Intel i7 CPU, and 16 GB RAM.The Colab environment was responsible for offline training and benchmarking the deep learning networks used in this work. Colab is a hosted Jupyter notebook service that requires no setup to use while providing access to high-performance GPUs . The cloThe software was developed in Python The detection and tracking service was responsible for event data processing, performing quadrotor detection and classification, and sending tracking information to the motion planning service.The event camera communication used a set of data acquisition functionalities  provided by the manufacturer to read event data.The event data contained information about the location of a pixel tree was used for tracking due to the tree structure’s efficiency for nearest-neighbor queries [Tracking quadrotors in real time is an essential step to bridge the gap between detections and motion planning. A  queries ,42. The x, y, k-d tree to keep the quadrotor positions over time, even in scenarios where detections were not possible.The event capture suffered from spurious noise effects that led to new IDs being assigned to quadrotor-like objects (such as quadrotor reflections on the ground). Additionally, when quadrotors would slow down or hover in place, the event camera reported fewer events (and generated indistinct images), causing lost tracks/IDs or mislabeling of quadrotors. These issues were addressed by developing a process called IDTrack. IDTrack leveraged nearest neighbor searches between quadrotor detection central points. Assigned quadrotor IDs were stored with the last known  architecture and ensured that each quadrotor could calculate its position and velocity while maintaining a collision avoidance distance of at least ous work . Given tt for each quadrotor i. The host machine sent these motion commands to each quadrotor through a WiFi connection. The linear and angular velocity commands were appropriately constrained (<80%) such that the motors of the quadrotors were never saturated. The roll, pitch, and yaw commands were implemented by the onboard flight controller of the quadrotor.As depicted in The detection and tracking service and the motion planning service were asynchronous, i.e., the detection and tracking service reported new positions as fast as possible, and the motion planning service requested the latest positions as and when it needed it. A quadrotor was instructed to land if the list of waypoints was empty, meaning it had completed its mission.https://github.com/radlab-sketch/event-quadrotor-mocap (accessed on 18 March 2022)Note: Readers are referred to the accompanying video for footage of the flight tests. Relevant Python functionalities have been open-sourced for the community at Github Link: Experiments performed in this work are grouped into ’offline’ and ’online’ categories determined by the GPU used to perform the computation. Offline experiments performed on the NVIDIA Tesla K80 GPU provided by the Google Colab environment were conducted to measure the speed and effectiveness of the CNNs. Online experiments performed on the host machine’s NVIDIA GTX 1080 GPU were conducted to study the practical performance of the motion capture system.The event camera was mounted on the ceiling directly above a 183 × 183 cm area. The area was covered with white foam mats to suppress light reflections on the glossy floor. For flights in outdoor environments, foam mats were not used. All quadrotor tests were performed at 80 cm AGL (above ground level). Quadrotors were instructed to follow a series of waypoints in one of four patterns, as illustrated in Detection Metrics: The detection performance was assessed using Precision Motion Planning Metrics: The motion planner’s performance was captured using two metrics: (1) waypoint navigation task via waypoint boundary violation rate The key metrics used in the study were divided into two categories as explained in the following:8300 (83%) images that depict quadrotors flying variable paths in an indoor arena;500 (5%) images that depict quadrotors flying variable paths indoors using an unstable camera;500 (5%) images that depict quadrotors flying variable paths indoors under low light conditions;700 (7%) images that depict quadrotors flying variable paths outdoors using an aerial camera.A key contribution of this work was the event dataset that involved multiple quadrotors flying in indoor and outdoor arenas under various lighting conditions . The datBoth low- and high-speed maneuvers were meticulously represented to ensure that sparse and dense event activity would result in valid detections. The input dataset was divided into three subsets, with 70% of the total dataset allocated to the training set, 20% for validation, and 10% for the testing set.Combinations of data augmentations such as rotations, distortions, mixup, cutout, and occlusions were randomly applied to the training dataset to train the YOLOv5 network. A learning rate of ironment ,44. The An expansive set of experiments and associated analysis was performed to compare the multi-quadrotor detection and run-time performance of four modern neural networks: YOLOv5 , YOLOv4 Detection results for experiments with YOLOv4, YOLOv5, Faster R-CNN, and RetinaNet on detection metrics are noted in Key Insight: Of note is the fact that, for small-form factor-embedded devices running object detection tasks, the size of the network weights should be considered when choosing an appropriate algorithm for detection applications. Faster R-CNN produced a weights file of 314.7 MB size, and RetinaNet produced a weights file of 120 MB size. Note that YOLOv5 produced a smaller weights file than the compared object detection networks, at 14 MB, while YOLOv4 produced a weights file of 22 MB size.Additionally, single image sampling (or inference) rates for all algorithms were evaluated and are depicted in On the host machine (GTX 1080), YOLOv5 performed 1.14× faster than YOLOv4, 4.8× faster than Faster R-CNN, and 12× faster than RetinaNet. On the Google Colab environment (Tesla K80), YOLOv5 performed 1.04× faster than YOLOv4, 6.14× faster than Faster R-CNN, and 13.5× faster than RetinaNet.As expected from a larger GPU with more processing power, the NVIDIA Tesla K80 provided by Google Colaboratory was capable of greater sampling rates than the host machine’s NVIDIA GTX 1080.Key Insight: Faster R-CNN performed the inference task slower than the YOLO variants due to more complex architecture. Additionally, as expected from a larger GPU with more processing power, the NVIDIA Tesla K80 provided by Google Colaboratory was capable of greater sampling rates than the host machine’s NVIDIA GTX 1080.Several experiments that involved flight tests were conducted to characterize the robustness of the motion-capture system to unfavorable ambient light conditions and event camera motion.As event-based cameras report brightness changes detected by each pixel, the performance of the camera is subject to environmental lighting conditions . Two ambKey Insight: Event-based cameras are sensitive to small changes in brightness, which proved sufficient to capture motion in the scene. Event-based detections prevailed in the face of low-light conditions. It should be noted that despite the reliable performance noted in varied lighting conditions, some external lighting is required for the camera to report changes in brightness. Event-camera-based systems usually fail to detect objects at night or in total darkness.Two experimental conditions were used to evaluate the effect of an unstable camera.In one case, a rope was affixed to the platform and harness attaching the event camera to the ceiling of the indoor experiment area. The rope was constantly pulled, introducing motion to the camera that could be observed as noise artifacts in the event frame. Readers are referred to the accompanying video for relevant footage.In the second case, the event-based camera was affixed to a quadrotor in a downward-facing orientation. Although the camera-equipped quadrotor was flown above the motion-coordinated quadrotors and instructed to hold the position in space, drift due to wind introduced instability to the quadrotor and camera.Additionally, quadrotor positions relative to the camera frame were subject to variation depending on the intensity of camera motion. In indoor scenarios with an unstable camera, precision and recall values of 0.94 and 0.98, respectively, were observed with YOLOv5. In outdoor operation scenarios, precision and recall values of 0.95 and 0.98 were achieved, respectively, with YOLOv5.Although quadrotor positions in frames fluctuated with camera motion, detection results were accurate within a range of 5 cm.Key Insight: Due to the microsecond temporal resolution of event-based cameras, minimal motion blur was observed during scenarios with an unstable camera, thus preserving the consistent appearance of quadrotors regardless of camera stability. Therefore, the main concern with unstable cameras was addressing the distance between any given quadrotor from one frame to the next within the tracking method. This is because IDTrack’s nearest neighbor search must assimilate a quadrotor’s position from one frame to the next to facilitate successful tracking across frames, despite a potential detection at up to 5 cm from the quadrotor’s previous position. Therefore, for a given quadrotor, the minimum Euclidean distance between successive detections should be sufficiently large so as to prevent the erroneous assignment of a new ID.These experiments focused on studying (1) the effect of varying Experiments were performed at 5 cm Flight tests featuring one or two quadrotors operating in indoor and outdoor arenas were conducted to study the effect of varying quadrotor paths on This study aimed to investigate whether event cameras and modern CNNs can provide suitable motion capture feedback for motion coordination of multi-quadrotor systems. The investigations provide reasonable evidence that event cameras can indeed provide a suitable motion tracking system for multi-quadrotor systems. In the following sub-sections, the strengths and limitations of this study are discussed.Experimental results indicate that YOLOv5 and YOLOv4 models performed well on GPU hardware for multi-quadrotor detection. YOLO architectures tested in this study outperformed two other comparable state-of-the-art CNN detector architectures. The performance of the proposed system displayed minimal deterioration under constrained lighting or camera instability.YOLOv5 ran natively on the Pytorch framework, making for a seamless development experience when interfacing with additional Python services. While YOLOv5 may be easier to bring quickly into production, YOLOv4 will still be used in circumstances where development in C using the Darknet neural network framework  is desirDetection performance remained consistently high across Runtime performance was unaffected by varying The resulting dataset from this study fills a much-needed gap in the aerial robotics community that is looking to work with event cameras.x and y directions. As such, it was not possible to test for scenarios with The minimum resolvable control distance for the quadrotor flight controller was 5 cm in the As this method of detection and tracking occurs in 2D space at a fixed perspective, there were some notable challenges. For example, quadrotors that flew close to the camera occupied a significant portion of the camera’s field of view and occluded other quadrotor activity. Taller ceilings or incorporating multiple camera sources to this method would expand the detection and tracking area of the indoor arena. Multiple event camera streams would allow quadrotors to follow three-dimensional paths within the experiment arena and introduce camera redundancy and resilience to occluded sensors.As depicted in Finally, with any supervised CNN approach, there is a need for sufficient training data, which can be a time-consuming (and hence expensive) process. The open-sourced dataset accompanying study will provide a strong starting point for the research community.This paper developed and experimentally validated an event-camera-based real-time motion-capture system for multi-quadrotor motion planning. Indoor and outdoor flight tests were performed to study the system’s performance. Multiple CNN detectors were evaluated in terms of precision, recall, and algorithmic runtime, with YOLOv5 proving to be the most effective architecture overall. The effect of a varying number of quadrotors, quadrotor paths, and collision safety distances on mission time and safety boundary violations was reported. The experimental results demonstrate the effectiveness of event cameras and neural-network-based object detection for multi-vehicle motion planning applications. Future work will extend this study to track and control multiple quadrotors in three-dimensional space using an asynchronous event stream. Additionally, quadrotor-mounted event cameras will be used to expand this work to a moving platform and follow the research approaches put forth in ,50,51.


A crashworthiness design of foam-filled local nanocrystallized thin-walled tubes (FLNTs) is proposed by using foam-filled structures and ultrasonic impact surface treatment. The crashworthiness and deformation modes of FLNTs are studied using an experiment and numerical analysis. A finite element numerical model of FLNTs is established, and the processing and test platform of FLNTs is set up to verify the numerical predication and analytical design. The results show that local nanocrystallization is an effective method to enhance crashworthiness for hexagonal FLNTs. The FLNTs with four circumferential continuous stripes of surface nanocrystallization exhibit a level of 47.12% higher specific energy absorption than the untreated tubes in numerical simulations for tubes with a 50% ratio of nanocrystallized area. Inspired by the strength mechanism, a novel nested foam-filled local surface nanocrystallization tube is further designed and studied in detail. Thin-walled structures have been widely used in passive vehicle safety systems as among the most typical crash energy absorber elements due to their light weight and high-energy absorbing capacity. Significant efforts have been taken to improve the energy absorbing ability of thin-walled structures under axial crushing. In recent decades, numerous studies were carried out to investigate the energy absorption capacity enhancement by experiments and numerical analyses ,6,7,8,9.Recently, much attention has been paid to foam-filled thin-walled structures. A large number of research works have been conducted to study the influence of aluminum foam on energy absorption of thin-walled structures. Meguid et al. ,13,14,15In the existing literature, thin-walled energy absorbers are mainly fabricated by steels due to their excellent mechanical properties and low costs. Therefore, it is difficult to improve their energy absorption capacities if there is no change in geometry and material. Fortunately, with the development of nanotechnology, surface nanocrystallization techniques have been proposed to improve the mechanical and chemical properties of materials. The principle of surface nanocrystallization involves making the surface of bulk metals or subsurfaces with severe plastic deformation by applying a load such that the grains are gradually refined to form nanograins . Lu and Currently the main energy absorbers are thin-walled structures with square sections. Researchers ,57,58,59For the above reasons, a foam-filled local surface nanocrystallized hexagonal thin-walled tube is proposed for axial crashing energy absorption. The technique of ultrasonic impact treatment (UIT) is employed to generate surface nanocrystallization with a 50% proportion of treated area in the structure. The material properties for thin-walled structures and aluminum foam are obtained by testing. Subsequently, the energy absorption capacity of foam-filled local nanocrystallization tube is carried out using an experiment and the interaction effect is analyzed. A finite element (FE) model is established to analyze the foam-filled local nanocrystallized tubes under axial crushing with different stripe numbers, with a 50% ratio of nanocrystallization area. The interaction effect of foam-filled local UIT tube is significantly enhanced compared with the untreated tubes. In addition, a novel nested UIT tube with foam-filled is designed and studied based on the strength mechanism.The UIT equipment is assembled on a three-dimensional platform for producing nanocrystallization patterns in an efficient manner. Ring columns with different masses are attached to the UIT head to enhance the effect of UIT and to avoid the waggle of impact head during processing. The impact head is made of W18Cr4V high speed steel, which can bear repeated impact at high frequencies. The UIT equipment is driven by an ultrasonic generator with a frequency range of 18–22 kHz, maximum displacement amplitude of 50 μm and maximum power output of 1 kW. The UIT equipment further contains a piezo-ceramic ultrasonic transducer and an ultrasonic horn made from strength material. The samples of 304 stainless steel are placed in a 0.6 mm groove of the specimen fixture.2. The tensile specimens are obtained by cutting the nanocrystallized specimens. The material properties of nanocrystallized specimens are measured by tensile test. Universal testing machine AGS-X 300 kN and Epsilon 3442 axial extensometer are utilized for tensile and compression testing. The strain is measured by Epsilon 3442 axial extensometer to ensure test accuracy. The tensile speed is 4 mm/min and the strain–stress relation of tensile specimens is shown in According to Yang et al. , the ela3 are fabricated for the uniaxial compression tests, as shown in pσ; and stage (III), densification region. From pσ is 2.8 MPa [Aluminum closed-foam is selected as the filled foam block. The deformation modes of hexagonal foam under quasi-static compression test are shown in  2.8 MPa .The local nanocrystallized tubes are fabricated by welding two plates. Firstly, two stainless steel plates of SUS 304 are locally nanocrystallized by UIT. Next, each plate is bent to 120° along the bending lines. Then, two bent plates are welded into one thin-walled tube by argon-arc welding. The location for welding lines is in the middle of one side. Lastly, the foam column is filled into the local nanocrystallized tube to form a foam-filled tube, as shown in The welding is the last process of the overall manufacturing process. The nanocrystallized steel plate is welded into tubes using the argon-arc welding machine. The welding material shows similar ultimate strength with the 304 steel which was also indicated in the published references ,64. In tThe quasi-static compression tests are conducted for untreated empty and foam-filled tubes, local nanocrystallized empty and foam-filled tube and the validity of the test result is verified a finite element analysis. The velocity of load is 4 mm/min during compression, and the whole buckling process is recorded using a digital camera. The compression displacement is set as 70% for the specimen height and all crashworthiness criteria are calculated within the range.W is the elastic strain energy, σx is the normal stress, εx is the linear strain and V is the volume of structures.As presented in The compression tests of the FUT (foam-filled untreated tube) and FLNT-2  are carried out and A comprehensive study by FE numerical simulations is conducted using Abaqus/Explicit to analyze, design and optimize local nanocrystallized hexagonal thin-walled tubes. The FE model details are shown in Based on the experimental data in The energy absorption of thin-walled structures is mainly dependent on its deformation during crush. To evaluate the crashworthiness of the structure, several widely used assessment parameters are selected, including energy absorption (EA), specific energy absorption (SEA), peak crushing force (PCF) and mean crushing force (MCF) .x0 is the crushing displacement and F(x) is the crushing force.EA can be calculated by integrating the displacement force under the load–displacement curve. It is given bySEA represents the energy absorption per unit mass and it can accurately measure the energy absorption capacity of the structure. It is given bym is the total mass of the structure. MCF is another effective criterion, which is defined as the average load during the crushing process. MCF is expressed asFE models for FUT and FLNT-2 are validated by comparing the deformation modes, load–displacement curve and crashworthiness assessments with an experiment. It is observed from A mesh sensitivity study for aluminum foam is presented in The result from the experiment and FE simulation shows that FLNT-2 has a higher SEA compared to FUT. In a previous work ,69, the In The load–displacement relation of FLNTs with different stripes and the corresponding deformation modes are presented in To verify the design parameters of FE numerical simulation, a test specimen for FLNT-4 is fabricated in As seen in The collapse mode for FLNT-4 under axial compression is shown in Inspired by the above investigation, two nested foam-filled local surface nanocrystallization tubes with four stripes are designed as shown in A new type of aluminum foam-filled hexagonal thin-walled structure treated with local nanocrystallization is proposed. The crashworthiness of FLNTs under axial crushing is investigated through an experiment and finite element simulation. The effects of different local nanocrystallization layouts on the energy absorption capacity for FLNTs is studied using numerical simulations. The numerical result concludes that: (i) Proper local surface nanocrystallization can significantly enhance the crashworthiness of hexagonal thin-walled tubes. Compared with UT and FUT, the SEA of LNT-2 is significantly increased by 46.19% and 26.61%, respectively. (ii) FLNT-4 is a better design for aluminum foam-filled local nanocrystallized hexagonal thin-walled tubes with respect to crashworthiness. By comparing the interaction effect for foam-filled tubes, FLNT-4 can dramatically improve the interaction effect from 176 J to 388 J. In addition to numerical analysis, a test specimen for FLNT-4 is fabricated and an experiment is conducted to verify the conclusions. Very good agreement between the experiment and FE numerical solutions has been reported. Moreover, a novel nested foam-filled local surface nanocrystallization tube is designed based on the interaction effect. The simulation result concludes that SEA for N-2-FLNT-4 is further enhanced with an 23.99% increase compared to FLNT-4. The proposed design idea is very promising for the further development of thin-walled energy absorption devices using other metallic and non-metallic materials.


The results did not change when participants with MBL were analyzed according to whether they had low-count or high-count MBL, acknowledging low power to evaluate high-count MBL.Included in the study were a total of 699 individuals with MBL (660 [94.4%] with low-count MBL and 39 [5.6%] with high-count MBL) and 2916 without MBL. Individuals with MBL were older  and more likely to be male (47%) compared to those without MBL  among those with MBL. After adjusting for age, the corresponding HR for MBL was 2.55 , and after adjusting for CCI, the corresponding HR for MBL was 2.61 . Thirty-four individuals died including 23 without MBL and 11 with MBL  [Despite the low risk of progression to CLL-requiring therapy, individuals with MBL have a distinct clinical phenotype with important clinical consequences , 9. In a≤ 0.001) . Similar≤ 0.001) .not associated with a higher risk of contracting SARS-CoV-2 infection but was associated with a 3-fold increased risk of hospitalization due to COVID-19, compared to individuals with no MBL. These findings were independent of age and co-morbidity status, suggesting that the presence of MBL per se is associated with increased risk for severe COVID-19 infection, although the effect attenuated when adjusting for other factors. The biologic underpinnings of an increased risk of serious infection in individuals with MBL are not completely understood. Although the rates of hypogammaglobulinemia are not significantly different between MBL and those without MBL [In the present study of Olmsted County residents, we found that a diagnosis of MBL was hout MBL , as obseOur study has several strengths. It is one of the first studies to examine the risk of contracting SARS-CoV-2 infection and developing severe COVID-19 requiring hospitalization in a large cohort of individuals with MBL in the United States. All study participants were followed systematically, with near complete data on reasons for death available for all. However, our study also has several limitations. Although we had access to all medical records from Olmsted County residents, ascertainment bias for hospitalization with COVID-19 infection outside the county is possible. Most study participants were White; hence, these results may not be generalizable. The findings reflecting the natural history of SARS-CoV-2 infection before broad-scale vaccination was approved is both a strength and a limitation of our investigation. Finally, despite including >3500 participants in this study, the total number of hospitalization and infection events was low, which limited our ability to adjust for other confounders and evaluate subgroups.In summary, in this study evaluating the risk and severity of COVID-19 infection among Olmsted County residents with and without MBL prior to the introduction of SARS-CoV-2 vaccinations, we found that the risk of acquiring SARS-CoV-2 infection was not different among individuals with or without MBL. However, once infected with COVID-19, those with MBL have a higher risk of hospitalization due to COVID-19, suggesting that immune dysregulation increases the risk of adverse outcomes. These findings add to the mounting evidence that MBL is associated with clinically significant impairment of immune function. Given that MBL is a largely unstudied condition affecting over 5-10% of adults over the age of 40 and the increasing incidence with aging, these findings have potentially large population health implications.Supplemental Table 1


Randomised controlled trials have shown that steroids reduce the risk of dying in patients with severe Coronavirus disease 2019 (COVID-19), whilst many real-world studies have failed to replicate this result. We aim to investigate real-world effectiveness of steroids in severe COVID-19.Clinical, demographic, and viral genome data extracted from electronic patient record (EPR) was analysed from all SARS-CoV-2 RNA positive patients admitted with severe COVID-19, defined by hypoxia at presentation, between March 13th 2020 and May 27th 2021. Steroid treatment was measured by the number of prescription-days with dexamethasone, hydrocortisone, prednisolone or methylprednisolone. The association between steroid > 3 days treatment and disease outcome was explored using multivariable cox proportional hazards models with adjustment for confounders . The outcome was in-hospital mortality.1100 severe COVID-19 cases were identified having crude hospital mortality of 15.3%. 793/1100 (72.1%) individuals were treated with steroids and 513/1100 (46.6%) received steroid ≤ 3 days. From the multivariate model, steroid > 3 days was associated with decreased hazard of in-hospital mortality (HR: 0.47 (95% CI: 0.31–0.72)).The protective effect of steroid treatment for severe COVID-19 reported in randomised clinical trials was replicated in this retrospective study of a large real-world cohort.The online version contains supplementary material available at 10.1186/s12879-022-07750-3. Currently, steroids are the main treatment for severe coronavirus disease 2019 (COVID-19) infection , 4 was tA guideline was issued by WHO on use of dexamethasone and other corticosteroids (hydrocortisone or prednisone) for treatment of severe and critically unwell COVID-19 patients in September 2020 [Guy’s and St Thomas’ NHS Foundation Trust (GSTT) is a multi-site inner-city healthcare institution providing general and emergency services predominantly to the South London boroughs of Lambeth and Southwark. NHS is the National Health Service in the UK. The acute-admitting site  has an emergency department with a large critical care service. A second hospital site  provides elective surgery, haemato-oncology, renal transplantation and other specialist services. There are also several community sites providing dialysis, rehabilitation and long-term care. Only COVID-19 cases admitted through the emergency department (ED) during March 13th 2020 and May 27th 2021 were included in this study. Patients dying or being discharged in the first 24h were considered most likely to have reached study endpoint independent of any steroid effect and were excluded from the primary analysis.GSTT has an on-site laboratory providing SARS-CoV-2 testing to all patients and hospital care workers (HCW). The policies and technologies employed for SARS-CoV-2 testing changed over time based on national and local screening guidance and improvements in diagnostics. Our laboratory began testing on 13th March 2020 with initial capacity for around 150 tests per day, before increasing to around 500 tests per day in late April during wave one, and up to 1000 tests per day during the second wave.Assays used for the detection of SARS-CoV-2 RNA include PCR testing using Aus Diagnostics or by the Hologic Aptima SARS-CoV-2 Assay. Testing commenced during the first wave on 13th March 2020 limited to cases requiring admission or inpatients who had symptoms of fever or cough, as per national recommendation; guidance suggested cases who did not require admission should not be tested. Cases without laboratory confirmation of SARS-CoV-2 infection were not included.Cases were identified by the first positive SARS-CoV-2 RNA test. The severe cases were measured by hypoxia upon admission to hospital. Cases were taken to be hypoxic if on admission they had oxygen saturations of < 94%, if they were recorded as requiring supplemental oxygen, or if the fraction of inspired oxygen was recorded as being greater than 0.21.Whole genome sequencing of residual samples from SARS-CoV-2 cases was performed using GridION (Oxford Nanopore Technology), using version 3 of the ARTIC protocol  and bioiClinical, laboratory and demographic data for all cases with a laboratory reported SARS-CoV-2 PCR RNA test on nose and throat swabs or lower respiratory tract specimens were extracted from hospital electronic patient record (EPR) data sources using records closest to the test date  Critical Care, DXC Technology’s MedChart, e-Noting and Citrix Remote PACS - Sectra). Data was linked to the Index of Multiple Deprivation (IMD), with 1 denoting the least deprived areas, and 5 the most deprived ones. Age and sex were extracted from EPR. Self-reported ethnicity of cases was stratified to be White, BAME  and Unknown according to the 18 ONS categories of White , Black , Asian , and Mixed/Other.Comorbidities, medication history, and medicine data were extracted from the EPR and e-Noting using structured queries with corresponding dictionaries. Comorbidities were extracted from any of the databases covering the pathway of the cases from arrival in accident and emergency through inpatient general ward and critical care unit, where applicable, to hospital discharge or death. If a comorbidity was not recorded, we assume that it was not present. Cases were characterised as having/not having a past medical history of hypertension, cardiovascular disease , diabetes mellitus, chronic kidney disease, chronic respiratory disease  and neoplastic disease . Additionally, checks on free text data were performed by a cardiovascular clinician to ensure the information was accurate.Steroid treatment was measured by number of prescription-days with dexamethasone, hydrocortisone, prednisolone or methylprednisolone. Duration of treatment with steroids was calculated as cumulative days throughout first hospital admission after the first SARS-CoV-2 PCR positive test through to discharge or death during that admission. Analysis for lengths of steroid use were conducted in multivariate model with steroid use ≤ 3 days versus steroid use > 3 days. The cut-off for the steroid treatment days were chosen according to the interquartile range of steroid-days (3 to 10 days) in RECOVERY trial. Sensitivity analysis was conducted with continuous steroid days as the variable input in the Cox proportional hazards model.The outcome was all-cause in-hospital mortality , with patients still hospitalised at the end of the cohort considered censored.The general statistics were summarised with mean and standard deviation (SD) for continuous variables if the distribution is normal and median and interquartile range (IQR) if the distribution is non-normal. Count and percentages were used for categorical variables. For the comparisons of the cohort statistics with different lengths of steroid use days (< 3 days vs. ≥ 3 days), Kruskal-Walllis test was used for continuous variables and Chi-squared test for categorical variables. The reference significant level was set to be p < 0.05.2, hypertension, cardiovascular disease, diabetes, respiratory disease, chronic kidney disease, sequenced SARS-CoV-2 variant and medications including steroids and tocilizumab/sarilumab were used as pre-defined covariates to adjust in multivariable models. As the distribution of steroid days is right skewed (steroid days ≥ 0), before modelling, the continuous steroid days were transformed with the log of steroid days plus one (log(steroid days + 1)). Missing values of the variant, BMI and ethnicity were imputed as a new category and cases with missing values in IMD were discarded. There were no missing values in other adjusted variables.Cox proportional hazards models were used for time-to-event survival analysis in which the time was starting from hospital admission and events as the defined outcomes. Adjusted hazards ratios for the primary and secondary outcomes using Cox proportional hazards models were presented. The adjusted variables used in the model were selected via literature review  and clinData management was performed using SQL databases, with analysis carried out on the secure King’s Health Partners (KHP) Rosalind high-performance computer infrastructure  running 1120 patients were identified with hypoxia on admission of which 1100 were included in the analysis after removal of 20 cases that stayed for less than 24h after admission. 23 cases with missing data in the IMD variable were imputed with median. In-hospital mortality of the whole cohort was 15.0%  compared to patients who received ≤ 10 days of steroids (13.7%) was observed ) for mortality. The protective effect of steroids was consistent when using steroids as a continuous variable (HR: 0.86 (95% CI: 0.76–0.96)) .Other variables Table includinThis study provides evidence for real-world effectiveness of steroids in reducing death amongst severe COVID-19 patients. The protective effect-size of treatment with steroids was similar to that reported in the RECOVERY clinical trial  for a coWe adjusted for potential confounders  as well as the characteristics of the virus (Alpha variant) and another treatment (tocilizumab) with the effect of steroids remaining statistically significant. Undoubtedly, we are unable to adjust for all confounders, including the vaccination status, other co-treatments and improvements introduced around the time of steroids e.g. thromboprophylaxis and proning which might compromise the practical use of the study findings even though the protective effects of steroids were significantly protective in the model. Vaccination could be a big confounder which was started from December 2020 and by the end of the study (17th May 2021), most of the adults had received one dose of vaccination. Regarding other co-treatments, during most of the study period, other drug therapeutics were not routinely deployed, and the effect size of newer treatments like tocilizumab were much less than steroids in clinical trials. No other SARS-CoV-2 variants that have been associated with altered severity of disease were circulating in our population during the study period.It is notable the study was done in an institution that had good overall comparative NHS outcomes and an standardized mortality ratio (SMR) of 0.5 in ICU patients, with guidelines and practice recommending longer courses of steroids for severe patients. Over 80% of the > 10 steroid-days group were treated deliberately with long steroids and the remaining were on long term steroids as therapeutic immunomodulation for other conditions. Whether longer course of steroids has an additional benefit is not known.Longer durations of steroids have not been systematically studied and might increase the risk/rate of adverse events, including delayed viral clearance . Some stIn this study we investigated the association of steroid days with outcomes, however our analyses are agnostic to the dose of steroids used. There may be reasons why duration of steroid treatment mediates effects on outcomes independently of cumulative dose, for instance if a sustained period of immunosuppression is needed to prevent immune-mediated inflammation. In addition, as this study is retrospective and observational the link between steroids and the outcome is only an association and causality should not be inferred.Many other studies on the real-world effectiveness of steroids have failed to reproduce the findings of clinical trials. Partly, this may be due to small sample size, heterogeneity of treatment and non-treatment groups, and incorrectly testing associations on individuals not expected to benefit, i.e. cases without evidence of hypoxia. Our study benefits from a wide time period for inclusion, allowing us to capture the changing treatment landscape before steroid use in COVID-19 was standardised in line with national and international guidelines. Additionally our adjustment accounts for many baseline variables which have previously been associated with severe outcomes. The validity of our analyses is supported by the findings that variables previously associated with severity, such as age and cardiovascular comorbidity retain significance in our modelling.Other studies have found the Alpha variant of SARS-CoV-2 to be associated with severe disease, especially mortality –18 and hLimitations of this study might include potential bias for patients who did not have a chance to receive steroids or received very short steroids because they were very severe and died soon after admission. This is an issue that is intractable with retrospective study, and we attempt to address this by excluding those who died in the first 24h after admission. Another limitation is that the choice of cut-offs for the steroid treatment days were chosen according to data from RECOVERY trial, our local recommendations, and WHO guidelines rather than pharmacological effect of steroids treatment in COVID-19.The protective effect of steroids in severe COVID-19 seen in our cohort is similar to that seen in clinical trials, confirming the real world effectiveness.Below is the link to the electronic supplementary material.Supplementary Material 1


The current study examines student satisfaction with e-learning, the adaption of online learning channels, digital competency of students' involvement, and academic achievement during COVID-19.The purpose of this study is to examine the online influencing components for learning among University students in Pakistan during the COVID-19 Pandemic.The study population comprised Pakistani University students in Punjab province who tooke online lessons throughout the epidemic. In accordance with the study's purpose, a questionnaire survey was employed to gather primary data. SPSS-23 is used for analyzing the demographic data, and cleaning and preparing data for testing hypotheses. SmartPLS 3.0 was used to investigate the suggested study framework using structural equation modeling (SEM).The analysis of the SEM model shows that all planned hypotheses  are confirmed.The results linked e-learning satisfaction to academic success and Pakistani students who utilized e-learning throughout the outbreak reported higher levels of academic satisfaction and achievement. The World Health Organization (WHO) considered COVID-19 a pandemic in 2020, impacting millions of children and educators globally, and various technological applications have been deployed in the epidemic age to facilitate learning  Pakistani University students who attended online courses during COVID-19 and (2) those who volunteered for this research. The convenient sampling technique was used to gather the primary data, and University students from six different cities of Punjab Province in Pakistan, namely, Lahore, Multan, Rawalpindi, Faisalabad, Bahawalpur, and Sialkot have fulfilled the inclusion criteria for this study.There are seven variables used in this study to investigate e-learning satisfaction and academic achievements of University students. The study confined one independent variable (COVID-19 Pandemic), four mediators , and two dependent variables (satisfaction with e-learning and academic achievement).The demographic questions were about gender , age , education , and year of attending the University .Due to COVID-19, the students' online learning impact was calculated by adopting five items developed questions. Studying e-learning from students' and instructors' perspectives on using and developing e-learning systems at public universities while dealing with the COVID-19 epidemic has become more critical than ever before  of SmartPLS 3.0 was used to examine the measurement and structural models as it analyses intricate models that include both observable and latent components  could be used to quantify concepts that had no connection to one another conceptually. Discriminant validation aims to show any evidence of discrimination based on the differences between all components  value for each connection . The impariables .p = < 0.0000) reveals that the adaptation of online education channels have positive impact on satisfaction about e-learning. H2 results  proved that COVID-19 pandemic has significant positive impact on the adaptation of online education channels. H3 results  statistically reveals that COVID-19 pandemic has positive association with digital competence. H4 statistically documented that  COVID-19 pandemic have positive effects on motivation for online learning. H5 supported that  COVID-19 pandemic positively influences on students' willingness for online learning. H6 reveals that  digital competence has positive association with satisfaction about e-learning. The result of H7 statistically documented that  motivation for online learning has positive influence on satisfaction about e-learning. H8 results shows that  satisfaction about e-learning has significant association with academic achievement and H9 reveals that  willingness for online learning has positive association with satisfaction about e-learning.The result  are confirmed. Many students may suffer from psychological repercussions due to the sudden move from face-to-face classrooms and interactions with peers and professors to online learning  statistically reveals the positive association in all. For future academics, several studies provide the directions and consequences for online education by identifying elements that impact online learning satisfaction at home as a result of the COVID-19 pandemic and getting information on difficulties in the teaching/learning process .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Evidence on the effectiveness of COVID-19 vaccines among people who recovered from a previous SARS-CoV-2 infection is warranted to inform vaccination recommendations. Using the territory-wide public healthcare and vaccination records of over 2.5 million individuals in Hong Kong, we examined the potentially differential risk of SARS-CoV-2 infection, hospitalization, and mortality between those receiving two homologous doses of BNT162b2 or CoronaVac versus those with a previous infection receiving only one dose amid the Omicron epidemic. Results show a single dose after a SARS-CoV-2 infection is associated with a lower risk of infection  and no significant difference was detected in the risk of COVID-19-related hospitalization or mortality compared with a two-dose vaccination regimen. Findings support clinical recommendations that those with a previous infection could receive a single dose to gain at least similar protection as those who received two doses without a previous infection. Its high transmissibility and extensive immune escape impose substantial challenge to global health amidst the ongoing Coronavirus disease 2019 (COVID-19) pandemic3. As of June 30, 2022, over 300 million cases and one million deaths have been recorded worldwide since the Omicron variant was declared a variant of concern4, and the global weekly new COVID-19 cases had reached 4,182,217, among which more than 90% were caused by the Omicron variant5.The Omicron variant of SARS-CoV-2 has rapidly emerged to dominate the global transmission since early November 20216, evidence shows that COVID-19 vaccines can still effectively reduce the risk of severe conditions associated with Omicron infection7. Likewise, a prior SARS-CoV-2 infection can also confer immunity against reinfection among survivors11, while some studies suggest that the combination of a previous infection together with vaccination, i.e., hybrid immunity, might be more effective against reinfection and associated severe conditions compared to either vaccine-induced or natural immunity alone14. As the Omicron variant emerged, a high risk of breakthrough infection and reinfection was observed in vaccine recipients and those who were infected before15. More people may be in need of further vaccination against the virus, including those who were infected with previously circulating variants. However, the recommendation on vaccination for people with previous SARS-CoV-2 infection varies by region and is often unclear. For instance, France, Germany, and Italy recommend only one priming dose for those who recovered from a previous infection and with a healthy immune system16, while the United States recommend two priming doses regardless of infection history17. To date, whether a previous infection plus one priming dose of the vaccine can achieve a similar strength of protection as the typical two-dose vaccine regimen remains unclear, especially against the Omicron variant. Individuals with a previous infection are largely left confused as to whether further vaccination is favorable or necessary, although its relatively high safety has been supported by previous research18. Real-world evidence on the effectiveness of COVID-19 vaccines among people who recovered from a previous SARS-CoV-2 infection is much warranted to inform vaccination policies and uptake as well as infection control measures.Both messenger RNA (mRNA) and inactivated COVID-19 vaccines are widely used as one of the most crucial public health measures to mitigate the impacts of the pandemic. Although the immune evasion of the Omicron variant hindered the vaccine effectiveness against infection19. The infection rate was largely kept under control in the previous four epidemic waves with daily new cases not exceeding 200 until the Omicron variant arrived in January 202221. As of June 30, a total of 763,551 laboratory confirmed cases and 469,615 self-reported cases using rapid antigen tests (RAT) were documented during the fifth wave22. This surge overwhelmed the city’s healthcare system and the mortality rate increased noticeably, with over 9000 COVID-19-related deaths recorded during the fifth wave22. The Hong Kong government has been promoting vaccine uptake to reduce severe conditions associated with infection and to alleviate the pressure on the local healthcare system. The government recommend people infected before to receive one priming dose instead of both doses to be considered as fully vaccinated23. Using population-based vaccination records linked with a territory-wide public healthcare database in Hong Kong, this study examined the potentially differential risk of SARS-CoV-2 infection, COVID-19-related hospitalization, and COVID-19-related mortality between individuals receiving the regular two-dose regimen of BNT162b2 (Pfizer-BioNTech) or CoronaVac (Sinovac) versus those with a previous infection receiving only one dose amid the Omicron outbreak.With 7.5 million population, Hong Kong has been implementing a dynamic zero-COVID policy since the virus started to spread in the community in January 2020In this study, vaccinated with previous infection refers to those who were infected with SARS-CoV-2 and then vaccinated with a single dose of the COVID-19 vaccine. The final cohort included 2,669,531 vaccine recipients, of which 7415 had prior SARS‑CoV‑2 infection , and 2,662,116 had no history of SARS‑CoV‑2 infection . Figure Within the observation period, 127,085 (8.137%) SARS‑CoV‑2 infections were observed among BNT162b2 recipients and 92,961 (8.392%) SARS‑CoV‑2 infections were observed among CoronaVac recipients. The incidence rate per 100,000 person-day in the BNT162b2 group was 15.38 among individuals with previous infection and 37.21 among individuals without previous infection. The incidence rate per 100,000 person-day in the CoronaVac group was 15.89 among individuals with previous infection and 40.16 among individuals without previous infection. The estimated incidence rate ratios (IRR) and the corresponding 95% confidence intervals (CI) are presented in Table Given that no differential risk of COVID-19-related hospitalization or mortality was detected between groups in the main analysis, we conducted a series of extended analyses to compare the risk between people infected before but with no subsequent COVID-19 vaccination and fully vaccinated people received two doses of BNT162b2 or CoronaVac with no prior infection. The results are presented in Table In this study, we found that individuals with a previous SARS-CoV-2 infection and received one priming dose of either BNT162b2 or CoronaVac vaccine had a lower risk of a subsequent infection during the Omicron outbreak compared with other regular vaccine recipients without previous infection after their second dose. The incidence rates of COVID-19-related hospitalization and mortality were low in both groups, and we did not find any differential risk of COVID-19-related hospitalization and mortality between the two groups. The results are reassuring to people in Hong Kong and other regions with similar vaccination recommendation who received only one priming dose due to previous infection. Our findings suggest that hybrid immunity induced by the combination of infection and vaccination provide stronger protection against subsequent infection and similar protection against COVID-19-related hospitalization and mortality as the immunity induced by two priming doses of the COVID-19 vaccine amid the Omicron outbreak.25. A cohort study in Israel similarly reported that people who received a single dose of the vaccine after previous infection had lower risk of infection compared with two dose recipients without any previous infection26. Existing data mostly focused on the antibody levels rather than COVID-19-related infection, hospitalization, and mortality. We were only able to identify several studies investigating the risk of SARS-CoV-2 infection and COVID-19-related hospitalization among individuals with hybrid immunity. A cohort study in Sweden compared previously infected individuals who received COVID-19 vaccine with those who did not receive any COVID-19 vaccine after infection and found that both one-dose and two-dose immunity were associated with a lower risk of COVID-19 hospitalization than natural immunity alone27. Another cohort study in Qatar reported that individuals with a previous SARS-CoV-2 infection and received two doses of the mRNA vaccines had a lower risk of reinfection compared to fully vaccinated individuals without history of infection28. However, it is well recognized that both SARS-CoV-2 infection and COVID-19 vaccines can induce immunity and a higher level of immunity tends to provide better protection.The finding is consistent with several studies suggesting that the antibody responses elicited by two doses of the mRNA vaccine are very similar to that elicited by a SARS-CoV-2 infection plus one dose of the vaccine29. Similar results were observed in several studies of different ethnicities where higher SARS-CoV-2 antibody levels in previously infected individuals after one dose of COVID-19 vaccine compared with infection-naive individuals after two doses32. A recent review comparing the efficacy and duration of natural and hybrid immunity also suggested that hybrid immunity appeared to confer the greater protection against SARS-CoV-2 infections, although there were still knowledge gaps on the underlying mechanism14. When we compared individuals who were previously infected but did not receive vaccination with fully vaccinated individuals without prior infection on the risk of COVID-19-related hospitalization, although the crude estimates suggested a significant lower risk in the infection group, the no significant difference between the two groups was detected after adjusting for potential confounders. We believe the difference in time since the last immune conferring event between groups contributed to the changes after adjustment. The group with a history of infection but unvaccinated were likely infected at a closer time before the Omicron outbreak and they were not yet eligible to receive a vaccine dose after recovery, while numerous vaccine recipients took their second dose early in time and the protection waned over time. Furthermore, the lower risk of infection remained among individuals with a previous SARS-CoV-2 infection and unvaccinated. This finding is consistent with a systematic review which revealed that the natural immunity in individuals recovered from COVID-19 was at least equivalent to the protection provided by complete vaccination in infection-naïve populations33. Among individuals who were previously infected with SARS-CoV-2, a population-based cohort study in Iceland identified a higher risk of reinfection in those who received two or more doses compared to one dose or fewer of vaccine, and the risk of reinfection increased with time from the initial infection, suggesting a differential level of protection against reinfection conferred by natural and hybrid immunity34. The natural immunity induced by a previous SARS-CoV-2 infection might generate a more extensive immune response to the SARS-CoV-2 proteins compared to that generated by the anti-spike protein immune activation induced by the COVID-19 vaccines37. However, the neutralizing antibody response in individuals recovered from COVID-19 varied largely38, and the role of different types of cellular immune remains unclear, which requires further investigations39. This finding may also reflect a ‘healthy survivor effect’ whereby individuals who survived a previous infection are likely to be healthier and therefore less likely to be reinfected. It is also possible that individuals with a previous infection are less likely to report their reinfection to the Department of Health especially when the symptoms are minor and do not require any medical treatment in the hospital. Additionally, previous SARS-CoV-2 infection may trigger behavioral changes including better personal hygiene practice, which reduces the risk of reinfection.The humoral response elicited by post-infection vaccination might explain the lower risk of reinfection. A previous study on humoral immune responses found that vaccination after infection elicited a greater humoral response compared with vaccine or infection alone, with a longer time between infection and vaccination associated with an increasing post-vaccine anti-spike-immunoglobulin G peak42, this study compared the risk of COVID-19-related infection, hospitalization, and mortality during an Omicron outbreak between those with a previous SARS-CoV-2 infection and received one priming dose and fully vaccinated individuals with BNT162b2 or CoronaVac. The use of population-based vaccination records and territory-wide public healthcare database ensures the population representativeness of the study cohort. A series of sensitivity analysis demonstrate the robustness of the results. The self-reported RAT positive cases obtained from the Department of Health were included as a sensitivity analysis. The Hong Kong government requires all RAT positive results to be reported within one day after the testing date through an online declaration system. Individuals who test positive using RAT are encouraged to report their results to receive more appropriate support from the health authority. The infection reported through the system affects the eligibility for vaccination and getting a Vaccines Pass that allows individuals to enter public places such as restaurants and sports facilities. Individuals fail to report their positive results may also face legal liabilities. Under these public health policies, the data from the Department of Health should covered most infection cases using self-administered RAT. Despite these strengths, this study has several limitations. First, we only used test results provided by the Department of Health to define SARS-CoV-2 infection. Despite better specificity, we may have omitted infection cases determined only by self-testing kits and with no record in the Department of Health. As the number of undetected and underreported cases were high in many countries and regions43, this issue might occur in similar studies and lead to an underestimation of the protection against subsequent infection conferred by natural or hybrid immunity. Nevertheless, in Hong Kong, these cases were mostly asymptomatic or minor cases that do not require any treatment in the hospital, so it should not affect our estimates on the risk of COVID-19-related hospitalization and mortality. Second, the low incidence rates and the relatively small sample size in the group with previous infection limited the numbers of events we could observe within the study period, resulting in wide confidence intervals in some estimates, especially for the mortality outcome. The small event number also limited the feasibility to further examine the time-varying effectiveness of the hybrid immunity as well as the effectiveness of the booster doses. Although we were unable to examine the waning of immunity over time, the time from the last immune conferring event to the index date was similar across different groups, and we did include it as a covariate in the models to control for the potential confounding effect. Third, several studies have shown that the vaccine-induced immunity appeared to wane faster than that induced by a previous infection14, but we were not able to control for this in the analysis. Fourth, the study outcome may also vary across different variants of the previous SARS-CoV-2 infection, but we were unable to ascertain the variant of the previous SARS-CoV-2 infection due to limited data availability. Fifth, we considered all hospitalization with positive PCR test within 28 days before admission as COVID-19 hospitalization. This might include hospitalization due to non-COVID-19-related conditions but with positive PCR result, which might not indicate severe COVID-19 cases. Finally, this study is observational in nature and the results are subject to bias introduced by unmeasured confounders such as behavioral factors. Future studies could incorporate a more comprehensive list of potential confounders and further examine the effectiveness of the subsequent booster dose among individuals with a previous SARS-CoV-2 infection.While most existing studies on vaccine effectiveness against SARS-CoV-2 infection excluded individuals with a previous SARS-CoV-2 infection44, future research could investigate whether the second dose for individuals with previous infection receiving one priming dose could be considered as the booster dose to achieve a similar level of protection as the regular vaccination regimen for individuals with no previous infection. Since we were only able to examine hybrid immunity within several months given the available data, the results might not be generalizable to time beyond our observation period. To our knowledge, there is only one study investigating the time-varying effects of vaccine-induced and hybrid immunity with a relatively small sample size45. Future studies should also examine the duration of hybrid immunity at a population level and develop an effective vaccination schedule for individuals with a previous SARS-CoV-2 infection to achieve an optimal level of protection over time.As the number of SARS-CoV-2 infections is still increasing worldwide, a sizeable fraction of the population needs to consider their history of infection in their subsequent vaccine uptake decisions, i.e., booster doses. Our data is apparently more in favor of clinical recommendations that patients who recovered from COVID-19 could receive one fewer dose of the vaccine to gain at least similar protection with people without an infection history. If substantiated by further research data and evidence, the government could consider an infection as a substitute for one dose of COVID-19 vaccines and prioritize vaccination for people without any previous infection, especially for countries and regions where vaccine supply is a concern. As hybrid immunity also tend to decline over timeIn conclusion, the hybrid immunity from single dose vaccine and previous variants of SARS-CoV-2 conferred a stronger level of protection against Omicron infection and a similar level of protection against hospitalization and mortality, compared with the immunity induced by two priming doses of the vaccine without previous infection. Our findings support clinical recommendations that individuals with a previous SARS-CoV-2 infection could receive a single dose to gain at least similar protection as those who received two doses without previous infection.48. This study was approved by the Hospital Authority Central Institutional Review Board (CIRB-2021-005-4) and the Department of Health Ethics Committee (LM171/2021).We obtained population-based vaccination record and positive SARS‑CoV‑2 PCR results from the Department of Health. Territory-wide de-identified electronic medical records (EMRs) between January 1, 2018 and March 31, 2022 were provided by the Hospital Authority (HA), the statutory body managing all public hospital services in Hong Kong. We performed data linkage between vaccination records, positive PCR results, and EMRs by matching unique pseudo-ID to protect patient privacy. Previous COVID-19 vaccine pharmacovigilance studies have been conducted using this databaseThis was a retrospective cohort study with the analysis stratified by vaccine type, i.e., BNT162b2 and CoronaVac. The study cohort consisted of all individuals aged 12 years or above and vaccinated before January 1, 2022, the month when the first local Omicron BA.2 case was identified, with either one dose of COVID-19 vaccine after a SARS‑CoV‑2 infection or with two doses of COVID-19 vaccines and with no history of infection upon receiving their second dose. Individuals with a previous SARS‑CoV‑2 infection and received two vaccines doses before the Omicron outbreak were not included in the study cohort. Those who received one dose of the vaccine and then got infected before the Omicron outbreak were also excluded from the study cohort. For individuals with a history of SARS‑CoV‑2 infection and received one dose of the vaccine, the index date was the date of taking the first dose of either BNT162b2 or CoronaVac. For individuals with no prior infection, the index date was the date of receiving the second dose of either BNT162b2 or CoronaVac. The observation started from the index date and ended upon outcome occurrence, death, receiving an additional dose of the COVID-19 vaccine, or the end date of data availability , whichever came the earliest. Considering the waning of protection conferred by the latest dose of the vaccines, the time from the index date to January 1, 2022 was included for multivariable adjustment .The exposure of the study was any SARS-CoV-2 infection defined by PCR-positive result before receiving the first dose of COVID-19 vaccine. The exposed group consisted of individuals who received their first dose of BNT162b2 or CoronaVac after a SARS-CoV-2 infection. The unexposed group included individuals who received two doses of BNT162b2 or CoronaVac and with no prior SARS-CoV-2 infection.The outcomes of this study were SARS-CoV-2 infection, COVID-19-related hospitalization, and COVID-19-related mortality during the Omicron outbreak in Hong Kong starting from January 1, 2022. SARS-CoV-2 infection was defined by PCR-positive result. COVID-19-related hospitalization and mortality were defined by a PCR positive test result within 28 days before admission or death.14. We hypothesized that a previous SARS-CoV-2 infection might provide similar protection against infection, hospitalization, and death as the two-dose vaccination regimen. We included the following covariates in the multivariable models for possible confounding effects: age, sex, number of days from the index date to the local Omicron outbreak , clinical history of chronic conditions before the index date  as the offset term. In an extended analysis, we also compared those who had a previous infection without vaccination (index date being the date of positive PCR test result) and those who received the regular two-dose regimen with no prior infection to compare the protection conferred by natural immunity and vaccine-induced immunity. Some evidence show that natural immunity offers equal or greater protection against SARS-CoV-2 infections compared to immunity induced by two doses of the COVID-19 vaccines, but the existing data are not consistent49, we postponed the index date by 14 days to better compare the optimal protection effects between the exposed and unexposed groups. Third, we included self-reported cases using RAT to define outcome events as these cases, although may not be confirmed by PCR, accounted for a considerable proportion of the total infection cases in the local Omicron epidemic. All analyses were conducted in R statistical environment . L.H. and V.K.C.Y. independently conducted the analyses and cross-checked to ensure the accuracy of the results.A series of sensitivity analysis were conducted to examine the robustness of the results. First, we changed the time window defining COVID-19-related outcome events from 28 days to 14 and 42 days, respectively, to cover fewer or more possible events. Second, as evidence showed that the vaccine recipients usually reached the optimal protection level about 2 weeks after being fully vaccinatedFurther information on research design is available in the Supplementary informationREPORTING SUMMARY