Nowadays, more and more vehicles are equipped with communication capabilities, not only providing connectivity with onboard devices, but also with off-board communication infrastructures. From road safety  to infotainment , there are a lot of applications and services that may be deployed in vehicular networks, where video streaming is the key factor. As it is well known, these networks suffer from high interference levels and low available network resources, and it is a great challenge to deploy video delivery applications which provide good quality video services. We focus our work on supplying error resilience capabilities to video streams in order to fight against the high packet loss rates found in vehicular networks. So, we propose the combination of source coding and channel coding techniques. The former ones are applied in the video encoding process by means of intra-refresh coding modes and tile-based frame partitioning techniques. The latter one is based on the use of forward error correction mechanisms in order to recover as many lost packets as possible. We have carried out an extensive evaluation process to measure the error resilience capabilities of both approaches in both (a) a simple packet error probabilistic model, and (b) a realistic vehicular network simulation framework. Results show that forward error correction mechanisms are mandatory to guarantee video delivery with an acceptable quality level , and we highly recommend the use of the proposed mechanisms to increase even more the final video quality. Video delivery over vehicular networks is a challenging task. On the one hand, video streaming requires high bandwidth and timely delivery, and, on the other hand, vehicular networks are error-prone environments with many challenges to deal with . Vehicular Ad-hoc NETworks (VANETs) are a subtype of Mobile Ad-hoc NETworks (MANETs), where the mobile nodes (vehicles) have certain restrictions regarding their mobility . In , we can Some of the video applications in VANETs may also be provided by other types of networks, like 4G and the future 5G technologies. However, these networks are not always a suitable option, in particular for those application with strict requirements  or when there is no fixed infrastructure available (remote areas). As an example, a real-time video-assisted overtaking application may require car-to-car direct communication, featuring very low delay and jitter values, a very high level of reliability, being always available even in areas without cellular network coverage.In order to provide error resilient video communications in VANET scenarios, we propose a combination of two different approaches. The first one improves bit stream error resilience at source coding level, i.e., adapting and adjusting video encoding in such a way that the encoded bit stream may provide, up to certain data loss degree, good video quality to the user. For this task we have proposed (a) a new frame partition scheme (a mix of tiles and slices), and (b) seven intra-refresh encoding modes (ranging from low to high intra-refresh rates). In order to enhance the video protection, the second approach proposes the use of well known Forward Error Correction (FEC) techniques to minimize the effect of packet losses. The former approach is based on the High Efficiency Video Coding (HEVC) standard  featuresThe rest of the paper is organized as follows. In Several works can be found in the literature about the challenges of video streaming over VANETs. In , the autIn a previous work , we evaIn this section, we will introduce and evaluate the first of the two complementary approaches proposed in this work, which is based on source coding techniques. The main goal is to increase the robustness of the video streaming by properly configuring some of the features provided by the selected video encoder. So, the robust bit stream will better fight against packet losses resulting in better perceived video quality. First of all, we will evaluate the proposed techniques in the absence of data losses. This is done to measure the compression efficiency of the different alternatives . This allows us to characterize the configurations in ideal conditions or, equivalently, in soft environments where the data losses can be completely neutralized with the FEC mechanisms proposed in Now, we will briefly introduce some concepts about the HEVC video encoding process (for a good overview refer to ). HEVC iIn the encoding process, each frame is divided into small square regions . Let’s call them blocks. These blocks can be encoded using one of three modes: (a) without any prediction, (b) using spatial prediction, or (c) using temporal prediction. Spatial prediction exploits redundancy within a single frame. In order to encode a block, it uses previously encoded regions of the same frame to search for similar pixel information to create a prediction. This prediction is subtracted from the current block to obtain the prediction residual. This method is also called intra-frame prediction. Temporal prediction uses previously encoded frames to estimate a block prediction by means of the search of a similar block in other frames , which have been previously encoded, decoded, and stored in a buffer. It exploits temporal redundancy, taking advantage of the fact that nearby frames usually contain blocks that are very similar to the current block and so the residuum of the motion compensation is close to zero. This method is also called inter-frame prediction.Slices are regions of an encoded frame that can be independently decoded, regarding other slices of the same frame. Slices are not a new concept in HEVC and were used in previous standards. The purpose of slices is providing error resilience to a frame. If one slice of a frame gets lost, then the rest of the slices of the same frame can be correctly decoded and only the region covered by the missing slice is lost. Slices consist of correlative blocks (Coding Tree Units—CTUs) in raster scan order see a. The maOne of the new features introduced in HEVC is the ability to divide a frame into rectangular regions, called tiles see b. The orIn the search for robust video streaming techniques, while attempting to keep the overhead introduced by slices low, we have devised a new element that we call tileslice. A tileslice is the combination of one tile and one slice. First, one frame is divided into tiles b, and thAs tileslices contain slice headers, overhead introduced by them is not zero. So, is it really worth using tileslices instead of typical slices for error resilience purposes? To answer this question, we have compared the overhead introduced by slices and tileslices. From this point forward we will use the term tile to refer to what we have defined as tileslice, i.e., one slice that contains one rectangular tile.We have compared the compression efficiency of tiles against slices for six different layouts: 1, 2, 4, 6, 8 and 10 slices (or tiles) per frame, using fourteen video sequences, of the HEVC “common test conditions” . They arIn LP mode, the first frame of the sequence is encoded as an I frame, and then P (predictive) frames are generated for the rest of the sequence. P frames are mainly formed by inter-coded CTUs, i.e., CTUs that are encoded by using motion estimation and compensation using other frames as reference . A P frame can also contain intra-coded CTUs . LP mode is much more efficient than AI mode as it generates a much smaller bit stream for the same quality level, but a simple error in a frame is propagated until the end of the sequence, even if the rest of the frames are correctly received. The compression rate of both modes can be selected by means of the Quantization Parameter (QP). This parameter is used by the quantization process, which entails the loss of precision in the transformed coefficients (lossy compression). When high QP values are used, the generated bit stream has a low bit rate and, correspondingly, low visual quality. When low QP values are used, the generated bit stream has a high bit rate with a resulting high visual quality. For the analysis of HEVC, 4 values for the QP have been used  as it is specified in the common test conditions.In AI—Encodes every frame as an I frame. It is considered our upper bound mode.LP—Encodes the first frame as an I frame and the rest of the frames as P frames. Four previous reference frames are available for temporal estimation and compensation for each P frame. It is considered our lower bound mode.IPx—Similar to LP mode but every P frame uses only the previous frame as reference frame, instead of having 4 reference frames.IPx25pctCTU—Similar to IPx mode, where 25% of the CTUs are forced to be intra refreshed.IPx25pctTIL—Similar to IPx mode, where 25% of the tiles are forced to be intra refreshed.IPxpattern—Similar to IPx mode, where 25% of the tiles are forced to be refreshed following a specific pattern, which covers all the tiles of a whole frame every 4 frames.IPPP—Similar to IPx mode, with an I frame inserted every 4 frames.LPI4—Similar to LP mode, with an I frame inserted every 4 frames.IPIP—I and P frames are inserted alternatively. Every P frame uses the previous I frame as reference.In the previous tests, two encoding modes have been used: All Intra and Low-delay P modes. AI offers better error resilience properties than LP because an error in one tile does not propagate to other frames. On the other hand, LP mode is very sensitive to lost tiles, because P frames are “infected” by erroneous reference frames, and these infected P frames, when used as reference frames, “spread disease” until the end of the sequence. There is also an unpredictable reaction in LP mode in the presence of packet losses. For example, when an LP encoded sequence loses the last frame, then the error affects only that frame. However, if the first frame (I) gets lost, then no frame can be correctly reconstructed at all. So, the position of the missing parts is also important when dealing with errors in LP mode. These two encoding modes have been determined as our upper and lower bounds regarding error resiliency. In this work, 7 new encoding modes with different intra-refresh rates have been proposed and evaluated. Intra refresh is an error resilience technique that forces to periodically intra encode (refresh) certain frame areas in P frames, in order to stop the error propagation mentioned before. The 9 encoding methods evaluated are the following ones:bbd_25), which exhibits an average coding performance among the 832 × 480 sequence set. In The 9 encoding modes can be classified into four groups, depending on the whole-frame refreshing rate: (a) LP and IPx modes only have an I frame at the beginning of the sequence, so no refreshing is performed (0%); (b) IPPP, IPx25pctCTU, IPx25pctTIL, IPxpattern, and LPI4 modes have an average frame-refresh rate of one intra frame every four frames (25%); (c) IPIP mode has an intra frame-refresh rate of one frame out of two (50%); (d) AI mode uses a full rate of intra-refreshed frames (100%). For the following tests, we have selected the BasketballDrill sequence  technique that will hide the missing information to the user in such a way that the received video quality will not be seriously damaged. A well-known EC technique  exploitIn this section, first a video quality analysis varying the Tile Loss Ratio (TLR) is carried out, and the reconstructed video quality (PSNR) is measured. In addition, then, the average proportion of packets per tile is presented and experiments varying the Packet Loss Ratio (PLR) are carried out. In these experiments, we measure both how the different configurations produce different TLR for the fixed PLRs, and also the final video quality (by means of the PSNR) provided by each configuration. The obtained results will be analyzed to find out the most appropriate source coding architecture that fulfills our goal.The compression performance of the proposed encoding modes and the overhead introduced by tiles and slices have already been evaluated. Now, we will evaluate the robustness of tiles and encoding modes for different TLR values. The combination of the 9 intra-refresh coding modes with the different tile partition layouts , under a simple probabilistic data loss model, will be evaluated. Six different TLR values have been fixed for the tests: 1%, 3%, 5%, 7%, 10% and 20%. For every one of these loss rates, 5 different seeds for the random number generator have been used, and the results have been averaged.First of all, we have evaluated the EC methods implemented in the decoder. The frame copy method has been always used in order to obtain a reconstructed video sequence with the same number of frames than the original video sequence. Also, the zero-MV technique has been tested and the PSNR differences when enabling this method are always positive. This means that the EC decoder version provides better PSNR values than the version without EC. The PSNR gains range from 0.14 up to 3.02 dB. However, the LP and IPx encoding modes (no intra-refresh) obtain PSNR values below the quality bounds which are considered the minimum aceptable, even when the EC decoder is enabled. As a product of these observations, from this point forward we will always use the EC decoder version for the reconstruction of video sequences.Regarding the error resilience attributes related to the encoding modes and the tile layouts, in One of the consistent results throughout all the tile loss percentages tested is that, for a fixed TLR, when the number of tiles per frame increases , the quality decreases. The reason is that the loss of small parts in many frames causes a worse effect than the loss of one big part in only one frame, even if the data loss percentage is the same. For 1% and 3% TLR, the most robust encoding modes are LPI4 and IPPP. At 5% TLR, these two modes and IPIP show very similar performance. In addition, at 7% TLR and above, IPIP is the most resilient mode. At 20% TLR, AI mode is over IPIP for 4 tiles per frame layout and above, but very low PSNR values are obtained at this point, being not effective at this TLR values.From these experiments, it seems that the 1 tile per frame is the most robust layout. However, in the “real world”, a particular PLR may produce very different TLRs, depending on the ratio between the tile size and the network Maximum Transfer Unit (MTU). As the tile size depends on several factors such as the frame type , the number of tiles per frame, and the selected QP parameter (the compression ratio), we will now evaluate the robustness of the combination of these parameters, for different PLR values.First of all, note that, if a tile is larger than the network MTU, that tile has to be divided into several network packets, and, if one of these packets gets lost, then the rest of the packets belonging to the same tile are completely useless because that tile cannot be decoded at all. The average number of network packets per tile in the encoded bit streams is shown in Now, we will show the video quality performance of the different tiles per frame layouts, at a fixed PLR. In From these results, some conclusions can be drawn. Of the 9 encoding modes evaluated, there are three that do not have good error resilience properties. Two of them  have shown the expected performance as they do not use any intra refreshing at all. However, the third one (IPx25pctCTU), which refreshes one of every four CTUs, does not have the expected intra-refresh effect in the bit stream. For the rest of the modes, two of them (LPI4 and IPPP) stand out with respect to the others when the percentage of loss remains low, and another method (IPIP) has a good performance when the percentage of loss increases. With respect to the effect of frame partitioning, we have confirmed in all experiments that the higher the number of tiles per frame, the higher the final video quality. However, it is recommended to use no more than 6 tiles per frame, since the benefits in video quality are negligible for higher values. Although some of the methods exhibit good error resilience properties, this is not enough to guarantee robust video streaming. In the next section, we will evaluate RaptorQ codes, and will search for the best setup to provide the desirable protection to the video packet stream.The second error resilience approach is based on channel coding techniques which provide data redundancy to correct the bit stream errors found during video delivery. These techniques are complementary to the source coding approaches of the previous section, enabling the recovery of lost packets and, as a consequence, improving the received video quality. In particular, we propose the use of RaptorQ codes, a well known Application Layer Forward Error Correction (AL-FEC) technique. In RaptorQ codes provide AL-FEC protection to video streaming in order to improve video quality by recovering as many lost packets as possible. The cost of using FEC is an additional network bandwidth to carry FEC repair data, and also an increased latency to support packet recovery from a block of packets. Increasing the FEC bandwidth (given a fixed FEC latency) leads to an increase in data protection. Similarly, increasing the FEC latency (given fixed FEC bandwidth) also leads to an increase in data protection.bbd_25 sequence, using 9 encoding modes, with 6 different til/frm layouts), we have generated several protected versions by combining different values of the following parameters: protection level, protection period, and symbol size. The properties of the protected versions will determine the most suitable configurations for the protection of the video bit streams. Three different levels of protection have been tested: 10%, 20% and 30%; seven protection periods have been used: 133, 166, 200, 250, 333, 500 and 1000 milliseconds; and three symbol sizes have been evaluated: 192, 450 and 1350 bytes.For the protection of the encoded bit streams, we have used the Qualcomm (R) RaptorQ (TM) Evaluation Kit . For eacIn For a fixed symbol size and a fixed til/frm layout, the overhead values for the different protection periods show monotonically decreasing behavior: the wider the temporal window, the lower the overhead percentage. As the size of the protection period has a direct effect on latency, the selection of the proper temporal window seems clear: the highest acceptable latency will determine the most efficient protection period. In this work, as a “design decision”, we have selected a protection period of 333 ms. Depending on the particular ITS video streaming application, the selection of the protection window can vary if the delay requirements are tight or more relaxed.In summary, the parameters selected for the protection of the video bit streams are a symbol size of 192 bytes because of efficiency (lowest overhead) and a protection period of 333 ms . We will maintain the three values for the protection level parameter in order to test their performance against packet losses. After tuning the parameters of the FEC module, we have analyzed the resulting bit rates to determine the overhead introduced by both FEC coding and the use of tiles. On the one hand, the overhead introduced by FEC is close to the protection level . The same behavior has been observed in all HEVC coding modes defined in this work. On the other hand, increasing the number of tiles per frame also produces an increase on the final bit stream size see . So, wheNow we will evaluate the behavior of the RaptorQ protection at different PLR values. The evaluations are formed by the following steps. Firstly, the video encoded bit streams are divided into network packets. In this step, tiles which do not fit into one network packet will be divided into several packets. After this, packets are sent to the RaptorQ encoding process, in order to protect them (adding the FEC trailer) and to generate the repair packets. Then, in the complete packet stream (protected packets and repair packets) some packets are discarded, using a simple probabilistic packet error model. In the next step, the surviving packets are sent to the RaptorQ recovery process, in order to recover as many missing packets as possible. After that, tiles are reconstructed by joining together all the packets which belong to the same tile. If one or more packets which belong to a tile are missing, the whole tile will not be decodable and so we will mark that tile as lost. At last, we obtain the TLR which is the rate of lost tiles versus the total number of original tiles.Now, we will present the results of our evaluations. In the case of a protection level of 10%, see a,b layouIn the case of a protection level of 20% see c,d, PLR In the previous tests we have been using a simple probabilistic loss model to simulate the packet losses in video delivery. As we have shown, real world details may change the behavior of the proposed error resilient techniques, so, in this section, we will use a realistic vehicular scenario to evaluate all the protection proposals presented before, to check their performance in this type of scenarios, where local network traffic interferences, source video signal strength, node mobility pattern, handover procedure, etc., determine the real packet loss patterns.Also, note that different layouts  produce very different packet streams , and this has influence on the final network conditions. For example, encoding a video sequence with a tile layout of 10 til/frm with LP encoding mode will probably generate many small network packets and, on the contrary, encoding a video sequence with a tile layout of 1 til/frm and with AI mode will produce packets with the maximum size allowed. Also, protecting a video sequence with a protection level of 30% will generate far more packets and, as a consequence, higher bandwidth requirements. All these characteristics, combined with the real network conditions , have influence on the PLR and in the distribution of packet losses. So, every different layout does not “work” under the same network conditions, so it will behave differently and will have to confront a different situation. In the previous sections, when we analyzed, for example, how a PLR of 3% affected a certain layout versus another one, we did not take into consideration that one of these layouts may probably have a lower impact in the network conditions than the other.For the experimental tests presented in this section, we use several simulators working together in order to reproduce the conditions of vehicular networks . In For the evaluation of error resilience proposals, we have used three main software blocks see . The firThe encoded bit streams are used to generate video trace files which are injected into the simulations. For this purpose, we developed a new module in OMNeT++. This module provides a new traffic source (video sender) which reads packets from a trace file to deliver them through a vehicular network scenario. Vehicles tagged as video receivers get video packets and write both a file with the correctly received packets and a file with several network statistics.*) receives that invitation and commutes to the specified service channel in order to receive the video stream. This vehicle travels along the avenue, receiving the video stream from the RSUs through the specified service channel. Another vehicle drives nearby the video client which can act as a background traffic source (labeled as T in The scenario for the simulations is an area of 2000 m × 2000 m in the city of Kiev (Ukraine). bbd_25), encoded with 9 different encoding modes  and with 6 different tile layouts . For each selected bit stream, 4 different versions have been generated. One of these versions has been encoded without any FEC protection. The other three versions have been FEC protected with RaptorQ codes at different protection levels, 10%, 20% and 30%. Each bit stream (with or without protection) has been delivered by the three RSUs and received by the video client under 3 different “network conditions”. The first group of tests has been conducted without injecting any background traffic. This group of tests is referred to as “ideal conditions” and permits the characterization of each one of the three zones mentioned before. After that, experiments have been performed under 2 further network conditions: one with moderate background traffic (62 packets per second of 512 bytes), and another with a high background traffic (125 packets per second of 512 bytes), both for protected bit streams and non-protected bit streams. The tests with the non-protected bit streams serve to evaluate the performance of the encoding modes and the frame layouts, regarding error resilience. The tests with the FEC protected bit streams serve to evaluate the specific level of protection added by RaptorQ codes.In the experiments, the combination of the bit streams from previous sections has been used. For the tests, we have selected the BasketballDrill sequence , has to wait in the sending buffers of the senders until the next service channel time slot is available. When the control channel time slot ends, all the nodes resume the transmission in the service channel so they have to contend for the wireless channel to transmit all the data accumulated in their buffers. In addition, the guard interval that has to be respected when the wireless network cards commute between the control channel and the service channel and vice versa, makes the real availability of the bandwidth for the service channel (in which the video is streamed) to be only 40% of the total. These issues are not taken into consideration by works which use, for example, standard WiFi protocols in real experiments or in simulations. Other challenges like the existence of shaded zones not covered by any RSU or the interruption of the communication when the network cards of two vehicles encounter an obstacle between them, also produce network packet losses, principally in a bursty pattern. By using realistic scenarios all these situations are modeled and this fact allows a better evaluation of the proposed approaches.In this section, we will show and analyze the results obtained for the different bit streams configured above. The first two sets of experiments in the vehicular scenario in  evaluateIn Zone I, under ideal conditions, every packet sent by the RSU is correctly received by the client vehicle for all the combinations of coding modes and tiles per frame. So, we have a PLR value of 0%. Consequently, no tile is missing in any of the sequences and the quality of the reconstructed video sequences is the quality directly provided by the encoding/decoding process shown in . The codIn Zone II, for the non-protected bit streams, we encounter a packet loss burst corresponding to the area where neither of the RSUs signals arrive. The average PLR in this zone has a narrow range (from 1.98 to 2.01%). The TLR has also a narrow range (from 1.95 to 2.39%). As opposed to the results shown in In Zone III, where the signals of two of the RSUs overlap, the PLR is around 22–23%. As the two RSUs are 1 km away from each other, they cannot “see” each other (because the coverage radius of the wireless devices is 500 m). These collisions produce a high loss rate in the overlapped area, which is wider than the shaded area in Zone II. Some bursts of data loss appear in this zone. As in Zone II, the TLR values are only slightly higher than the PLR values. However, in this zone, the TLR values are very high and this fact produces very low values for PSNR. The same behavior as that for Zone II is observed regarding the tiles per frame layouts: all of them show very similar performance. Therefore, we can conclude that when bursty losses occur, the til/frm layout does not have any influence on the PLR value, nor on the PSNR value.Now, we will analyze the performance of the FEC protection in Zone II and Zone III under ideal conditions (without any background traffic load). In order to evaluate FEC recovery efficiency, the After-Recovery Packet Loss Ratio (ARPLR) metric measures the percentage of the source video packets missing over the total source video packets of the sequence, once the FEC recovery process has been carried out. In In Zone II, the PLR values of the protected bit streams are very similar to the non-protected ones. The ARPLR values for this zone do not show a good efficiency of the RaptorQ packet recovery process. RaptorQ codes have good recovery properties when dealing with isolated losses, but a minimum number of packets need to be received, which is not the case when bursty losses are encountered. The recovery of missing packets from a burst of packet losses would need a very long protection window for any of the FEC techniques available (which would introduce a long delay) or else the use of other complementary techniques like interleaving, which converts bursty losses into isolated losses, but which also introduces a non-negligible delay in the transmission process. So, shaded areas (where no packet can be received) are not suitable for live video delivery and a readjustment of the RSUs location in order to eliminate these “black zones” may be the most appropriate action. In this zone, as happened with the non-protected bit streams, the ARPLR values do not either generate high TLR values.In Zone III, the PLR is around 23–24%. The bursty nature of losses in this zone makes FEC recovery unfeasible, as in Zone II. Furthermore, there is a striking fact in Zone III measurements: the ARPLR values are higher than the PLR values. How can this happen? The PLR metric takes into consideration the total number of packets in the FEC-encoded packet stream, which includes protected packets and repair packets. If losses are bursty and FEC recovery is not effective, only protected packets (which include video data) will be translated into source video packets (by just taking away the trailer). As repair packets may recover a few (or no) source video packets they are not useful and the proportion of missing source video packets over the original number of source video packets may produce a higher value for the ARPLR than for the PLR. As a consequence of the high TLR values in this zone, the reconstructed video sequences can be considered useless. A possible solution to the problem found at overlapping areas resides in using different service channels for each one of the overlapping RSUs and incorporating a seamless horizontal handover mechanism to guarantee a nearly uniform coverage area.In this section, we present the results of the tests for a moderate and a high background traffic loads.In In Zone I, the PLR obtained is exclusively due to background traffic . Only when using six or more tiles per frame with a 62 pps background network load, the TLR values keep under reasonable limits. The quality of video sequences for this zone at moderate background traffic loads is depicted in In Zone II and Zone III, the PLR value results from the combination of isolated packet losses and bursty packet losses. The high TLR values for these two zones clearly show that all the bit streams received in these two zones are completely useless, since the received packets produce very low quality video sequences.Regarding the FEC protected bit streams, in In Zone I, under moderate traffic conditions, a protection level of 30% gets almost 100% of packet recovery for all the layouts of tiles per frame . Encoding a video sequence at 10 tiles per frame produces a bit stream around 9–12% larger than encoding it at 1 tile per frame. If we protect the bit stream with a protection level of 30%, the tile-layout overhead can be avoided in Zone I under these precise conditions. Also, a protection level of 20% nearly completely recovers all the missing source video packets, so the layouts with medium and low numbers of tiles per frame can be used, avoiding extra overhead and still obtaining the best video quality. If a protection level of 20% or 30% is used, then the most efficient encoding mode (LP) provides the best quality, and, therefore, it is the best candidate to be used under these network conditions. Please note that, for a certain til/frm layout, all FEC protection levels produce very similar PLR values .In the experiments where a more dense background traffic is used (125 pps) the PLR values obviously increase. Under these conditions, a protection level of 30% can restore almost all the missing packets for every layout, and a protection level of 20% also obtains good ARPLR values, mainly for layouts with a high number of tiles per frame. If Regarding the encoding modes performance, in In For Zone II, the PLR, ARPLR, and TLR values for background traffic conditions of 62 pps and 125 pps are higher than those in Zone I. By correlating TLR values and PSNR of many tests, we have observed that with the four most robust modes , TLR values under 15% may produce PSNR values over 29 dB. Taking this observation into consideration, we can state that for moderate network load conditions (62 pps), the 10% protection level is considered enough protection for layouts equal or greater than 4 tiles per frame, as can be seen in Two complementary approaches for the protection of video streaming over vehicular networks have been proposed. The first one is based on source coding, by using and fine tuning some intrinsic features of the video encoder (HEVC). In this approach seven new encoding modes have been presented, which introduce intra refresh into the encoded bit stream in order to avoid the propagation of errors in the decoding step. Also, a new element called tileslice has been introduced, which offers the good encoding efficiency of tiles and the good error resilience features of slices. The second approach is based on a well-known FEC technique, RaptorQ codes. We have analyzed the RaptorQ performance with our video encoded streams, defining the configuration parameter set that introduces the lowest overhead with the highest level of protection.To the authors’ knowledge, no other works in the literature have evaluated the proposed tools in a realistic VANET scenario as the one proposed here, including state-of-the-art video coding/decoding, packetization, channel coding/decoding with Raptor codes, and network delivery using realistic simulation tools that provide accurate network and traffic models.Even though it makes the video bit stream more robust against losses, the source coding approach, by itself, cannot guarantee the minimum level of visual quality in these networks, especially when moderate and high background traffic conditions are encountered. However, it can significantly improve the video quality (PSNR), if the appropriate combination of encoding mode and tiles/frame layout is selected, for a particular network conditions. From all the encoding modes proposed, the IPPP and LPI4 show the best performance in most of the situations. For isolated packet losses, the selected til/frm layout has a direct influence on the PLR and TLR values. As the number of tiles per frame increases (a) the PLR value decreases and (b) for the same PLR value, a lower TLR value is obtained. When packet losses are bursty in nature the layout has little influence because (a) the PLR is very similar for all the til/frm layouts and (b) the TLR value is in line with the PLR value because missing packets probably belong to the same tile(s). For moderate network load conditions the 4 and 6 til/frm layouts have in general a good performance, and for tight network conditions a high number of tiles per frame  are needed to provide robustness to the video bit stream.Even though, FEC channel coding is necessary in video streaming over vehicular networks because the protection provided by source coding is not enough to guarantee video delivery. The combination of the two protection approaches (RaptorQ codes and source coding) offers good performance when RaptorQ codes are not able to recover all the missing packets and the source coding protection mitigates the propagation and multiplication effect of errors.In order to select a suitable setup for the delivering of video in urban vehicular networks, we recommend the use of an encoding mode which has both good compression efficiency while providing a certain level of robustness. To this extent, the LPI4 mode is the best positioned mode. When it comes to select a til/frm layout, the combination of low overhead, good error resilient features, and a low avalanche effect (when translating PLR into the resulting TLR), values from 4 to 6 til/frm are the best tradeoff. At last, if we are protecting video streams delivered under full coverage areas, a protection level of 10% (in combination with a good encoding mode and til/frm layout), may provide the required robustness against isolated losses, to guarantee an appropriate user experience.The two main research efforts in which we are targeting our future work are: (a) the application of Quality of Service methods to the prioritization of data packets through the vehicular network, by means of 802.11p priority queues, in order to provide more robustness to those video packets which are more relevant in the final quality of the reconstructed video, and, (b) the introduction of feedback mechanisms in the network to measure the real time packet loss ratio and network delay, in order to create an adaptive version of the protection mechanisms presented in this work.


Temporary drinking abstinence campaigns have emerged globally in recent years. In Western countries, campaigns usually challenge drinkers to abstain for one month. In Thailand, the campaign called the Buddhist Lent Abstinence Campaign has been organized annually since 2003. The campaign encourages Thai people to abstain from drinking for three months during the Buddhist Lent period, which coincides with the monsoon season in Southeast Asia (around July–October). This study aimed to estimate the proportion and number of drinkers changing their drinking behaviours during the 3-month Thai abstinence campaign and to examine the determinants of abstinence.The 2016 Buddhist Lent Abstinence Evaluation Survey was analysed. The survey was a national representative survey of Thai populations aged ≥15 years. Weighted data were employed throughout the analysis. The number and proportion of drinkers changing their drinking behaviours were estimated. The determinants of alcohol abstinence during the campaign were explored using weighted logistic regression.The prevalence of drinking in the Thai population was 34.3% (95% CI: 32.2–36.4%). A third of the current drinkers, equal to almost six million drinkers, abstained completely during the 3-month period. Another six million drinkers partially changed their drinking behaviours . The factors associated with abstinence included religion, occupation, drinking frequency prior to the campaign, type of beverages consumed, perceived harm from alcohol, exposure to campaign media, and making a public commitment.This study demonstrated the effectiveness of a temporary abstinence campaign in Thailand. The work is part of the growing global evidence on the effectiveness of this type of intervention. Temporary abstinence campaigns could be a potential approach to controlling alcohol consumption and related harms. Further research should focus on the long-term effects of such campaigns. Recently, campaigns challenging individuals to temporarily refrain from drinking have emerged in many countries. Examples of those campaigns are “Dry January” in the UK, run by Alcohol Concern, with the first campaign in January 2013; “On The Dry” in Ireland, run by the Irish Heart Foundation, with the first event in January 2015; and “Dry July” in Australia, run by the Dry July Foundation, with the first public event in July 2008 . All camThe temporary abstinence challenge might be considered a new type of social influence or mass media campaign. Conventional campaigns with methods such as resistance skills training and normative education attempt to alter attitudes towards drinking. The effects of such campaigns are controversial and have been regarded as ineffective strategies for reducing harm from drinking , 6. The One study assessed the effects of Dry January in the UK. The study reported that campaign participants had decreased their drinking frequency, intoxication episodes, and drinking amount per drinking day in the sixth month after the campaign. It also observed an increase in drink refusal self-efficacy among campaign participants. The frequency of intoxication episodes prior to the campaign was the only predictor of successful completion of the one-month abstinence . Hence, A similar campaign has been implemented in Thailand since 2003, long before it emerged in Western countries. The campaign is called the Buddhist Lent Abstinence Campaign. The campaign organizers exploit the fact that the vast majority of Thais 93.6%) are Buddhists . The cam3.6% are There are two main activities the campaign uses to promote alcohol abstinence. The first is a mass media campaign that advertises the campaign through TV broadcasting, radio, newspaper, billboard, and social media . The mass media campaign is carried out nationwide. The other activity is a community-based campaign. Local staff of the StopDrink Network Thailand who work with the community encourage community members to participate in the campaign. In some communities, there is a campaign kick-off ceremony that provides an opportunity for campaign participants to make a public commitment to the campaign, allowing other community members to monitor their drinking behaviour throughout the campaign period. Nevertheless, the campaign targets all drinkers and requires no formal registration. The campaign is mentioned under Strategy 2  and Strategy 4 (promoting community-based solutions) of Thailand’s National Alcohol Strategy . The camTo date, only two studies, mentioned earlier, have assessed the effectiveness of temporary abstinence campaigns, and both focused on the UK Dry January campaign. This study is the first to address temporary abstinence campaigns in Asian and middle-income countries, where drinking contexts and habits are different from those in high-income Western countries, using a national representative survey . The objThis study used data from the 2016 Buddhist Lent Abstinence Evaluation Survey. The survey was conducted by the Research Centre for Social and Business Development and funded by the Center for Alcohol Studies for to the evaluation of the campaign. The survey used the multistage sampling method. First, the provinces were stratified into five strata: the Bangkok metropolitan region, central region, northern region, north-eastern region, and southern region. Three, two, two, three, and two provinces were selected from each stratum, respectively; a number of provinces from each stratum, and the probability of a province being selected within its strata was proportional to the population sizes of the strata and provinces. In the second and third stages, districts and sub-districts were randomly selected with a probability proportional to their size. In the fourth stage, subjects were randomly selected from among the sub-district civil registrations. Face-to-face interviews were conducted by trained interviewers during 1st–16th October 2016.Eligibility criteria for the survey subjects included an age ≥ 15 years and the ability to communicate in Thai. The sample size was computed to estimate the proportion of Thai people aged ≥15 who participated in the campaign using a formula for a finite population. The proportion was assumed to be 0.5 with a margin of error of 0.02. The 2015 population size of 52,618,286 was used in the sample size calculation . A desigThe survey questionnaire was divided into three parts. The first part included exposure to campaign media and opinion towards the campaign. The second part included drinking behaviours prior to and during the campaign. The third part included demographic data. Important questions about drinking behaviours include drinking status (drinking during the 12 months prior to the campaign and not drinking during the 12 months prior to the campaign), drinking frequency prior to the campaign , and beverage types drunk in the 12 months prior to the campaign . Thai frappe cocktail is a type of alcoholic beverage made by blending liquor with flavoured syrup, water, and ice. It is popular among teenagers and women .Questions related to the campaign included changes in drinking behaviour, making a commitment to the campaign publicly, perceived impacts of abstinence, and exposure to campaign media. The drinking behavioural change variable comprised four levels: complete abstinence (abstained for three months), partial abstinence (abstained for a certain period), decreasing quantity (a reduced number of drinks per drinking occasion), and drinking as usual. Campaign kick-off ceremonies, which allowed members to make a commitment to the campaign publicly, were available only in some communities. Thus, the variable of making a public commitment included three levels: yes, no, and not available. Some who were exposed to the campaign media also forwarded the message to their family and peers. Survey subjects had three options when answering the question about exposure to campaign media: no exposure; yes, and forwarded the message to others; and yes, but did not forward the message to others. The questionnaire is included as a supplementary file —namely, the SES index—was created by adding scores from two variables: education  and monthly income . The SES index therefore had five levels, with 0 indicating the lowest SES level and 4 indicating the highest SES level. The SES index was used as an independent variable instead of the education or income variables in the regression analysis.The drinking behaviour change variable was further dichotomised and used as a dependent variable of the regression. A participant with complete abstinence or partial abstinence was classified as an “abstainer”. A participant with a decreasing quantity or drinking as usual was classified as a “drinker”. The decreasing quantity group was grouped with the drinking as usual group to avoid social desirability bias . The detThe total number of survey subjects was 4296. Among those, 1486 had drunk in the 12 months prior to the campaign (current drinkers). Using weighted data, 17,922,215 Thai drinkers were used, and the estimated drinking prevalence of the Thai population was 34.3% (95% CI: 32.2–36.4%). Only current drinkers were included in the further analysis.Table As shown in Table Table p-value = 0.293; results not shown).Table This study demonstrates that the 3-month abstinence campaign in Thailand led to the complete abstinence of approximately six million drinkers (one-third of the current drinkers) and the partial abstinence of three million other drinkers. Drinking behaviours prior to the campaign (frequency and types of consumed beverages), campaign activities (making a public commitment and campaign media exposure), perception about harm from drinking, and demographic characteristics were associated with drinking behaviours during the campaign period.The 3-month abstinence campaign in Thailand achieved a higher rate of participation (via participants stating that they had abstained or reduced their consumption) compared to the UK “Dry January” campaign : the participation rate was 67.2% in Thailand and 11% in the UK 2016 campaign . DrinkinOne distinct feature of the Thai campaign is that it incorporates the Buddhism concept of the Five Precepts and Buddhist Lent into the campaign. This religious aspect is designed to enhance participation and compliance with the campaign by taking advantage of the fact that most Thais are Buddhists . The finMaking a public commitment to the campaign boosted the chance of abstinence. Making a commitment either privately or publicly has been shown to be an effective strategy for short- and long-term changes in behaviours regarding household conservation  . The expAn association between drinking behaviours prior to the campaign period and successful abstinence was reported in a previous study from the UK . Those dThe primary strength of this study is the usage of data from a national representative survey. This study is also the first systematic attempt to assess and report a temporary abstinence campaign from an Asian and middle-income country. This study has some limitations. The assessment of drinking behaviours was based on self-report and was likely suffer from social desirability effects, given the involvement of religion. To mitigate this effect, those reducing their consumption (but not abstaining) were grouped with those who were drinking as usual. The survey contained no follow-up data to assess whether the abstainers consumed more than usual after the campaign ended (rebound effect). Regarding this point, findings from the UK study indicated that such a rebound effect affected only a small portion of campaign participants .In conclusion, this study, together with findings from previous UK studies , 30, sheAdditional file 1. Survey questionnaire. English translation of the questionnaire used in the survey


Glassware design may affect drinking behaviour for alcoholic beverages, with glass shape and size influencing drinking speed and amount consumed. Uncertainty remains both about the extent to which these effects are restricted to alcohol and the underlying mechanisms. The primary aim of the current study was to examine the effect of differently shaped glasses on time taken to drink a soft drink. The secondary aim was to develop hypotheses about mechanisms concerning micro-drinking behaviours and perceptual effects.In a single-session experiment, 162 participants were randomised to receive 330ml of carbonated apple juice in a glass that was either inward-sloped, straight-sided, or outward-sloped. The primary outcome measure was total drinking time. Secondary outcome measures included micro-drinking behaviours , and perceptual measures .r(162) = -.45, p < .001). The direction of effects suggested sips were larger from the outward-sloped and inward-sloped glasses, compared to the straight-sided glass . There were no significant differences between glasses in mean sip or interval duration. Bias in midpoint estimation was greater for the outward-sloped glass  than for the straight-sided glass, although the degree of bias was not associated with total drinking time (Pearson’s r(162) = 0.01, p = .87).Participants drank 21.4% faster from the outward-sloped glass than from the straight-sided glass  in adjusted models. They were also 18.2% faster from the inward-sloped glass than the straight-sided glass, but this did not reach statistical significance with wide confidence intervals also consistent with slower drinking . Larger sips were associated with faster drinking times (Pearson’s Individuals drank a soft drink more quickly from an outward-sloped glass, relative to a straight-sided glass. Micro-drinking behaviours, such as sip size, are promising candidates for underlying mechanisms. Overconsumption of sugary drinks and alcohol is a major public health concern, contributing to rising levels of obesity and premature preventable mortality and ill health –3. InterPopularised in the book ‘Nudge’, some examples of such interventions include introducing chevrons on roads to reduce driving speed, adding flies on urinals to improve aim, and increasing the time taken for lift doors to close to encourage stair use . To chanWhen we drink, we nearly always come into contact with a drinks container. Glassware, a modifiable cue in our drinking environment, is therefore a good candidate for interventions that aim to change drinking behaviour at a population level. It is widely documented that the design of a drinks vessel can influence subjective ratings of its contents, including flavour, perceived volume, liking for the drink, the amount an individual is willing to pay , andThere is some evidence that glass shape influences drinking speed. Attwood and colleagues  found thad libitum consumption; for a review see 21]) may Taken together, the evidence suggests that micro-drinking behaviours might be important for understanding the effects of glass shape on drinking behaviour, but it is not clear which particular aspects of glass shape or size might cue differences in micro-drinking behaviours , nor which micro-drinking behaviours warrant further investigation .The present study investigated whether glass shape predicts total drinking time for a soft drink, using inward-sloped, straight-sided, and outward-sloped glasses. Although previous research was inconclusive as to the effect of glass shape on drinking time for a soft drink, with an effect found only for an alcoholic drink , the soff = .25) of glass shape on total drinking time, with 80% power, at an alpha level of .05, a sample of 159 was needed. To allow for equal numbers of males and females, a total sample size of N = 162 was sought.Participants were recruited from the students and staff at the University of Cambridge, as well as the general population, using flyers, mailing lists, and word of mouth (forming a convenience sample). To take part, it was required that individuals: were over 18 years old; had English as a first language or an equivalent level of fluency; were prepared to consume a drink that contained sugar ; and had no known allergies to Appletiser® . We calculated that in order to detect a medium overall effect size (n = 54), as well as equal proportions of males and females in each group  inward-sloped, b) straight-sided, or c) outward-sloped. Randomisation was constrained to ensure equal group sizes . ,26. n = www.havens.co.uk . The straight-sided glass was designed and supplied by LSA International https://www.lsa-international.com/ . The outward-sloped glass was designed by Libbey and supplied by www.drinkstuff.com . See The inward-sloped glass was designed by Dartington and supplied by Total drinking time (min) was measured using video recordings of the drinking sessions. These video recordings were coded using a program to extract the time at each sip initiation and sip end (indicated by key press). Total drinking time was calculated by taking the difference in time between: (1) the initiation of the first sip (when the glass touches the lips for the first time), and (2) the endpoint of the last sip .Micro-drinking behaviours were measured from video recordings of the drinking sessions, coded manually using a program with key presses signalling the initiation and endpoint of sips.Mean sip size (ml), or the average volume consumed per sip, was measured by dividing 330ml  by the number of sips (extracted from the coded video data).Mean sip duration (sec), or the average time spent sipping, was calculated by taking the average of all sip durations (the difference between the initiation and end point of a given sip), again derived from the coded video data.Mean interval duration (sec), or the average time spent in between sips, was calculated by taking the average of all inter-sip intervals (the difference between the endpoint of a given sip and initiation of the next sip), again derived from the coded video data.Bias in midpoint estimation was determined from six poured estimates of the midpoint of the drink (165ml),averaged to provide a single estimate for each participant. This was then subtracted from 165ml (the true midpoint), to determine bias. Negative values reflect underestimation of the true midpoint (pouring too little liquid into the glass), while positive values indicate overestimation of the true midpoint (pouring too much liquid into the glass).Drink enjoyment was measured using two questions, rated on a ten-point scale anchored at one end by 1  and at the other end by 10 (Extremely), which asked how ‘pleasant’ and ‘tasty’ the drink was. Together, these ratings formed ‘drink enjoyment’ (Cronbach’s a = .90). These two questions formed part of a ‘taste perception’ task in which participants rated the drink along 10 different descriptors . The results of all other ratings were not analysed. This rating task and measure of ‘drink enjoyment’ has been used previously in a laboratory study investigating drinking behaviour [ehaviour .Participants completed a computer-based word-search task. They were asked to find as many words as possible in 4-minutes. This was included to obscure the true aim of the study, and to make the cover story (that we were investigating the impact of glucose on cognitive performance) more believable. The data from this filler task were not analysed.Participants were asked to indicate what they thought the main purpose of the study was, choosing from nine possible answers. Those who correctly identified ‘To investigate the impact of glass design on drinking rate’ were coded as aware.The study was approved by the University of Cambridge Psychology Research Ethics Committee (reference: PRE.2017.018). Eligible participants were invited to attend a single study session, scheduled between 8 am and 8 pm, Monday to Saturday. As a cover story, participants were informed they were taking part in a study on the impact of glucose on cognitive performance. On arrival, participants completed self-reported eligibility screening, and gave written informed consent. They then answered questions about their age, gender, level of education, and thirst. During this time, the experimenter removed a 330ml can of Appletiser® from the fridge, and brought it into the testing room along with the glass the participant had been randomised to drink from. The full 330ml can was then poured into the glass, immediately prior to serving to ensure consistent carbonation across study sessions. The experimenter then placed the drink on a coaster and informed the participant to consume the drink at their own pace, whilst watching a nature documentary . Before leaving the room, the experimenter turned on the documentary and switched on the video camera. Participants were asked to open the door when they had finished the drink.Next, participants completed the computer-based four minute word-search, followed by the ‘taste perception’ task, in which they rated the drink along 10 descriptors. For the final task, assessing bias in midpoint estimation, the experimenter first placed the 330ml glass of Appletiser® directly in front of the participant—the same glass as they had been randomised to drink from. Participants were then instructed to pour half of the liquid in the glass into a jug containing 660ml of Appletiser®, which was placed behind the glass. After the experimenter had weighed the glass to determine the participant’s poured estimate, the participant was instructed to pour another midpoint estimate, this time from the jug into an empty glass. Pours were attempted six times in total . For reference, they were presented with a full 330ml glass of Appletiser® (a ‘Reference Glass’), placed to their left throughout the task.Finally, participants were asked to indicate what they thought the aim of the study was, to examine the effectiveness of the cover story in blinding participants to the behavioural measures and the true nature of the study. A basic debrief was provided, and a full debrief was sent via email once testing for the study was complete. Participants received £7 in cash for taking part.The primary analysis was a multiple linear regression to determine whether glass shape  predicted drinking rate. Using linear regression, two dummy-variables  were entered, with straight-sided as the reference variable. Analyses were run to adjust for differences in gender, age, thirst, maximum daily temperature, and time of day. Secondary analyses were conducted to determine whether glass shape predicted micro-drinking behaviours , as well as perceptual factors . Sensitivity analyses excluded participants who correctly guessed the true purpose of the study. Reliability analyses, with an independent coder, were conducted on 20% of the videos.https://osf.io/fwmg9/ (DOI: 10.17605/OSF.IO/FWMG9).The data for this experiment are available from the Open Science Framework: SD = 6.72, range = 18 to 69), and all participants had at least AS/A Level education . Baseline characteristics, split by condition, are given in One hundred and sixty two individuals took part in the study . The mean age was 24.0 years . Where means are reported, they are geometric (back transformed) with 95% CIs ,29. We nMdn = 5 minutes 48 seconds, IQR = 5 minutes 53 seconds), fastest from the outward-sloped glass  and between these two speeds from the inward-sloped glass . See Individuals drank slowest from the straight-sided glass  and 6 minutes 22 seconds  respectively, CI: 0.21%, 38.02%), p = .048. Total drinking time from the inward-sloped glass was 18.22% faster than from the straight glass, although this was also consistent with slower drinking , p = .098. See Adjusting for gender and other pre-specified covariates, total drinking time from the outward-sloped glass was 21.36% faster than from the straight-sided glass,  indicated positive skew. Log(10) transformations improved the shape of these distributions so analyses were conducted on the transformed data for all three micro-drinking behaviours. For micro-drinking behaviours, where means are reported, these are Geometric (back transformed) with 95% CIs. See Geomean = 17.79ml, 95% CI: 16.07ml, 19.68ml) than males , t(161) = 3.29, p = .001). To adjust for these differences, we included gender in the model for sip size.Females took smaller mean sips  = 5.01, p = .002. After adjusting for the effect of gender , estimates suggested that individuals took sips that were 19.40% larger from the inward-sloped glass, compared to the straight-sided glass, although the data were also consistent with smaller sips , p = .057. Sips were estimated to be 15.08% larger from the outward-sloped glass than from the straight-sided glass, although the data were again also consistent with smaller sips , p = .13.Glass shape and gender explained 8.7% of the variability in log(10) mean sip size, F = 0.91, p = .40), or log(10) mean interval duration  = 0.39, p = .68).Unlike mean sip size, gender did not predict mean sip duration or mean interval duration. It was therefore not included in these models. Glass shape did not meaningfully predict log(10) mean sip duration (r(162) = -.45, p < .001), see r(162) = -.09, p = .25).There was a medium-sized negative association between mean sip size and total drinking time, suggesting that larger mean sip sizes were associated with shorter total drinking times  = 11.54 , p < .001). All glasses were associated with an underestimation of the true midpoint , p = .82. Bias in midpoint estimation was not associated with total drinking time (Pearson’s r(162) = 0.01, p = .87).Glass shape predicted bias in midpoint estimation, explaining 12.7% of the variance,  = 0.18, p = .83); means and standard deviations are given in r(162) = -.04, p = .62).We found no evidence that glass shape influenced how much the drink was enjoyed (n = 5) who correctly identified the purpose of the study (to investigate the impact of glass shape on drinking rate) did not alter the main conclusions. Unadjusted and adjusted estimates suggested outward-sloped glasses led to faster drinking than straight glasses , p = .06, respectively), although the confidence interval for the outward-sloped vs. straight-sided glass comparison now crossed zero.Removing participants  = 1.0, p < .001, sip duration (32) = .99, p < .001, and interval duration (32) = .99, p < .001.Inter-rater reliability was high for the video coded data. Single measures intra-class correlation indicated strong and positive associations for total drinking time (32) = .98, Geomean = 2.47 seconds, 95% CI: 2.12, 2.88) than the straight glass , p = .004. First sip durations from the inward-sloped glass  did not differ from the straight-sided glass, p = .386. The pattern of sip durations over time (line gradient) also differed significantly between the outward-sloped glass and straight-sided glass (p < .0001). Note: means and 95% CIs are back-transformed from log10 scale and p-values are Kenward-Roger adjusted.To understand the dynamic pattern of drinking rate across time, or ‘drinking tempo’, we explored the coded video data further. We plotted individual log(10) sip durations as a function of time, separated by condition. Time was normalised to each participant’s total drinking time, with 100% reflecting each individual’s total drinking time see . Visual The present study found an effect of glass shape on drinking rate for a soft drink: faster drinking was observed from outward-sloped glasses than from straight-sided glasses. Findings for inward-sloped glasses were inconclusive, although suggestive of faster drinking when compared with straight-sided glasses. To our knowledge, this study is the first to show an effect of glass shape on total drinking time for a non-alcoholic drink, with a previous study suggesting the effect might have been limited to alcohol . The finmean sip size’, which is derived from dividing the amount consumed by the total number of sips, may not capture the complexity of sipping behaviours, and miss true differences where they exist. These might be better examined dynamically, or across the drinking period. Given that we did not measure individual sip sizes, to examine the dynamic changes in drinking rate, we explored individual-level (rather than average) sip durations, and plotted these as a function of time. From the outward-sloped glass, participants took longer initial sips, which then got shorter over time. By contrast, from the straight-sided and inward-sloped glasses, sip durations gradually increased over time. Using sip duration as a proxy for sip size, we speculate that sips were initially larger from the outward-sloped glass due to an automatic cueing of sip size. When full, and given the same angle of tilt, an outward-sloped glass affords a faster flow of liquid than the other glasses. These larger initial sips might therefore be a key determinant in the faster overall drinking rate seen from outward-sloped glasses. Taken together, these findings suggest that drinking glasses may cue specific patterns of micro-drinking behaviours, depending on both glass shape and relative fullness which in turn may influence total drinking time.The micro-structure of drinking behaviour—micro-drinking behaviours—was considered as a possible mechanism underlying the effect. Mean sip size was negatively associated with total drinking time, with larger sips associated with faster consumption. Individuals tended to take larger sips when drinking from the inward-sloped and outward-sloped glass, as compared to the straight-sided glass. However, the confidence intervals for these effects crossed zero. Thus, it remains unclear whether differences in sip size taken from smaller vs larger cups shown in previous studies ,23 are sIn addition to micro-drinking behaviours, we explored perceptions as another set of possible mechanisms underlying the observed effect. While the ability to estimate volume was poorest in the outward-sloped glass, as previously shown ,30, midpincrease desire for the drink [decrease pleasantness ratings [There were several strengths to the study. To our knowledge, this study is the first to find an effect of glass shape on drinking behaviour for a soft drink. This extends prior knowledge, suggesting that effects might not be limited to alcoholic drinks. We also used an objective measure of drinking, as well as subjective ratings and perceptual measures . There he drink , and oth ratings . However ratings ). FutureGiven that the effect of glass shape on total drinking time in the current study was substantially smaller than has been previously found using alcohol , future The current study provides evidence that glass shape influences drinking rate for a soft drink with consumption faster from outward-sloped glasses than from straight-sided glasses. Changes in the micro-structure of drinking–for example, sip size–may be a promising candidate for understanding this effect. In line with a shift of focus towards choice architecture interventions, this study, in conjunction with other experimental and field data, can aid the design of effective interventions to reduce consumption of sugary and alcoholic drinks.


T staging and administration of RAI therapy. Background. Regarding the rare but aggressive insular thyroid carcinoma (ITC), the value of prophylactic central compartment neck dissection (CCND) for clinically node-negative (cN0) disease is unclear. We aimed to provide the first evidence. Methods. N0 and pN1a ITC patients were identified from the Surveillance, Epidemiology, and End Results database. These patients were divided into thyroid-surgery + CCND group (pN0/pN1a patients confirmed by CCND) and thyroid-surgery group (cN0 patients without CCND). Differences in overall survival (OS) and disease-specific survival (DSS) between the two groups were evaluated. Subgroup analyses were also conducted. Results. Of the overall 112 patients, 44 (39.3%) received CCND. On multivariate analyses, the lobectomy ± isthmusectomy/total-thyroidectomy (Lob/TT) group demonstrated poorer OS and DSS than the Lob/TT + CCND group (P < 0.05). When we separately analyzed patients treated by TT, multivariate analyses showed the TT group still revealed compromised OS and DSS than the TT + CCND group (P < 0.05). Furthermore, absence of CCND independently predicted decreased OS no matter whether radioactive iodine (RAI) was administered. Similar results were obtained for T3/T4 patients. Moreover, for T1/T2 patients receiving CCND, 0/12 died during the study period, while for T1/T2 patients without CCND, 8/23 (34.8%) died, 5/23 (21.7%) due to ITC. Conclusion. Regardless of T staging and RAI treatment, cN0-ITC patients without CCND had decreased survival compared with pN0/pN1a patients receiving CCND. Therefore, if a cN0 patient is diagnosed with ITC, prophylactic CCND may be considered as a secondary procedure (postoperatively diagnosed) or a primary procedure (preoperatively/intraoperatively diagnosed). Prospective studies are expected to validate the conclusion.For the rare but aggressive insular thyroid carcinoma (ITC), there's no clear evidence to determine whether prophylactic central compartment neck dissection (CCND) is necessary for cN0 disease. This study provides the first evidence that treating cN0 ITC without prophylactic CCND is associated with decreased survival regardless of  First described by Carcangiu et al. in 1984 –4. Data The value of prophylactic central compartment neck dissection (CCND) in clinically node-negative (cN0) differentiated thyroid cancer (DTC) has been a hot topic for decades but is still a matter of debate. With regard to PDTC found postoperatively, the previous National Comprehensive Cancer Network (NCCN) guideline (2017 version 2) lists CCND as a consideration for cN0 patients . HoweverAlthough the 2015 ATA guideline recommends prophylactic CCND for T3/T4 cN0 patients, lobectomy without CCND is regarded appropriate for cN0 patients with T1/T2 tumors , 12.Due to the aggressive behavior of the insular subtype, whether cN0 ITC necessitates CCND, especially for T1/T2 patients or those undergoing RAI therapy, deserves further attention in clinical practice. However, given the paucity of data, this problem has never been investigated. In this study, we sought to explore this issue using data from the Surveillance, Epidemiology, and End Results (SEER) database.T staging and known tumor size. We excluded patients without information on whether CCND was performed and those less than 18 years. The selection process is presented as a flow diagram  diagnosed with ITC as the first malignancy using the International Classification of Diseases for Oncology, 3rd edition (ICD-O-3) code 8337; (2) diagnosed between 1999 and 2014, as the first ITC patient recorded in the SEER program was diagnosed in 1999; (3) received thyroid surgery; (4) without distant metastasis at diagnosis; (5) with N0 or pN1a record of diagnosis; (6) with definite record of Institutional Review Board (IRB) approval is not necessary for this study, because the SEER program is a publicly available cancer database with deidentified data.T and N stages, multifocality, extrathyroidal extension (ETE), tumor size, thyroid surgery, radiation, and presence/absence of CCND. As small ITCs were sometimes found after lobectomy, in the present study, thyroid surgery included both lobectomy ± isthmusectomy (Lob) and total thyroidectomy (TT) to better reflect the real clinical situation. Radiation was divided into three categories: “no evidence,” “external beam radiotherapy (EBRT),” and “RAI,” just like the previous studies [T and N staging were identified according to the American Joint Committee (AJCC) eighth edition.Data extracted for each case included age at diagnosis, sex, race,  studies , 5. As othyroid-surgery + CCND group (pN0/pN1a patients confirmed by CCND) and thyroid-surgery group (cN0 patients without CCND).The N0 patients included pN0 patients confirmed by CCND and cN0 patients without prophylactic CCND. It is worth noting that, in the SEER database, if a patient has a pathologic stage, the record of clinical stage will be covered and not available to users. Consequently, the cN staging (cN0/cN1) of pN0 and pN1a patients was unknown, the CCND of whom might include both prophylactic CCND and therapeutic CCND. Subsequently, the overall cohort was divided into the Overall survival (OS) and disease-specific survival (DSS), respectively, defined as the interval from initial pathologic diagnosis to the date of all-cause or ITC-specific death were compared between the two groups.T stage was not incorporated because the two factors, tumor size and ETE, which constituted the T staging were both included in the model. The Type-B model included the best subsets of covariates identified by the smallest Akaike Information Criterion (AIC) value, which reflected the minimal loss of information [The differences of baseline characteristics across the two groups were compared by chi-squared test or Fisher's exact test, as appropriate. The survival curves were plotted by Kaplan––Meier estimates, and the differences were compared by log-rank tests. Multivariate Cox proportional hazards regression models were employed to identify the prognostic factors after adjusting for potential confounders, and the results were presented as hazard ratios (HRs) with 95% confidence intervals (CI). To better evaluate the prognostic relevance of CCND, for all multivariate analyses (MVA), two types of Cox regression models were parallelly performed. The Type-A model was defined as the Cox regression model including comprehensive baseline variables , ormation –15.P value < 0.05.Log-rank tests and multivariate Cox analyses were conducted by SPSS Statistics v23.0 . Kaplan–Meier survival plots were generated by GraphPad Prism 7.0 . The AIC values were calculated by R version 3.4.3 . Statistical significance was defined as a two-tailed n = 72) had tumors larger than 4 cm, nearly three fourths of the tumors were unifocal , and approximately one third  had ETE. 98 (87.5%) patients underwent TT, and 70 (62.5%) received RAI therapy  and the median age at diagnosis was 58 years (range: 21–94). 64.3% of patients ( therapy .n = 44). The 68 cN0 patients without CCND constituted the Lob/TT group. Baseline characteristics across the two groups are also shown in P=0.038).102 patients were recorded as N0, 34 were pN0 receiving CCND, and 68 patients were cN0 not receiving CCND. 10 patients were diagnosed with pN1a disease (possible reasons were discussed in Discussion), and none of these patients had level VII  metastases. The pN0 and pN1a patients undergoing CCND constituted the Lob/TT + CCND group (P=0.048). No significant difference in DSS was observed in the log-rank test (P=0.078).The survival difference between the Lob/TT + CCND group and the Lob/TT group was investigated for the overall cohort. Among 44 patients in the Lob/TT + CCND group, 5 (11.4%) died during the study period, 3 (6.8%) of which were due to ITC. Among 68 patients in the Lob/TT group, we observed 25 (35.8%) deaths and 16 (23.5%) were due to ITC . The logP=0.005) and DSS (HR = 5.707 (95% CI 1.362–23.907), P=0.017) compared with the Lob/TT + CCND group , P=0.018)  In the Type-A Cox models, the Lob/TT group was independently associated with worse OS (HR = 4.640 (95% CI 1.576–13.659), ND group . (ii) ThP=0.015) and DSS (HR = 5.087 (95% CI 1.089–23.751), P=0.039) compared with the TT + CCND group , P=0.030)  In the Type-A Cox models, the TT group demonstrated significantly inferior OS (HR = 4.175 (95% CI 1.325–13.158), ND group . (ii) InP=0.021; DSS : HR = 15.796 (95% CI 1.380–180.789), P=0.026) and Type-B Cox models incorporating age at diagnosis, multifocality, ETE, and CCND determined by the smallest AIC (OS : HR = 7.350 (95% CI 1.440–37.520), P=0.016; DSS : HR = 14.933 (95% CI 1.442–154.649), P=0.023), as shown in Supplementary Tables For patients who underwent TT and RAI, absence of CCND still predicted compromised survival in both Type-A (OS : HR = 7.137 (95% CI 1.354–37.636), P=0.082; DSS : HR = 2.977 (95% CI 0.786–15.539), P=0.095) (Supplementary P=0.035) in the Type-B model controlling for variables identified by the smallest AIC, as shown in Supplementary For patients who did not receive RAI ablation, the prognostic difference failed to meet statistical significance in Type-A Cox regression models (OS : HR = 3.951 (95% CI 0.803–19.945), P=0.023; DSS : HR = 4.414 (95% CI 1.084–17.967), P=0.038; Type-B : OS: HR = 3.318 (95% CI 1.107–9.946), P=0.032; DSS : HR = 4.178 (95% CI 1.040–16.780), P=0.044).For patients with T3/T4 disease, similar results were obtained. As shown in Supplementary Tables For patients with T1/T2 disease, there were no significant differences in age at diagnosis, sex, race, multifocality, thyroid surgery, and radiation (Supplementary n = 0) in the TT + CCND group.Furthermore, we focused on T1/T2 patients undergoing TT plus RAI and all-cause mortality in the TT + CCND group and TT group was 0/9 (0.0%) and 6/17 (35.3%), respectively, while disease-specific mortality in the two groups was 0/9 (0.0%) and 4/17 (23.5%), respectively Figures . SimilarBiologically and morphologically, ITC occupies an intermediate position between well-differentiated thyroid carcinoma (WDTC) and anaplastic (undifferentiated) thyroid carcinoma (ATC) . KazaureNevertheless, neither the 2015 ATA nor the latest NCCN guideline pays special attention to the indication of prophylactic CCND for ITC patients , 10, posn = 52) were identified to be RAI-refractory [Although Conzo et al. reported that TT without routine prophylactic CCND, followed by RAI ablation also revealed low recurrence rate , we founfractory . For patP values of less than 0.05 could not be achieved because of the small samples, the non-negligible difference in the number of deaths between T1/T2 patients with and without CCND also prompted us to attach importance to the value of CCND in early-stage ITC patients.Another important result derives from the subgroup analyses for T1/T2 disease. Although As a most common subtype of PDTC, accurate preoperative diagnosis of ITC is crucial for planning optimal surgical management. However, its cytomorphological features are not well characterized, making it easily to be missed in fine-needle aspiration biopsy (FNAB) . At presn = 43) or “N1b/N1-NOS/NX” (n = 33), suggesting that ITC tends to involve more extensive regions and disease confined to the central compartment is not as common as it is in PTC.A noteworthy point is that there are only 10 eligible pN1a patients in our study cohort despite the aggressive behavior of ITC. In a study using NCDB data, only 27 of 405 M0 ITC cases were diagnosed with N1a disease [thyroid-surgery + CCND group might undergo therapeutic CCND. Second, surgical margins were not recorded, although the thyroid-surgery + CCND group revealing better prognosis had a significantly higher proportion of patients with extrathyroidal extension, which sometimes lead to a difficulty in R0 resection. Third, dose of RAI, BRAF mutational status, and comorbidities were not provided in the database. Fourth, age at diagnosis of the CCND and No-CCND groups was not totally the same, which possibly influenced prognosis to some extent. However, in this study, the various multivariate Cox analyses for the overall cohort or subgroups might reduce the confounding effect of patients' age to a large extent. Fifth, this study is limited by its retrospective nature.Some limitations should be acknowledged. First, as mentioned above, whether the pN0/pN1a patients were cN0 or cN1 was unknown in SEER database, so some patients in the T staging and administration of adjuvant RAI therapy. Therefore, if a cN0 patient is diagnosed as ITC, after fully weighing the benefits and risks, prophylactic CCND may be considered as a secondary procedure (if postoperatively diagnosed) or a primary procedure (if intraoperatively/preoperatively diagnosed), especially for experienced high-volume surgeons. If possible, prospective studies are expected to validate our findings.In conclusion, treating cN0 ITC without CCND is associated with decreased survival compared with pN0/pN1a patients receiving CCND irrespective of


Cdkn1a (p21) gene, and used different bromodeoxyuridine (BrdU) paradigms for cell fate mapping. The acute apoptotic response of NPCs in the subgranular zone of the dentate gyrus was independent of p21 after irradiation. In nonirradiated mice, p21 knockout resulted in an increase in neuroblast proliferation and neurogenesis. At 9 weeks after 5Gy, NPCs in the subgranular zone demonstrated increased p21 expression. Loss of newborn type-1 cells and disruption of hippocampal neurogenesis was evident at 9 weeks after irradiation, and these effects were independent of p21 genotype status. Within the developmental milestones of NPCs, irradiation resulted in loss of early intermediate NPCs (type-2a cells) in wild-type mice, whereas the principal effect of irradiation with p21 loss was culling of proliferating late intermediate (type-2b cells) and neuroblasts. These results suggest that p21 exerts differential effects on cell fate of NPCs after irradiation. p21 may serve to protect proliferating late NPCs but does not alter the ultimate inhibition of new neuron production after DNA damage.Inhibition of hippocampal neurogenesis is implicated in neurocognitive impairment after cranial irradiation. We recently demonstrated that disruption of neuronal development after DNA damage was regulated by p53. The cyclin-dependent kinase inhibitor 1 or p21, a downstream effector p53, mediates cell cycle arrest in response to DNA damage. There is evidence that p21 negatively regulates proliferation of neural progenitors (NPCs). Here we characterized the effects of p21 on disruption of neuronal development in the hippocampal dentate gyrus after irradiation. We irradiated young adult mice wild type (+/+) or knockout (−/−) of the  In the adult mammalian brain, the dentate gyrus of the hippocampus represents an area where NPCs continue to generate new neurons which become integrated into the neuronal circuitry3. Neuronal development in the adult hippocampus is disrupted in various pathologic conditions and brain injuries2 including after ionizing radiation4. Neurogenesis is associated with hippocampal function of learning and memory6. Inhibition of neurogenesis is implicated in neurocognitive decline following radiation treatment for brain tumors4. How DNA damage following ionizing radiation leads to impaired neuronal development in the adult hippocampus remains unclear7.Many physiologic conditions such as an enriched environment and exercise have been reported to result in enhanced adult neurogenesis2. They give rise to transient amplifying or intermediate NPCs  which differ by their potential for proliferation and increasing neuronal differentiation8. NPCs in the adult mouse hippocampus are known to undergo apoptosis after irradiation9, a response mediated by the tumor suppressor p5311. Despite the absence of NPC apoptosis, p53 loss resulted in increased ablation of newborn type-1 cells and profound inhibition of adult neurogenesis after irradiation12. Activation of p53 after irradiation results in upregulation of its downstream effector, the cyclin-dependent kinase inhibitor 1 or p21. There is evidence that p21 negatively regulates NPC proliferation13. Here we asked whether p21 might play a role in disruption of hippocampal neuronal development after irradiation. Using mice wild type (+/+) or knockout (−/−) of the p21 gene, p21 was found to have differential effects on cell fate of NPCs, and specifically on disruption of the intermediate NPC stages of neuronal development after irradiation. Loss of p21 however did not alter the extent of inhibition of production of new neurons after irradiation.In the commonly accepted model of hippocampal neuronal development, radial glial-like cells or type-1 cells are thought to be the neural stem cells11. Two apoptosis radiosensitive NPC subpopulations, proliferating type-2 cells and nonproliferating neuroblasts (type-3 cells) have been described14. We first determined whether p21, a downstream effector of p53, plays a role in radiation-induced apoptosis. Using nonbiased stereology, we compared the number of apoptotic cells at 8 h, the peak apoptotic response after 5Gy in the dentate gyrus of p21−/− mice with p21+/+ mice. Apoptotic cells with characteristic morphology on 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining and positive (+) for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) or caspase-3  to label neuroblasts, we observed no difference in the number of TUNEL+/DCX+ and caspase-3+/DCX+ cells in p21−/− mice compared to p21+/+ mice at 8 h after irradiation  was given every 2 h for four doses, and animals were given a single dose of 5 Gy immediately after the final BrdU injection. No change in the number of BrdU+/GFAP+/nestin+ cells with characteristic radial glia morphology (type-1 cells) in irradiated p21+/+ mice and p21−/− mice at 24 h was observed compared to their respective 0-Gy controls , type-2b (Tbr2+/DCX+), and type-3 (Tbr2−/DCX+) cells. Of the p21+ subgranular cells, 9.2 ± 1.6% were Tbr2+, and 10.4 ± 3.7% were DCX+. Of the type-2a, type-2b, and type-3 cells, 32.5 ± 10.3%, 56.7 ± 3.3%, and 14.3 ± 6% respectively demonstrated p21 immunoreactivity after irradiation ion Fig. .p < 0.001; p21 genotype, p = not significant; two-way ANOVA; Fig. p < 0.01; p21 genotype, p = not significant; Fig. 13, an increase in DCX+ cells and BrdU+/NeuN+ cells was observed in control nonirradiated p21−/− mice compared to wild-type mice. Irradiation resulted in loss of DCX cells  at 4 weeks after 0 or 5 Gy, and BrdU-labeled NPC subpopulations were quantified at 2 h, 2 days, 1, and 5 weeks after BrdU. Using this BrdU paradigm, cells that demonstrated BrdU incorporation at 2 h were primarily proliferating cells. BrdU+ cells at 2 days represented a blend of proliferating and newborn cells, and those at 1 week and 5 weeks after BrdU were expected to be mostly newborn cells. Tbr2 and DCX immunohistochemistry was used to differentiate BrdU+ intermediate NPCs into type-2a (Tbr2+/DCX−), type-2b (Tbr2+/DCX+), and type-3 (Tbr2−/DCX+) cells Fig. .Fig. 5Pup < 0.05; time after BrdU, p < 0.0001; interaction, p = not significant; two-way ANOVA; Fig. p < 0.05; time after BrdU, p < 0.0001; interaction, p = not significant; Fig. p < 0.001; time after BrdU, p < 0.0001; interaction, p < 0.01; Fig. p = not significant; time after BrdU, p < 0.01; Fig. p = not significant; time after BrdU, p < 0.01; Fig. The number of BrdU-labeled cells declined over the 5 weeks after BrdU injection in nonirradiated mice and irradiated mice regardless of genotype Fig. . In 0-Gyp < 0.05; time after BrdU, p < 0.001; interaction, p = not significant; p21−/− mice: irradiation, p < 0.001; time after BrdU, p < 0.0001; interaction, p = 0.01; Fig. p < 0.05; time after BrdU, p < 0.05; interaction, p = not significant; Fig. p = not significant; time after BrdU, p < 0.01; Fig. p = not significant; time after BrdU, p = not significant; Fig. p = not significant; time after BrdU, p = not significant; Fig. A dose of 5 Gy resulted in loss of total BrdU+ cells in both p21+/+ mice and p21−/− mice  but decreased % BrdU+ type-2b cells , and % BrdU+ type-3 cells appeared unperturbed .To further assess the role of p21 on inhibition of NPC proliferation after irradiation, we determined the number of BrdU doublets, type-1 (GFAP+/nestin+) BrdU doublets Fig.  and type18. How DNA damage following ionizing radiation results in disruption of neuronal development remains unclear. The tumor suppressor p53 is known to regulate cellular response after DNA damage19. It negatively regulates proliferation and self-renewal of neural stem cells22. We recently showed that p53 loss resulted in increased ablation of neural stem cells and profound inhibition of neurogenesis in mouse dentate gyrus after irradiation despite lack of NPC apoptosis12. Damage of the neurovascular niche plays a role in impaired neurogenesis after irradiation25. Impaired neuronal differentiation of NPCs transplanted into the irradiated hippocampus was not altered by p53 genotype of recipient mice12. Thus the effect of p53 on disruption of neuronal development after irradiation is likely to be independent of damage of the neural stem cell niche.Inhibition of hippocampal neurogenesis is implicated in neurocognitive decline following radiation treatment for brain tumors21−/− mice demonstrated limited in vitro self-renewal13. It has been proposed that p21 serves to keep adult neural stem cells in relative quiescence for life-long maintenance of self-renewal. Here, p21 loss was associated with increased cell proliferation in the dentate gyrus, including enhanced proliferation of type-1 cells and neuroblasts. We also observed an increase in newborn neurons, consistent with an inhibitory effect of p21 on neurogenesis in the dentate gyrus.How p53 regulates DNA damage response in NPCs remains unclear. A well-described downstream effector of p53 is p21. In young adult mouse brain, p21 loss is associated with increased NPC proliferation but neurospheres from p26. Adult stem cells are known to be resistant to apoptosis after irradiation. p21 may play a role in the regulation of DNA damage response of stem cells27. In hematopoietic and mammary stem cells in vitro, irradiation was shown to result in activation of p21 independent of p53, and subsequent inhibition of basal activity of p53. This p21-dependent response in stem cells after DNA damage has been postulated to result in inhibition of apoptosis28. Neural stem cells are known to be apoptosis resistant after irradiation14. Here, irradiation resulted in an increase in p21 immunoreactivity in type-1 cells and intermediate NPCs. There was no evidence of acquisition of apoptosis radiosensitivity in neural stem cells with p21 loss, and we also observed no altered apoptosis radiosensitivity in proliferating type-2 cells and neuroblasts in the absence of p21. In irradiated p21−/− embryonic mouse cortex, there was decreased not increased apoptosis29. Whether these differences in p21-dependent responses post-DNA damage are related to differences in tissue-specific stem cell radiobiology, developmental or in vitro conditions, remain to be investigated.Stem cells may have distinct mechanisms from progenitors to mitigate DNA damage to preserve self-renewal and differentiation30. Our results here provide evidence for differential effects of p21 on NPC cell fate after irradiation. In wild-type mice, the predominant effect of irradiation appears to be death of type-2a cells and/or inhibition of their proliferation. In contrast, the major effect of irradiation with p21 loss was increased culling of proliferative type-1, type-2b, and type-3 NPCs. There is in vitro evidence that activation of p21 in certain stem cells results in induction of symmetric self-renewing divisions after irradiation28. Although we did not address self-renewal directly, the absence of any increase in type-1 cells at 2 days compared to at 2 h after BrdU provided no evidence to suggest induced symmetrical division of type-1 cells after irradiation. We also did not observe any p21-dependent effects on type-1 cell fate after irradiation.Neural stem cells and NPCs are known to exhibit differential responses to physiologic stresses and external insults31. These different fate mapping approaches may contribute to the various models of NPC behavior proposed. Here we used BrdU injection paradigms to assess neuronal development. We chose a dose of 5 Gy in our study as this dose resulted in loss of about 50% of newborn neurons12 and was considered optimal to discern the effect of genotype on inhibition of neurogenesis after irradiation. Although our results appeared to internally consistent, no BrdU+ type-2 cells were observed at 5 weeks using the single BrdU injection paradigm. Further information may be obtained using other fate mapping methods and additional sets of phenotypic markers for NPCs.Several methods including thymidine analogs, retrovirus, and transgenic animals have been used to study adult hippocampal neurogensisIn conclusion, there is increased hippocampal neurogenesis associated with p21 loss which may be due to enhanced NPC proliferation and/or neuronal differentiation particularly in the latter stages of intermediate NPC development. Irradiation results in inhibition of neurogenesis and loss of newborn neural stem cells. These effects appear to be independent of p21. In wild-type mice, the most pronounced effects of irradiation is death and/or inhibition of proliferation of type-2a cells. In contrast, there is increased ablation of proliferative type-1, type-2b, and type-3 NPCs after irradiation with p21 loss. Our results are thus consistent with differential effects of p21 on NPC cell fate after irradiation. Loss of p21 however does not alter the ultimate inhibition of production of new neurons after irradiation.p21 gene were obtained from the Jackson Laboratory . Mouse colonies were generated by littermate inbreeding and genotyped by PCR12. To avoid the potential confounding influence of sex on neurogenesis32, only male mice were used in the study. All animal procedures (protocol number: 18-156) were approved by the institutional animal care committee and performed according to the Canadian Council on Animal Care guidelines.C57 mice wild type (+/+) or knockout of the 33. Control (0 Gy) mice were sham-irradiated. A single dose of 5 Gy was used to evaluate the effects of the p21 genotype on neuronal development after irradiation. Three different BrdU incorporation schedules were used for cell fate mapping as described in the Results section. BrdU was administered by intraperitoneal injection.Animals were irradiated at the age of 10 weeks. They were anesthetized using an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (6 mg/kg) and positioned in a customized jig with the entire hippocampus irradiated using an anterior-posterior and posterior-anterior pair of 160 kV X-ray beam  defined by an 8-mm diameter lead cut-outUnder anesthesia with ketamine and xylazine, mice underwent intracardiac perfusion with 0.9% saline followed by 4% paraformaldehyde in PBS. The brain was dissected out, post-fixed for 2 days and cryoprotected in PBS with 30% sucrose. Coronal sections containing the hippocampus were cut at 40-μm thickness and stored in tissue cryoprotectant solution in 96-well plates at −20 °C prior to immunohistochemistry.17. The apoptotic response was further characterized using TUNEL  and caspase-3  immunohistochemistry as previously described11.Apoptotic cells were identified based on characteristic nuclear condensation and fragmentation of apoptosis using DAPI11. Primary antibodies included those against DCX , GFAP , nestin , NeuN , and Tbr2 . Secondary antibodies were conjugated to Cy2, Cy3  or Alexa Fluor 647 . Colocalization of phenotypic markers with BrdU  or Ki67  in selected sections were evaluated using a confocal laser scanning microscope . A BrdU doublet was defined as two abutting DAPI-stained nuclei that demonstrated BrdU incorporation12.NPCs, immature and mature neurons were identified by phenotypic markers using immunohistochemistry11. Cell counting was performed using a Zeiss Imager M1 microscope (Carl Zeiss AG Corporate) with the Stereo Investigator software . The observers were blinded to the experimental groups. Details of stereology were as described previously11. Apoptotic cells and NPCs were counted using a counting frame and a sampling grid of 75 × 75 µm2 at a magnification of ×630. Every seventh section was used as the periodicity of sections sampled. For all stereology data, the coefficient of error ranged from 0.03 to 0.06.Apoptotic cells and cells labeled using different phenotypic markers were counted within the dentate gyrus including a 50-µm margin of the hilusp21-genotype, or p21-genotype and time after BrdU on the various end-points was determined using two-way ANOVA. Pair-wise comparisons were based on post hoc analysis with Bonferroni correction for multiple comparisons. Differences were considered significant for p < 0.05. Statistical analyses were performed with the GraphPad Prism 5 .Cell population analysis represented data from 3 to 5 mice per genotype per dose per time point. All data represented the mean ± SEM. The significance of two independent variables such as irradiation and Supplementary Figure 1Supplementary Figure 2Supplementary Figures Caption