Video delivery in Vehicular Ad-hoc NETworks has a great number of applications. However, multimedia streaming over this kind of networks is a very challenging issue because (a) it is one of the most resource-demanding applications; (b) it requires high bandwidth communication channels; (c) it shows moderate to high node mobility patterns and (d) it is common to find high communication interference levels that derive in moderate to high loss rates. In this work, we present a simulation framework based on OMNeT++ network simulator, Veins framework, and the SUMO mobility traffic simulator that aims to study, evaluate, and also design new techniques to improve video delivery over Vehicular Ad-hoc NETworks. Using the proposed simulation framework we will study different coding options, available at the HEVC video encoder, that will help to improve the perceived video quality in this kind of networks. The experimental results show that packet losses significantly reduce video quality when low interference levels are found in an urban scenario. By using different INTRA refresh options combined with appropriate tile coding, we will improve the resilience of HEVC video delivery services in VANET urban scenarios. Among the potential applications that may be supported by Vehicular Ad-hoc NETworks (VANETs), video delivery is one of the most resource-demanding. Several application scenarios may require video delivery services in either on-demand or real-time live video streaming, using unicast, multicast or broadcast communications. We may find scenarios where video delivery is required, like the ones related to accidents/rescue assistance, V2X real-time video, context-aware video broadcasts, security surveillance services, driving assistance, etc. However, multimedia streaming over VANETs is a very challenging issue mainly due to the high mobility of vehicles, bandwidth limitations, and the high loss rates typically found in wireless communications. In addition, video transmission requires a high bandwidth with a bounded packet delay, especially when real-time restrictions are mandatory. So, when video suffers from packet losses and/or highly variable packet delays, the user perceived video quality is seriously reduced.In this work, we analyze the performance of video delivery in a typical VANET urban scenario by means of a simulation framework named Video Delivery Simulation Framework over Vehicular Networks (VDSF-VN) , which aInside VDSF-VN, the selected source video sequence is encoded with the High Efficiency Video Coding (HEVC) standard . In partAlthough the simulation framework provides a lot of useful performance metrics , our main performance metric is the video quality delivered to the user, since it will be used to determine the minimum quality levels that a video delivery application should provide. After analyzing several simulation runs we notice that the received video quality becomes unacceptable when the first interferences appear in the network. So, we proceed to study some coding options implemented in the HEVC video encoder, in order to improve the final video quality delivered to the user.These options are based on INTRA refresh and tile-based video coding techniques. On the one hand, INTRA refresh technique will help to stop the error propagation produced by a single packet loss in the prediction process of future (next) video frames. So, we have analyzed the performance of several INTRA refresh approaches in order to determine the ones which deliver a higher video quality to the user with an acceptable bit rate overhead. On the other hand, TILE-based coding allows partitioning each video frame in blocks (Tiles), which are completely independent regarding coding/decoding. This may be useful to mitigate the impact of packet losses when compared with no-tiling approaches. When no-tiling approaches (the default coding option) are used, an encoded frame is typically fragmented into several network packets. To decode the frame, all those packets need to be received. If just one packet is lost, the frame is undecodable although the rest of packets had been correctly received. If tiling is used, a frame can be partially decoded (not completely lost) improving the final video quality at the same packet error rate.A thorough experimental process was developed in order to identify the above mentioned HEVC coding options which offer the best video quality to the user. The simulation results obtained from the experimental tests will show the behavior of the HEVC coding options under different network impairment degrees. This knowledge will provide us with the ability to decide which level of INTRA refreshing is required and how many tiles per frame should be used to guarantee the minimum video quality required by the video delivery application.The rest of the paper is structured as follows. Many different techniques have been proposed in the literature to improve video streaming over not reliable medium like the wireless channel, in general, and VANETs, in particular .Error Concealment (EC) methods try to minimize the impact of packets loss in the perceived quality of the received video sequence ,9,10. IfError Resilience (ER) is a group of techniques which aim to provide robustness to video sequences, which are applied at the sender side. One of these is the Feedback Channel (FC), in which the receiver can request the retransmission of some missing part . Other mAnother group of ER techniques are the so-called ’intra refresh’ methods. They encode the bit stream in such a way that it prevents fast quality degradation in the presence of data losses. An inter-coded frame, a frame which it is predicted using other frames as references, although correctly received, can be affected by a reference frame with errors, and it can propagate the error to other inter-frames which use it as reference. A proper insertion of intra-coded frames together with an adequate selection of reference frames may increase the error resilience of an encoded bit stream. So, defining appropriate INTRA refresh encoding modes will make bit streams more robust. There are several works in the literature that exploit these techniques to improve video robustness like the ones found in ,21,22.To evaluate the performance of source coding techniques we need a simulation framework to analyze their effects in the final video quality experienced by a VANET user. To measure video quality we will use the Peak Signal-to-Noise Ratio (PSNR) metric. So, the error protection of our encoder will be based on those encoding options which provide a better received video quality, taking into account the bit rate overhead which they typically provide.There are several simulation frameworks proposed in the literature that follow our requirements, like EvalVid , which aAnother example is HEVStream , which iDespite of the different existing video frameworks , none of them allows the transmission and quality evaluation of video sequences with the combination of the OMNeT++ simulator, the Veins framework and the SUMO traffic mobility model for vehicular networks. Some analyzed frameworks lack of an updated video codec module, being not trivial to change it with another one, because the packetizer needs to be properly adapted to the intrinsic features of the target bit stream syntax. Also, node mobility models are too simple in most approaches to fit with the particularities of vehicular networks . In addition, finally, the different modules of the framework need to be completely integrated in order to launch global simulations specifying the detailed configuration of every module, and performing, on the fly, all the processes, from the video encoding at source node to the decoding process at the receiver end, passing through the network simulation of the video delivery in a realistic vehicular network scenario. These aspects have motivated us to develop the VDSF-VN  simulatiIn The VDSF-VN framework coordinates and manages all the necessary tasks involved with this kind of experiments, such as, source video encoding, trace files generation, video decoding tasks, running the simulation sequences and obtaining the desired plots from the simulation statistic results. VDSF-VN was also designed to be multithread-aware, so several tasks or simulation runs may be launched in parallel depending on the available hardware resources.Pre-process: This step is in charge of encoding the desired video sequences and then perform the Real-time Transmission Protocol (RTP) packetization of the encoded bit stream and generate the corresponding video trace file. This step is a time-consuming task, because the video encoding requires a great amount of computation and the video sequence may be encoded with different configuration parameter sets.Simulation: This step includes the actions to prepare the network scenario and run the simulations with OMNeT++, Veins and SUMO. The trace file from the pre-process step is loaded by the video servers to simulate the video broadcasting. During the simulation, the client nodes write the correctly received video packets into an output trace file, which will be used in the post-process step. This is done in an OMNeT++ project named “ppp_qos”, which references the original Veins project, but it is implemented in a separate project for convenience. This project includes the “TraCIDemo11p” application module provided in Veins, which has been extended with: (a) the support of HEVC video trace files, (b) statistics at application level , and (c) statistics related to the mobility of the vehicles . The “ppp_qos” project also extends the MAC layer with many statistics like the Channel Busy Ratio (CBR), that may be local statistics , global network statistics or statistics grouped by Access Category (AC).Post-process: This step is taken to get all the statistics defined at different network levels: Physical, MAC, and Application, with the corresponding plots. To compute the application performance metrics like Frame Loss Rate (FLR) and PSNR, the bit stream needs to be conformed with the received packets and then decoded to obtain the reconstructed video which will be rendered to the user. Many works show that PSNR, under some circumstances, does not properly assess the quality perceived by users. In the literature, we can find quality assessment metrics that better correlates with human perceived quality like MS-SSIM . These vAs depicted in This section describes the methodology used in the simulation and the experiments conducted with the proposed VDSF-VN framework for evaluating the video streaming in an urban VANET scenario. Now, we will describe the simulation setup which includes the network scenario, the network simulation parameters, the video source, and the video encoding options.2 , which act as video servers delivering the same video sequence in a synchronized and cyclic way. The parameters of the network cards are set with the values shown in SimpleObstacleShadowing instead of the SimplePathLoss model.Three fixed Road Side Units (RSUs) are placed along a central avenue (node[0]), which receives the video sequence sent by the video servers. Then, ten vehicles acting as background traffic nodes follow the first one (node[1..10]), and send packets at different bit rates as background traffic load. To represent different levels of network loads, each background traffic node injects packets with a size of 512 bytes in a sequence of the following rates: {0,12,25,50,75} packets per second (pps). Therefore, on average, the total aggregate background traffic is {0,120,250,500,750} pps, respectively.On the other hand, several mobile vehicles are defined, which travel together along the simulation time. The first one is the video client , although their velocity is not constant, as the traffic simulator takes into account intersections, traffic-lights, the presence of other vehicles, etc. SUMO is a microscopic vehicular simulator that slows down or accelerates each vehicle according to the traffic conditions. The mobility model used is the default one in SUMO, that is, a modified version of the Krauss model  and rsu[0] (Zone B), as well as the overlapping area between rsu[0] and rsu[2] (Zone C).The simulation time is 340 s, which is time enough for all the vehicles to travel from the beginning to the end of the avenue, receiving the video sequences sent by the three RSUs. The selected video sequence is ’BasketballDrill’, which belongs to the HEVC Common Test Conditions set . As showThis video sequence was encoded with the HEVC reference software HM (HEVC Test Model) . The resAnother coding parameter to define is the Quantization Parameter (QP), which is used to adjust the compression level. A low QP value implies a soft quantization that will result in larger bit streams with higher video quality. In our experiments, the video quality, measured in terms of PSNR, for all the generated bit streams was fixed to the same value by adjusting the compression level  in each case, targeting to a desired PSNR value of approximately 36 dB.Apart from the compression mode and QP value, the video encoder accepts many other parameters. For example, HEVC allows splitting a frame into several independent regions (tiles or slices). This will be useful for our purposes since it will provide an extra robustness to video delivery when packet losses start to damage the video packet streaming. However, these partitioning modes lower the encoding performance since spatial correlation is limited to the slice/tile domain, and their use requires extra bit stream signaling information .Encoding modes (×9): {AI, IPIP, I3P, I7P, I15P, IPx, IPx25pctCTU, LPI4, LP} see .QP values (×1): the QP value used for each encoding mode was fixed to achieve the desired video quality (PSNR ≈ 36 dB) when using 1 tile per frame see .Number of tiles per frame (×7): seven values were considered to encode each bit stream: {1, 2, 4, 6, 8, 10, 16}. The size of these partitions can be specified with the number of rows and columns (uniform), or with the number of Coding Tree Units (CTUs) per each row and column (non-uniform). In this work, the following uniform tile patterns were considered: {1×1, 1×2, 2×2, 2×3, 2×4, 2×5, 4×4}, respectively.As a result, many combinations of parameters need to be considered. In this work, the selected encoding modes, QP values and number of tiles per frame are the following:So, all of these combinations make a total of 63 different bit streams (9×1×7). As it can be appreciated in Once video encoding is done, we proceed to build a trace file from the encoded bit stream . A traceWe have modified the HM software to include RTP bit stream packetization . In addiAt last, once the video sequence is reconstructed we compute, among other application metrics, the PSNR value relating to the original video sequence, which is the most commonly used metric for measuring the video quality.The purpose of this paper is to find out how different encoding modes affect the video delivery in vehicular networks, that is, to discover which of the generated bit streams achieves the best trade-off between reconstructed video quality and resulting bit rate. So, we start with an evaluation of the different INTRA refresh modes without using tile partitioning . Then we analyze the effect of increasing the number of tiles per frame with the All-Intra (AI) coding mode. In addition, finally we will see in action both INTRA refresh modes and the use of tiles to evaluate their overall behavior on error resilience, video quality and coding bit rate.Goodput (GP): the total number of received video packets at the client application layer divided by the application runtime .End-to-End (E2E) Delay: the average time each packet requires in order to be delivered to the destination application (running at a particular node).Jitter: the average E2E delay variation between two consecutive packets received at the destination node.Packet Delivery Ratio (PDR): the relation between the total number of video packets received by the client with respect to the video packets sent by the server. As all the video servers (RSUs) transmit the same video sequence in a synchronized and cyclic way, in this work, only the number of packets sent by the associated RSUs is considered.Packet Loss Ratio (PLR): the percentage of lost packets out of the total number of packets of the video sequence.Tile Loss Ratio (TLR): the percentage of tiles that have not been completely received out of the total number of tiles of the delivered video sequence. In case of using a 1 tile per frame layout , this value corresponds to the Frame Loss Ratio (FLR).n by averaging the squared intensity differences between each pixel Peak Signal-to-Noise Ratio (PSNR): the measure of the objective video quality. The PSNR is computed frame-by-frame for the luminance (Y) component as shown in Equation Although there are much more metrics available, we have selected the following application layer metrics:The metrics are collected in both scalar  and vector format. Scalar metrics allow to study the overall performance of the network, whereas the vector data allows studying the evolution of such metrics over the entire simulation. To drastically reduce the huge amount of data needed to store the vector data, some of these metrics are also collected in vector format but averaged every second only; in this way, it is possible to analyze the behavior of the network in any zone defined by two given simulation times.rsu[0]).Zone A (t = 180..190s): an area where the client node has full coverage of one of the RSUs (rsu[1] and rsu[0]), where none of them are “visible” for the client node.Zone B (t = 100..110s): a shadow area between two RSUs (rsu[0] and rsu[2]).Zone C (t = 240..250s): an area where the coverage of two RSUs are overlapped . Therefore, only results for Zone A are shown below.In this work we have analyzed the selected simulation metrics in 3 different fragments of the whole simulation of 10 s in length each:In this section the effect of different INTRA refresh modes is analyzed. Since we want to determine the potential of INTRA Refresh alone, only one tile per frame is used, that is, the experiments do not use tile partitioning.As can be seen in The End-to-End (E2E) Delay shows similar behavior for all the encoding modes while increasing the background traffic c. The AIRegarding Jitter, In These results demonstrate the benefits of using INTRA refresh encoding modes for improving the reconstructed video quality. However, the price we have to pay for is the increased bit rates, which can cause a greater loss of packets when the network gets saturated.After analyzing the INTRA refresh coding modes with their pros and cons, we proceed to study another error resilience technique that may efficiently work together with INTRA refresh. We propose the use of frame partitioning that allow fragmenting each frame into independently encoded/decoded tiles. The idea is to significantly reduce the number of frame losses. In this section we will study the effect of using 1, 2, 4, 6, 8, 10 and 16 tiles per frame with the AI coding mode.As it was explained in the introduction section, using tiles introduces an overhead in the bit stream as required signaling in form of headers, so higher bit streams are produced. From In As shown in From Now, we are going to analyze the combined effect of both INTRA refresh coding modes and tiles. So, we will show the PLR, TLR and PSNR metrics for all the encoding modes as a function of the number of tiles for every coding mode for a particular background traffic level. From the previous experiments, only 12, 25, and 50 pps background traffic levels will be explored in As it can be seen in From this study, we may conclude that the coding modes with higher "intra refresh" degree are the ones that better stand up in front of packet losses, but they are the ones that higher network bandwidth will require. On the other hand, when using tiles, the behavior is very similar for all the coding modes, resulting in PSNR improvements. However, the consequence of using a higher number of tiles, as for high intra refreshing coding modes, is also a bit stream size increase. So, a trade-off should be defined between error resilience and bandwidth (network resources), what should be finally determined by the application requirements. We may propose a selection criteria of using a frame partitioning in no more than 6 tiles (no significant improvements are found beyond), and the coding mode that, remaining over the application quality threshold, requires the lowest bit rate.In this work, we have introduced VDSF-VN, a simulation framework based on the OMNeT++ network simulator, the Veins framework and the SUMO mobility traffic simulator, which was designed to study and evaluate the robustness of a video delivery stream in vehicular ad-hoc network scenarios. Using the proposed simulation framework we analyzed different coding options, available at HEVC video encoder, to improve the robustness of the resulting video stream when it is faced with error-prone network scenarios like urban vehicular networks. We have tested different INTRA refresh coding modes and frame partitioning schemes by means of tiling, so we can determine the configurations that better performance and robustness provide at different network loads.The experimental results show that error resilience improves as the INTRA refresh level increases. So, the best coding mode would be AI followed by IPIP, I3P, and LPI4. Notice that these encoding modes require high bit rates with respect the others, so a trade-off between error resilience and final bit rate should be defined, which strongly depends on the application requirements and the available network resources. With respect to frame partitioning, we have observed that the use of tiles has a positive impact when combined with INTRA refresh coding modes. So, acceptable video quality levels are achieved from low to moderate background traffic loads, reinforcing the properties of the INTRA refresh coding modes. Again, a trade-off should be applied since the use of tiles increases the bit stream, so the recommendation is not to use more than 6 tiles per frame since when using more tiles the improvements are, in general, negligible.The main conclusion is that by using different INTRA refresh options combined with appropriate tile coding we will improve the protection of video streaming in VANET urban scenarios, allowing to keep acceptable video quality at high packet loss rates. We can use these error resilience techniques at encoding/decoding sides to protect the video streams. However, we need to explore additional techniques, like channel coding (FEC), the use of QoS at MAC level, and efficient error concealment, which, combined with the ones proposed here, minimize the introduced bit stream overhead and maximize the final error resilience. The final goal is focused to provide robust and efficient video streams which are able to fight against moderate to high packet network error conditions, keeping the video quality over the threshold demanded by the application. As future work, we are planning to use the available MAC QoS features, and later we will extend our error resilience architecture with packet-based FEC proposals, and advanced error concealment strategies.


The relationship between malnutrition and tuberculosis (TB) severity is understudied. We investigated the effect of malnutrition on radiographic findings and mycobacterial burden.2, moderate malnutrition as 16–18.4kg/m2, and “normal”/overweight as ≥18.5 kg/m2.Subjects included newly diagnosed, smear-positive, culture-confirmed, pulmonary TB cases enrolled in the Regional Prospective Observational Research for TB (RePORT) cohort. Multivariate regression models were used to evaluate the relationship at start of treatment between body mass index (BMI) and chest radiograph (CXR) findings of cavitation and percentage of lung affected and mycobacterial growth indicator tube (MGIT) time to positive (TTP). Severe malnutrition was defined as BMI<16 kg/mOf 173 TB cases with chest x-ray data, 131 (76%) were male. The median age was 45 years (range 16–82); 42 (24%) had severe malnutrition and 58 (34%) moderate malnutrition. Median percentage of lung affected was 32% (range 0–95), and 132 (76%) had cavitation. Individuals with severe malnutrition had, on average, 11.1% [95% CI: 4.0–13.3] more lung affected, compared to those with normal BMI, controlling for diabetes and cavitation. In multivariable analyses, cases with severe malnutrition had a 4.6-fold  increased odds of cavitation compared to those with normal BMI, controlling for smoking. Median MGIT TTP was 194.5 hours. Neither severe  nor moderate  malnutrition was associated with MGIT TTP.We found that malnutrition was associated with increased extent of disease and cavitation on CXR. These findings may reflect the immunomodulatory effect of malnutrition on pulmonary pathology. By contStudies of the effect of malnutrition on mycobacterial burden are similarly limited. Analysis of data from the Latvian national MDR-TB database found that malnourished individuals >18 years of age had 2.7 times the odds of having a 3+ grade culture compared to those that had normal or overweight BMI, but multivariable analyses were not done . To our Understanding how malnutrition affects radiographic findings and mycobacterial burden is important, as these factors affect transmissibility to others and predict treatment outcomes and long-term pulmonary damage ,11,12. FA secondary analysis was conducted using data collected for the Indo-US Regional Prospective Observational Research in TB (RePORT) study. Details of the study design have been previously reported ,11. In bm2, malnutrition as BMI 16–18.4, and normal/overweight ≥18.5, as per WHO categorization ) compared to those with moderate malnutrition (26/58 [45%]) or normal/overweight BMI  more lung affected than those with normal BMI in univariate analysis . Male seThe median MGIT TTP was 194 hours (range 39–1008); among severely malnourished, malnourished, and normal BMI individuals, the median MGIT TTP was 205, 185, and 199, respectively. Severe and moderate malnutrition were not associated with MGIT TTP in univariate analysis. Current smokers had a decreased risk of a long TTP MGIT  response and granuloma formation [The finding that malnutrition is associated with more extensive radiographic disease likely reflects an immunomodulatory effect of malnutrition. Malnutrition is the leading cause of acquired immunodeficiency and has been labeled nutritionally acquired immune deficiency syndrome . Containormation . In animormation , as wellormation . Similarormation ,20. It iThe increased percentage of affected lung that is associated with malnutrition may have implications for chronic sequelae of TB and worsened respiratory health. Studies have shown that more extensive disease on CXR is associated with a decrease in forced expired volume (FEV1) up to 16 years after treatment  and thatOur data suggest that diabetes mellitus is associated with decreased percentage of lung affected. Studies have reported atypical radiological findings among diabetics ,27, wherThe strengths of this study include use of clearly defined data from a prospective cohort enabling adjustment for potential confounders, including tobacco use, among others which have not been controlled for in previous studies. The inclusion of HIV-uninfected, new TB cases allows us to remove the potential impact of HIV and retreatment on chest x-ray findings. The major limitation of this study is that the data are cross-sectional; therefore no inferences about causation can be drawn. It is possible that some of the malnutrition is due to TB itself, however, studies are quite clear that malnutrition is a strong driver of TB risk ,6,29. FuEnd TB goals [Our finding that severe malnutrition is associated with increased cavitation and extent of disease in pulmonary TB underscores the fact that malnutrition needs to be addressed in areas of the world where the conditions are co-prevalent. Future studies are needed to determine if severe malnutrition correlates with worse TB treatment outcomes and if nutritional interventions for malnourished TB patients might improve the radiographic findings. As we move toward the TB goals , all potTB goals . InterveS1 TableaRisky alcohol use based on AUDIT-C score. bMGIT TTP = mycobacterial growth indicator tube time to positive.(DOCX)Click here for additional data file.S1 DatasetSheet one provides the data for the large cohort used for comparison in supplemental table . Sheet t(XLSX)Click here for additional data file.


Introduction: Given the changing global nutrition landscape, the double burden of malnutrition is a major public health challenge in many developing countries. The main aim of this study is to estimate the double burden of malnutrition among children in low- and middle-income countries (LMICs). Methods: This study used cross-sectional data from Demographic and Health Surveys (2001–2016). A meta-analysis was conducted to estimate the prevalence of malnutrition indicators in 595,975 children under five years from 65 LMICs. Significant heterogeneity was detected among the various surveys (I2 >50%), hence a random-effect model was used. Sensitivity analysis was also performed, to examine the effects of outliers. Results: The pooled estimate for stunting, wasting, underweight, and overweight/obesity was 29.0%, 7.5%, 15.5%, and 5.3% respectively. Countries with the highest coexistence of undernutrition and overweight/obesity were: South Africa ; overweight/obesity 13.3% ), Sao Tome and Principe ; overweight/obesity 10.5% ), Swaziland ; overweight/obesity 10.8% ), Comoros ; overweight/obesity 9.3% ), and Equatorial Guinea ; overweight/obesity 9.7% ). Conclusions: There is an urgent need to strengthen existing policies on child malnutrition to integrate and scale up opportunities for innovative approaches which address the double burden of malnutrition in children under five years in LMICs. Child malnutrition in all its forms is a major public health challenge in low- and middle-income countries (LMICs). Malnutrition refers to a lack of proper nutrition either from insufficient nutrient and/or energy intake (undernutrition) or excessive nutrients and/or energy intake (overnutrition). Undernutrition and overnutrition have different effects on the child’s development and cognitive performance. Child undernutrition including stunting, wasting, and underweight adversely affects survival, physical and cognitive development, and reproductive and economic productivity of individuals as well as increasing susceptibility to acute and chronic diseases in adulthood ,2.However, childhood undernutrition is closely linked to overweight and obesity later in life. According to the World Health Organisation (WHO), children who have suffered from undernutrition and were born with low birthweight or stunted are at far greater risk of being overweight and obese when faced with energy-dense diets and a sedentary lifestyle later in life . ChildhoThe WHO double-duty actions aim to integrate and harness the synergetic effect of multiple interventions, programs, and policies to simultaneously address the burden of both undernutrition and overnutrition as well as diet-related noncommunicable diseases . The appTherefore, to properly operationalize the WHO double-duty actions and maximize pre-existing policies linked to nutrition in the Sustainable Development Goals (SDGs), baseline data on the double burden of child malnutrition are urgently needed, especially in LMICs. Hence, the aim of this study is to estimate the double burden of malnutrition among children under five years in LMICs to identify high-priority countries for implication of the WHO double-duty actions, thus making proper use of limited resources.This study used data from the most recent Demographic and Health Survey (DHS) of 65 LMICs. The datasets are publicly available from the DHS website . DHS areChild malnutrition was measured by stunting, wasting, underweight, and overweight/obesity in children under five years. The DHS manual  indicateAnalyses were performed using Stata version 14.0 . The syntax “metaprop” was used to generate forest plots for each of the malnutrition indicators. Each forest plot showed the prevalence of an indicator in individual countries, its associated 95% confidence intervals (CI), and corresponding weight. A test of heterogeneity of the DHS data obtained for the different countries showed a high level of inconsistency (I2 > 50%), hence a random-effect model  which inThis study estimated the double burden of child malnutrition in LMICs. Countries with the highest burden of child undernutrition were Timor-Leste, Burundi, Yemen, Nigeria, Madagascar, India, Guatemala, Niger, and Eritrea. Child undernutrition has consequences on morbidity, mortality, human capital development, and economic productivity . ResearcCountries with the highest burden of child overweight/obesity were Albania, Azerbaijan, Sao Tomo and Principe, South Africa, and Armenia. The food environment has been a primary driver of the rise in childhood overweight/obesity worldwide over the past half-century ,27. StudCountries experiencing the highest coexistence of child undernutrition and overweight/obesity were South Africa, Sao Tome and Principe, Swaziland, Comoros, and Equatorial Guinea.Optimal child nutrition is a key determinant in achieving global health targets. The growing double burden of malnutrition in LMICs presents a major global public health concern, which, if not properly addressed, might prevent the SDGs from being met. Existing nutrition interventions mostly target single forms of malnutrition  and thisAs highlighted in the United Nations Decade of Action on Nutrition 2016–2025 , the intThis study had some strength. First, the DHS are nationally representative and population-based surveys with large sample sizes. Second, the variables used by DHS are comparable across all countries . HoweverThe burden of child malnutrition varies within LMICs; therefore, interventions to address the double burden of malnutrition should be appropriate to the country setting. Such interventions should simultaneously target shared drivers of both undernutrition and overweight from shared platforms. Additionally, there is an urgent need to assess existing policies and their alignments with the Sustainable Development Goals 2 (Zero Hunger) and 3 , and to evaluate impact, dissemination strategies, and scaling up opportunities of innovative approaches to prevent the double burden of child malnutrition in LMICs.


The articles in this volume reflect the continued popularity of video in professional development and raise important questions about how to situate video productively in varied contexts. Together, they highlight the complexity of expanding video-based professional development beyond designers and the challenges that designers and facilitators experience as they prepare others to lead teacher learning in these settings. In this commentary, we examine how these papers advance research on facilitation of professional development, with a particular focus on the issues for using video productively with teachers. We conclude by raising several issues for future research. The articles in this special issue reflect the continued popularity of video in teacher preparation and professional development and raise important questions about how to situate video productively in varied contexts. Like others, we see video as a powerful tool that has the potential to support teacher learning. Achieving this potential, however, demands attention to the system in which video is situated and to the details of how video is used  according to evidence of cognitive demand. Thus, rather than simply identify the level of cognitive demand in the videos, the facilitator now aims to have participants identify contrasting features of the videos. As such, facilitators need to learn to select clips that reveal such differences. In the final sessions, the goal shifts to “labeling” key factors involved in maintaining cognitive demand, with clips being selected that elucidate instructional practices for sustaining high quality tasks. What the authors demonstrate is that using video productively is inextricably tied to the purposes of teacher learning and that the selection of clips is an integral step in this process. In addition, because video is used over the course of the program to achieve different aims, which videos are selected and how they are sequenced has consequences for the kinds of discussions that can occur and the extent to which video can help to achieve the goals of the program.Borko et al.  also addThe STeLLA program also provides insights into the range of considerations that are necessary when selecting a segment of video from a full lesson. Initially, facilitators use videocases that have been prepared by the development team. Over time, however, facilitators transition to using videos from participants’ classrooms. To scaffold this work, participants implement STeLLA lesson plans. In addition, the Lesson Analysis Protocol guides video analysis, and we suspect, is also useful in directing the selection of video clips. Similar to Tekkumru-Kisa and Stein’s  work, beFive Practices for Orchestrating Productive Mathematics Discussions framework  or what should be the objects of focus in the videos for discussions to achieve different learning aims .Five Practice for Orchestrating Mathematics Productive Discussions to facilitation implies that leaders will need tools and resources to lead discussions with video. A central tool in the original framework is a chart for monitoring student contributions (see Smith and Stein, Tools will also be needed to help leaders orchestrate discussions with video. For example, adapting the A second issue concerns facilitation of highly specified video-based programs in contrast to facilitation of more emergent and adaptive forms of video-based professional development (van Es et al., because the video is from the participants in the group and necessitates setting particular norms that may be different from viewing others’ videos (see van Es et al., While video serves as the anchor for teacher learning across these contexts, we conjecture that the facilitation demands vary. In particular, we suspect that the facilitation challenges discussed above may need to be adjusted for highly adaptive settings. For instance, in a less structured environment such as TeachingChannel or video clubs, it may be the case that designers and facilitators collaborate from the start to define the goals for using video and to design tasks that will leverage the affordances of video to achieve these goals. Additionally, when videos come from participants’ classrooms, we suspect that facilitation of conversations is different than when the videos come from others’ classrooms and that facilitators have to attend to aspects of facilitation that are relevant A final concern for future inquiry relates to the trajectory of learning for facilitators. These manuscripts reveal the work entailed in planning for and leading video-based discussions. We believe it would also be valuable to investigate the process by which facilitators come to learn to engage in these practices and how they develop knowledge for leading professional development. The question for us is, thus, not only what do facilitators need to know, but how does this learning proceed and how does the design and enactment of these programs foster this learning. Borko et al.  illustra


However, the physiological function of SLC41A1 remains to be elucidated. In this study, cellular Mg2+ transport assays combined with zebrafish slc41a1 knockdown experiments were performed to disclose SLC41A1 function and its physiological relevance. The gene slc41a1 is ubiquitously expressed in zebrafish tissues and is regulated by water and dietary Mg2+ availability. Knockdown of slc41a1 in zebrafish larvae grown in a Mg2+-free medium resulted in a unique phenotype characterized by a decrease in zebrafish Mg content. This decrease shows that SLC41A1 is required to maintain Mg2+ balance and its dysfunction results in renal Mg2+ wasting in zebrafish larvae. Importantly, the Mg content of the larvae is rescued when mouse SLC41A1 is expressed in slc41a1-knockdown zebrafish. Conversely, expression of mammalian SLC41A1-p.Asp262Ala, harboring a mutation in the ion-conducting SLC41A1 pore, did not reverse the renal Mg2+ wasting. 25Mg2+ transport assays in human embryonic kidney 293 (HEK293) cells overexpressing SLC41A1 demonstrated that SLC41A1 mediates cellular Mg2+ extrusion independently of sodium (Na+). In contrast, SLC41A1-p.Asp262Ala expressing HEK293 cells displayed similar Mg2+ extrusion activities than control (mock) cells. In polarized Madin-Darby canine kidney cells, SLC41A1 localized to the basolateral cell membrane. Our results demonstrate that SLC41A1 facilitates renal Mg2+ reabsorption in the zebrafish model. Furthermore, our data suggest that SLC41A1 mediates both Mg2+ uptake and extrusion.Solute carrier family 41 member A1 (SLC41A1) has been suggested to mediate magnesium (MgThe online version of this article (10.1007/s00424-018-2234-9) contains supplementary material, which is available to authorized users. Of the 2400 mg of Mg2+ filtered in the kidney on a daily basis in humans, 95–99% is recovered through tubular Mg2+ reabsorption [2+ handling results in a heterogeneous group of disorders that are characterized by hypomagnesemia (serum Mg2+ < 0.7 mmol/L) [2+ wasting has resulted in the elucidation of the molecular mechanisms that regulate renal Mg2+ reabsorption [Magnesium  and thick ascending limb of Henle’s loop . The remainder is reabsorbed in the distal convoluted tubule (DCT), where Mg2+ reabsorption takes place in a transcellular manner via transient receptor potential melastatin type 6 (TRPM6) Mg2+ channels [TRPM6 cause hypomagnesemia and secondary hypocalcemia (HSH) [2+ reabsorption. However, the basolateral Mg2+ extrusion mechanism remains subject of debate, and no proteins have been characterized facilitating this process [Mgchannels . Mutatiobule PT, % and thiia (HSH) . Its memule PT, 2% and thiia (HSH) , 23, 26. process .2+ extrusion by operating as a Na+-Mg2+ exchanger [2+ transporter and is widely expressed in the human body [2+ was shown to regulate renal Slc41a1 gene expression in mice [2+ transport, SLC41A1 has not been linked with the regulation of the Mg2+ balance in vivo, remaining unclear whether SLC41A1 is relevant for renal Mg2+ reabsorption.Recently, the solute carrier family 41 member A1 (SLC41A1) was suggested to facilitate cellular Mgxchanger , 20. SLCman body , 28. In man body , and tarman body . In addi in mice . Despite2+ transport experiments.In this study, we aimed to elucidate the physiological role of SLC41A1 by knockdown experiments in the zebrafish model. Moreover, the molecular function of SLC41A1 was studied by cellular MgSlc41a1 transcript was amplified from a cDNA clone with GenBank accession number NM_173865  using Phusion polymerase. The primer sequences contained AscI and XbaI and are provided in Table AscI and XbaI restriction sites. The p.Asp262Ala mutation in mouse SLC41A1  was introduced using the QuikChange site-directed mutagenesis kit . For experiments in the zebrafish model, the SLC41A1 constructs were subcloned into pT7Ts vectors using the AscI and XhoI restriction enzymes. All constructs were verified by sequence analysis. Throughout the manuscript, we followed the guidelines from HUGO, MGI, and ZFIN on gene nomenclature for human, mouse, and zebrafish genes, respectively.The full-length mouse slc41a1 in adult zebrafish tissues, three females and three males were dissected following anesthesia (0.1% (v/v) 2-phenoxyethanol ). Brain, ovary, gills, testis, heart, spleen, kidney, gut, operculum, scales, and liver tissues were collected and stored at − 80 °C until analysis. The expression of slc41a1 in the pronephric tissue was determined in 120 hours post-fertilization (hpf) zebrafish which were grown in E3 medium . After anesthesia with tricaine/Tris pH 7.0 solution, the pronephros was isolated from zebrafish larvae as previously reported [Zebrafish from the Tupfel long-fin (TLF) strain were used for experimentation. For the study of the tissue distribution of reported . Samplesslc41a1 during zebrafish development, zebrafish were grown in E3 medium and sampled at 6, 12, 24, 48, 72, 96, and 120 hpf. For sampling, animals were anesthetized with tricaine/Tris pH 7.0 solution. Each sample was constituted of 10 embryos/larvae and was stored at − 80 °C until further analysis.For the study of the expression of slc41a1 gene expression by dietary and water Mg2+ in zebrafish gills and kidney, fish Mg2+ balance was challenged by feeding different Mg2+ diets  Mg) or by exposure to different water Mg2+ concentrations  as previously reported [v/v) 2-phenoxyethanol . After anesthesia, death of animals was induced by spinal transaction and organs were collected, immediately frozen in liquid nitrogen, and stored at − 80 °C until analysis.To study the regulation of slc41a1 gene expression and of the zebrafish orthologs of human magnesiotropic genes expressed in the DCT  in 120 hpf zebrafish, zebrafish embryos were grown in different Mg2+-containing E3 mediums from fertilization until 120 hpf, time at which sampling took place. The different Mg2+-containing E3 mediums had the following electrolyte concentrations (in mmol/L): low Mg2+ water, 5 NaCl, 0.17 KCl, 0.33 CaCl2; control Mg2+ water, 0.33 MgSO4, 5 NaCl, 0.17 KCl, 0.33 CaCl2; high Mg2+ water, 25 MgSO4, 5 NaCl, 0.17 KCl, 0.33 CaCl2. At 120 hpf, zebrafish were anesthetized with tricaine/Tris pH 7.0 solution and samples (10 larvae/sample) were collected and stored at − 80 °C until further analyses.To study the regulation of slc41a1 transcripts were used: a translation blocking MO 5′-TGATCTTTCAACCAGAGTACCATGC-3′ and a splice-site blocking MO 5′-GAAGGACTCAATGGTCTCACCTAAA-3′ . In parallel with these slc41a1-MOs, a standard mismatch MO directed (control-MO) against a human β-globin intron mutation, 5′-CCTCTTACCTCAGTTACAATTTATA , was used in control zebrafish. MOs were diluted in deionized, sterile water supplemented with 0.5% (w/v) phenol red and injected in a volume of 1 nL into the yolk of one- to two-cell stage embryos using a pneumatic PicoPump pv280 . WT embryos (uninjected) were also included in the experiments to control for the effects of the injection procedure per se. After injection, embryos from the same experimental condition were placed in three Petri dishes and cultured at 28.5 °C in Mg2+-free E3 medium  or normal E3 medium containing 0.33 mmol/L MgSO4, which was refreshed daily. Three different types of experiments were carried out: a dose-response experiment , a time-course experiment , and an experiment to study the gene expression of slc41a1, trpm6, slc12a3, slc41a3, cnnm2a, cnnm2b, pro-egf, and fxyd2 . During samplings, animals were anesthetized with tricaine/Tris pH 7.0 solution, and morphologic phenotypes were analyzed under the microscope . Two morphologic phenotypes were distinguished: normal and malformed . After phenotyping, only morphologically normal fish were sampled, each sample constituted of 10 larvae, which were stored at − 80 °C until further analyses.Zebrafish eggs were obtained from natural spawning of wild-type (WT) TLF zebrafish which were bred and raised under standard conditions (28.5 °C and 14 h of light:10 h of dark cycle) in accordance with international and institutional guidelines. Two non-overlapping morpholinos (MOs) targeting slc41a1-MO as described above. At 120 hpf, zebrafish were anesthetized with tricaine/Tris pH 7.0 solution, and morphologically normal zebrafish were sampled. Samples, constituted by 10 larvae/sample, were stored at − 80 °C until further analyses.In vivo cRNA rescue experiments were performed with mouse WT SLC41A1 and mutant SLC41A1-p.Asp262Ala cRNAs. Constructs were subcloned into the pT7Ts expression vector, suitable for rescue experiments in zebrafish , and cRN2+ and Ca2+. The washing procedure was repeated twice. Fish were then dried and digested as described previously [Sample processing started by quickly washing the samples with nanopure water in order to avoid contamination of remaining water Mgeviously . Within-RNA was isolated from zebrafish adult tissues, the pronephros, and total embryos/larvae using TRIzol reagent  according to the manufacturer’s instructions, in which glycogen  was used in order to maximize the RNA recovery. One microgram of RNA (200 ng in the case of the pronephric samples) was subjected to DNase treatment to prevent genomic DNA contamination and subsequently used to perform the reverse transcriptase reaction.elongation factor-1α (elf1α) [−ΔΔCt). Primer sequences are shown in Table Changes in target genes mRNA levels were determined by relative real-time quantitative polymerase chain reaction (RT-qPCR) as previously described  and in a (elf1α) . Relativslc41a1-MO, cDNA from 120 hpf control (injected with 16 ng control-MO) and morphant (injected with 16 ng splice-site blocking slc41a1-MO) larvae was used for PCR analysis. Three different primer pairs flanking exon 5 were used (Table w/v) agarose gel. The efficacy of the splice-site blocking MO targeting exon 5 (145 bp) in the slc41a1 gene was measured by RT-qPCR following the methods described above. The specific primers used are shown in Table slc41a1-MO. Thus, the resulting amplicon quantification reflects the expression levels of the functional, non-spliced slc41a1. In addition, slc41a1 expression of the total slc41a1 transcript, which entails non-spliced and aberrantly spliced variants, was measured by RT-qPCR (primers used are shown in Table To determine the knockdown mechanism induced by the splice-site blocking v/v) fetal calf serum , 2 mmol/L L-glutamine, and 10 μg/mL non-essential amino acids, at 37 °C in a humidity-controlled incubator with 5% (v/v) CO2. The cells were transiently transfected with the respective DNA constructs using Lipofectamine 2000  at 1:2 DNA:Lipofectamine ratio for 48 h unless otherwise stated.HEK293 cells were grown in Dulbecco’s modified Eagle’s medium  containing 10% (v/v) fetal calf serum , 2 mmol/L L-glutamine, ciproxin, and 10 μg/mL non-essential amino acids, at 37 °C in a humidity-controlled incubator with 5% (v/v) CO2. The cells were stably transfected with SLC41A1 using Metafectene pro (Biontex) in 1:3 DNA:Metafectene ratio. After 48 h, transfected cells were placed on selection medium containing 0.8 mg/mL G418 and 0.15 mg/mL hygromycin for 14 days.Madin-Darby kidney (MDCKI) cells were cultured in DMEM with 5% (6 cells/12 wells) and transfected with empty (mock), WT, and mutant SLC41A1 constructs for 48 h and seeded on poly-L-lysine (Sigma)-coated 12-well plates. For extrusion experiments, the cells were transferred to Mg2+-free medium supplemented with 1 mmol/L 25Mg2+ during the last 24 h . Before the start of the experiment, cells were washed three times in basic uptake buffer  and subsequently placed in basic uptake buffer with 0.5 mmol/L Mg2+  for 5, 10, or 15 min. For experiments using NMDG, NaCl was replaced in the buffer by NMDG (pH 7.4 with HCl), and in the Cl-free experiments, NaCl, KCl, and CaCl2 were replaced by Na-gluconate, K-gluconate, and Ca-gluconate (pH 7.4 with NaOH). The buffer was removed and subjected to inductively coupled plasma mass spectrometry (ICP-MS) analysis. Uptake experiments were performed as described previously [25Mg content of the cells was determined at time point 0 and was subtracted from the other time points when calculating net fluxes. In the experiments using pharmacological inhibition, 100 μM quinidine, 100 μM ouabain, and 100 μM 2-APB were added 30 min before the start of the uptake. The coefficient of variation (CV) for intra-assay and inter-assay variability has been calculated to determine the variability of our assay. The intra-assay CV is 4% and the inter-assay CV is 12%.HEK293 cells were seeded in 12-well plates  primary antibody and peroxidase-conjugated sheep anti-mouse secondary antibodies .w/v) paraformaldehyde solution in PBS, permeabilized in 0.3% (v/v) Triton in PBS for 15 min, and quenched for 15 min in 50 mM NH4Cl. After blocking with goat serum (16% (v/v) for 30 min), cells were incubated with primary antibody rat anti-HA  or anti-E-Cadherin  for 60 min. After rinses in PBS, cells were incubated with Alexa Fluor 488-conjugated donkey anti-rat IgG or Alexa Fluor 594-conjugated donkey anti-rabbit IgG secondary antibodies  and DAPI for 45 min. After PBS and ethanol rinses, the cells were mounted on slides using Mowiol. Fluorescence microscopy was performed with a Zeiss inverted microscope, and images were taken with ImageJ software.SLC41A1-expressing MDCKI cells were seeded on 0.4 μm transwell filters (Corning Costar) and cultured for 7 days. Subsequently, the cells were rinsed with PBS and fixed for 30 min with 4% (slc41a1 expression in the pronephros), an unpaired Student’s t test was used. Statistical significance was accepted at P ˂ 0.05.All results are depicted as mean ± standard error of the mean (S.E.M.). Statistical analyses were conducted by one-way ANOVA. Tukey’s post-test was used to identify significantly different groups. When only two experimental groups were present X4PX6D, which form the Mg2+ selective pore [To examine the involvement of SLC41A1 in Mg nephron , 19, 30.ive pore , are preslc41a1 gene is ubiquitously expressed in adult tissues , similarly to the orthologs of mammalian DCT-specific genes such as trpm6, slc41a3, and cnnm2a  slc41a1 transcripts , the morphology of slc41a1 morphants was also comparable to controls  [2+ extrusion in cells expressing SLC41A1-p.Asp262Ala was comparable to mock  . Indeed,ock Fig. b. Cells ing Fig. c. Plasmaing Fig. d. To exaons Fig. e. To exc2+ extrusion was not Na+-dependent in our setup, we examined whether SLC41A1 can facilitate Mg2+ uptake despite high extracellular Na+ concentrations using physiological conditions of 1 mM Mg2+ and 140 mM Na+ in the extracellular buffer. Indeed, SLC41A1 increases 25Mg2+ uptake fivefold compared to mock-transfected cells  Mg2+ wasting since the pronephros is the only exiting route for Mg2+ in developing zebrafish (0–120 hpf). In this time frame, there is no intestinal Mg2+ excretion since zebrafish do not eat and also do not drink as they are hyperosmotic with respect to the surrounding environment. In addition, by growing the zebrafish in a Mg2+-free E3 medium, skin Mg2+ uptake is abolished, so the lower Mg content in slc41a1 morphants compared to controls can only be explained by deficient tubular Mg2+ reabsorption. In agreement with a defect in renal Mg2+ reabsorption, the total Mg content in slc41a1 morphants was decreased after full maturation of the pronephros (96 and 120 hpf) but not at earlier time points [The low Mg content in zebrafish e points .2+ extrusion function. As SLC41A1 is expressed in the DCT [2+ occurs, it can be reasoned that SLC41A1 may contribute to the extrusion of Mg2+ in this segment of the nephron. In line with these findings, mRNA expression of genes involved in DCT Mg2+ reabsorption was upregulated in slc41a1 morphants , fu2+ leak in slc41a1 morphants could not be explained by glomerular dysfunction and/or disturbances in renal fluid flow, because enlarged pericardial cavities (fluid retention) and pronephric cysts were absent in the fish where the total Mg content was measured. These kidney defects have been, however, associated with slc41a1 knockdown in a study by Hurd et al. [slc41a1 morphants.The renal Mgd et al. . Differe2+ wasting phenotype in slc41a1-knockdown zebrafish was rescued by expression of mouse WT SLC41A1 but not by expression of SLC41A1-p.Asp262Ala, harboring a mutation in a key residue for SLC41A1 function [2+ transport function of SLC41A1 is required to rescue the phenotype and that zebrafish Slc41a1 is functional equivalent to mammalian SLC41A1.The renal Mgfunction . This im2+ transporter that facilitates cellular Mg2+ uptake as well as Mg2+ extrusion dependent on the concentration gradient. In contrast to previous studies suggesting that SLC41A1 functions as a Na+-Mg2+ exchanger, our results do not support a Na+-dependent mechanism [2+ in Na+-free (NMDG-containing) buffer which cannot be blocked by Na+-channel blocker quinidine and Na+-K+-ATPase inhibitor ouabain.In line with SLC41A1 function in vivo, we demonstrate that SLC41A1 is a Mgechanism , 20. Our2+ transport remains elusive. SLC41A1 function is not altered by ouabain, in line with an electroneutral transport mode and a Na+-independent mechanism. Our experiments exclude Cl− as co-transported ion, but we were not able to identify the counter/co-ion. Based on the strong capacity of SLC41A1 to mediate both Mg2+ uptake and extrusion, the Mg2+ gradient seems to be the main determinant of Mg2+ transport. Previous studies have suggested the presence of a Mg2+-Mg2+ exchange mechanism in erythrocytes, where Mg2+ can compete with Na+ extracellularly for common binding sites in the erythrocyte Na+-Mg2+ exchanger [2+ exchange and its physiological relevance is questionable, we cannot exclude this possibility based on our data. It is interesting to note that the fivefold increase in Mg2+ uptake by SLC41A1 is high compared to previous experiments measuring CNNM2-dependent Mg2+ uptake (2–3-fold). In contrast to CNNM2-dependent Mg2+ uptake, the effects cannot be blocked by 2-APB, supporting that SLC41A1 mediates Mg2+ uptake and is not dependent on TRPM7.The molecular mechanism of SLC41A1-mediated Mgxchanger , 14. AltSLC41A1 mutation has been previously associated with a nephronophthisis-like phenotype [SLC41A1 mutation that developed renal failure, eventually requiring renal transplantation [2+ transport impairment. However, it must be indicated that kidney failure typically results in Mg2+ conservation [2+ reabsorption defects in these specific SLC41A1 patients. In contrast to the patient’s phenotype, no signs of renal cysts or dilatations were observed in our zebrafish model though our experiments were restricted to developing zebrafish (0–120 hpf). Furthermore, Mg2+ supplementation in the medium where zebrafish morphants were grown did not restore the Mg content to comparable levels as in controls. Similarly, in patients with inherited Mg2+ reabsorption disturbances and without renal cysts, Mg2+ homeostasis is not restored upon Mg2+ supplementation [An exon-skipping henotype . This stantation . These pervation , an effeslc41a1 knockdown with both splice-site blocking and translation blocking MOs, a feature that was rescued by expression of mammalian SLC41A1. These results clearly demonstrate the specificity of the slc41a1 knockdown in zebrafish to yield Mg2+ deficiency and discard these results as off-target effects of the slc41a1 MO methodology used in the present study. A limitation is the use of HEK293 cells for our functional assays. However, Mg2+-transporting DCT cell lines are not available and our approach is more physiological than previous experiments. All measurements were performed in physiological Mg2+ concentrations (0.5–1 mM). Previous experiments were dependent on fluorescent probes and required super-physiological Mg2+ concentrations to load the cells (10 mM) [2+ stores. An additional limitation of our study is that we cannot exclude differences in buffer capacity of the cells induced by overexpression of SLC41A1.A strength of our study is that the reduced Mg content was present when inducing the  (10 mM) , 20. The2+ balance by transporting Mg2+ in the kidney. Further characterization of SLC41A1 in renal cells is required to determine the molecular mechanism of SLC41A1-mediated Mg2+ transport. Based on the SLC41A1 function disclosed, patients with hereditary hypomagnesemia should be screened for SLC41A1 mutations.In conclusion, our data indicate that SLC41A1 regulates the body MgESM 1(PDF 629 kb)


Molecular chaperones play important roles in preventing protein misfolding and its potentially harmful consequences. Deterioration of molecular chaperone systems upon ageing are thought to underlie age-related neurodegenerative diseases, and augmenting their activities could have therapeutic potential. The dementia relevant domain BRICHOS from the Bri2 protein shows qualitatively different chaperone activities depending on quaternary structure, and assembly of monomers into high-molecular weight oligomers reduces the ability to prevent neurotoxicity induced by the Alzheimer-associated amyloid-β peptide 1-42 (Aβ42). Here we design a Bri2 BRICHOS mutant (R221E) that forms stable monomers and selectively blocks a main source of toxic species during Aβ42 aggregation. Wild type Bri2 BRICHOS oligomers are partly disassembled into monomers in the presence of the R221E mutant, which leads to potentiated ability to prevent Aβ42 toxicity to neuronal network activity. These results suggest that the activity of endogenous molecular chaperones may be modulated to enhance anti-Aβ42 neurotoxic effects. Gefei Chen et al. show that a mutated BRICHOS molecular chaperone domain from the dementia associated Bri2 can reduce toxicity of amyloid formation in mouse hippocampus in vitro. Upon mutating Arg221 to glutamate, Bri2 BRICHOS forms stable monomers that block a source of neurotoxicity during Aβ aggregation and promote disassembly of wild-type oligomers. Surveillance by human molecular chaperones may decline during aging, resulting in increased cellular stress and eventually leading to neurodegenerative diseases and other disorders4. Thus, pharmacologically augmenting or stimulating chaperone capacity could have therapeutic potential6, and strategies to increase the specificity and activity of the interactions of molecular chaperones with their target substrates are sought for8.Molecular chaperones are ubiquitously expressed and responsible for the maintenance of protein homeostasis by transiently binding their substrates to assist them in folding and trafficking, and to prevent them from aggregating and exerting cytotoxic effects10. AD is the most common form of dementia and is developing into a global challenge since no disease-modifying treatment exists. The development of AD treatments has been outstandingly unsuccessful, with a failure rate above 99% in clinical trials11. Several observations support that amyloid-β peptide (Aβ) aggregation contributes to the pathogenesis of AD. The Alzheimer plaques are composed of Aβ, mutations in the Aβ precursor protein (AβPP) that increase the production or augment aggregation propensity of Aβ lead to early onset AD and to AD-like phenotypes in animal models. In contrast, a mutation that lowers Aβ production is protective14. Proteolytic processing of AβPP generates Aβ peptides of different lengths, whereof Aβ42 is the most aggregation prone and toxic15. The kinetics of Aβ42 aggregation follows recently defined nucleation-dependent microscopic events16. During primary nucleation Aβ42 monomers associate and form a nucleus, from which a fibril can start to elongate. During secondary nucleation, monomers attach to the surface of a fibril, which catalyses the formation of a new nucleus, leading to exponential fibril growth. This monomer-dependent secondary nucleation autocatalytic pathway is the predominant source of toxic Aβ42 species17. Key challenges in finding a treatment of AD is to specifically reduce Aβ42 neurotoxicity, rather than focusing on overall aggregation and plaque formation19. Many compounds have been suggested to interfere with amyloid formation18, including molecular chaperones23 as well as chaperone-like proteins27. The effects of chaperones on Aβ42 fibrillization are diverse when it comes to effects on different nucleation events; DNAJB6 from the heat shock protein 40 (Hsp40) family for example prevents predominately primary nucleation23, while αB-crystallin and calcium-binding protein nuclebindin-1 interfere with both elongation and secondary nucleation events25.Proteins can misfold and self-assemble into highly ordered fibrillar amyloid structures with toxic effects, a phenomenon that is linked to ~40 human diseases, including type-2 diabetes, the neurodegenerative disorders Parkinson disease, and Alzheimer’s disease (AD)29. The BRICHOS domain has been suggested to prevent amyloid formation of aggregation prone regions (clients) of the respective proprotein during biosynthesis32. Recombinant human (rh) BRICHOS domains from proSP-C and Bri2 are efficient inhibitors also of amyloid formation of non-client proteins, such as medin, islet amyloid polypeptide (IAPP), Aβ40, and Aβ4235. Bri2, mutations of which give rise to amyloid formation and familial British and Danish dementias, is produced in the central nervous system (CNS), with expression in neurons of the hippocampus and cortex in humans37, and colocalizes with senile plaques in AD patients39. Under physiological conditions, the BRICHOS domain is released by proteolysis from the Bri2 precursor protein40. Rh Bri2 BRICHOS is efficient in inhibiting Aβ42 fibril formation in vitro and in alleviating the related neurotoxicity to hippocampal slice preparations and in Drosophila models34. Rh proSP-C BRICHOS specifically impedes the secondary nucleation step in Aβ42 fibril formation19. Rh Bri2 BRICHOS modulates both elongation and secondary nucleation events, but different assembly states of Bri2 BRICHOS affect Aβ fibril formation in different ways27. Bri2 BRICHOS monomers are most potent in preventing Aβ42-induced disruption of neuronal network activity, while dimers most efficiently suppress Aβ42 overall fibril formation and oligomers inhibit non-fibrillar protein aggregation26. The Bri2 BRICHOS monomers are not long-term stable and form high-molecular weight oligomers in a concentration-dependent manner in phosphate buffer or in mouse serum in vitro, which is accompanied by reduced potency against Aβ42 fibril formation26. Conversion of Bri2 BRICHOS monomers to high-molecular weight oligomers may be relevant for AD, as increased amounts of different Bri2 forms were found in AD brain compared with healthy controls38. These observations imply that modulating the distribution of Bri2 BRICHOS assembly states so that the amount of monomers is increased is a concept to combat Aβ42 neurotoxicity.The BRICHOS domain has been found in several human precursor proteins, initially in Bri2, chondromodulin-1, and prosurfactant protein C (proSP-C)Here, we design a single point mutant of rh Bri2 BRICHOS that stabilizes the monomeric state. This mutant monomer is potent in preventing Aβ42 neurotoxicity, specifically suppresses secondary nucleation during fibril formation and, importantly, it potentiates wild-type protein against Aβ42 neurotoxicity.30, the only available high-resolution structure of a BRICHOS domain, shows a homotrimer in which residues from helix 2 point into a pocket of the neighbouring subunit γ, where m(0) is the initial Aβ42 monomer concentration, is similar in the absence and presence of both rh Bri2 BRICHOS R221E species  and secondary (monomer-dependent k2 and monomer-independent k−) nucleation as well as elongation (k+)51. To identify microscopic processes in the fibrillization pathways of Aβ42 that are most affected by different rh Bri2 BRICHOS R221E species, we determined the combined rate constants 53. We fitted the kinetic model globally at different Aβ42 concentrations and a constant rh Bri2 BRICHOS R221E concentration  data showing that neither monomers nor dimers of rh Bri2 BRICHOS R221E interact with Aβ40 monomers  and fibril-end elongation (k+).Perturbations of the individual microscopic rate constants 50. Under these conditions, the fibrillization traces typically follow a concave aggregation behaviour . We found that both the monomer and dimer bind abundantly and with similar densities to the Aβ42 fibril surfaces Fig. , which s19. The results shown in Fig. 19, while the dimers do not.Relative inhibition of Aβ42 secondary nucleation and elongation may be linked to the reduction of formation of toxic low-molecular weight species, as the number of new nucleation units is decreased by inhibiting secondary nucleation but increased by inhibiting elongationTo investigate whether the abilities of rh Bri2 BRICHOS R221E to reduce Aβ42-associated neurotoxicity correlates with the generation of nucleation units, we tested the efficacies of rh Bri2 BRICHOS monomers and dimers in preventing Aβ42-induced reduction of γ oscillations in mouse hippocampal slices. We found that addition of 50 nM rh Bri2 BRICHOS R221E monomer to 50 nM Aβ42 prevented Aβ-induced neurotoxicity and the power of γ oscillations reached the levels of non-treated controls Fig. . In contMonomeric Bri2 BRICHOS species most efficiently prevent Aβ42-induced neurotoxicity, and hence their assembly into oligomers result in reduced anti-Aβ42 neurotoxicity. In this study, we designed the R221E Bri2 BRICHOS mutant that forms stable monomers and make rh wild-type Bri2 BRICHOS oligomers release monomers. Rh Bri2 BRICHOS R221E monomers efficiently alleviate Aβ42-induced neurotoxicity by selectively blocking secondary nucleation, which has previously been shown to constitute a main source of toxic species during Aβ42 aggregation. Moreover, the capacity of rh Bri2 BRICHOS R221E monomers to disassemble wild-type oligomers results in improved ability to delay Aβ42 fibrillization and, importantly, reduces Aβ42 toxicity to hippocampal slice preparations , such as direct binding to receptors or membrane destruction55. Interestingly, it has been shown that both intra- and extracellular chaperones, including clusterin, Hsp70, and αB-crystallin, can bind to oligomers of several different amyloidogenic peptides, including Aβ, and thereby induce their assembly into larger species that shield reactive surfaces56. It remains to be studied whether this mechanism applies to the BRICHOS domain as well, but electron microscopy data suggest that rh Bri2 BRICHOS monomers interact with oligomeric Aβ42 species26. In the current study, we found that the Bri2 BRICHOS R221E monomer predominantly attenuates the secondary nucleation rate, while the dimer substantially affects both the elongation and secondary nucleation rate , release of smaller species  and concomitantly increased chaperone activity71. The chaperone activity of alkyl hydroperoxide reductase C from Pseudomonas aeruginosa was enhanced by a point-specific mutation that results in the formation of large oligomers, and the mutant confers heat tolerance to E. coli72. Recently, conjugation of a small-molecule inhibitor of α-synuclein aggregation to Hsp70 was shown to augment the anti-aggregation activity8. In our current study, augmentation of rh Bri2 BRICHOS activity against Aβ42-induced neurotoxicity is shown to occur as a result of formation of monomeric species from larger oligomers  plasmid as polymerase chain reaction (PCR) template Bri2 BRICHOS R221E (Addgene ID: 138134) was obtained with QuikChange II XL Site-Directed Mutagenesis Kit , and the DNA sequence was confirmed . As described previously41, the proteins were expressed in SHuffle T7 competent Escherichia coli cells. Briefly, the cells were incubated at 30 °C in LB medium with 15 µg mL−1 kanamycin until an OD600 nm ~0.9. For overnight expression, the temperature was lowered to 20 °C, and 0.5 mM Isopropyl β-D-1-thiogalactopyranoside was added. The induced cells were harvested by centrifugation  and the cell pellets were resuspended in 20 mM Tris pH 8.0. After sonicated  for 5 min on ice, the lysate was centrifuged  for 30 min and the target protein was purified with a Ni-NTA column. To remove the His6-NT* part, the target proteins were cleaved with thrombin  at 4 °C overnight and loaded over a second Ni-NTA column. Different rh Bri2 BRICHOS R221E species were isolated and analysed by Superdex 200 PG, 200 GL, or 75 PG columns  using an ÄKTA system .For generating rh Bri2 BRICHOS R221E the primers 5′-CACCTGGGTTTCTTTATTTATGAACTGTGTCATGACAAGGAAAC-3′ and 5′- GTTTCCTTGTCATGACACAGTTCATAAATAAAGAAACCCAGGTG-3′ were synthesized. With the wild-type NT*-Bri2 BRICHOS .For specific immunodetection, an AU1 tag (DTYRYI) or a mCherry domain was fused to the C-terminal of rh wild-type Bri2 BRICHOS by PCR amplification. The construct coding for His2 with a flow rate of 8 mL h−1. Data analysis was performed using Waters MassLynx 4.1 software.Prior to ESI-MS analysis, rh Bri2 BRICHOS R221E monomers isolated by SEC were buffer exchanged using BioSpin microcentrifuge columns  into 200 mM ammonium acetate pH 7.5. The final concentration of monomeric subunit was 20 μM. Spectra were recorded on a Waters Synapt G1 mass spectrometer . Rh Bri2 BRICHOS R221E monomers were introduced using in-house produced gold-coated borosilicate capillaries. Instrument settings were: sample cone voltage 30 V, capillary voltage 1.5 V, collision trap voltage 50 V, extraction cone voltage 4 V, and transfer voltage 10 V. The source pressure was 7 mbar, trap gas was NCircular dichroism spectra were recorded in 1 mm path length quartz cuvettes at 25 °C from 260 to 190 nm in an Aviv 410 Spectrometer  with protein concentration of 12 µM. The bandwidth 1 nm, averaging time 0.3 s, wavelength step was 0.5 nm, and time constant 100 ms. The spectra shown are averages of three consecutive scans. One µM, calculated for the monomeric subunit, of different rh Bri2 BRICHOS R221E species in 20 mM Tris pH 8.0 were incubated at 25 °C with 2 µM bis-ANS --5,5′-disulfonic acid dipotassium salt) for 10 min, and the fluorescence emission spectra were recorded from 420 to 600 nm after excitation at 395 nm with an SLM-Aminco AB-2 spectrofluorimeter and thermostated cuvette holder . Citrate synthase from porcine heart  was diluted in 40 mM HEPES/KOH pH 7.5 to 600 nM and then equilibrated at 45 °C with and without different concentrations of various rh Bri2 BRICHOS R221E species. The aggregation kinetics were measured by reading the apparent increase in absorbance at 360 nm under quiescent conditions using a microplate reader .3) at 37 °C. Samples were taken out after 0, 1, 4, and 24 h, and analysed for assembly states by sodium dodecyl sulfate–PAGE under reducing and non-reducing conditions. Ten µM rh Bri2 BRICHOS R221E oligomers and monomers were incubated in ThT assay buffer at 37 °C overnight and analyzed by native PAGE. Twenty µM rh Bri2 BRICHOS R221E dimers were also incubated in 20 mM sodium phosphate, pH 8.0 with 0.2 mM EDTA, and 0.02% NaN3 at 37 °C overnight and analyzed by native PAGE.Rh Bri2 BRICHOS R221E oligomers, dimers, and monomers at 20 µM concentrations (referred to the monomeric subunits) were incubated in ThT assay buffer  cells and purified by ion exchange26. The purified Aβ42 proteins were lyophilized and redissolved in 7 M Gdn-HCl, and the monomers were isolated in 20 mM sodium phosphate pH 8.0 with 0.2 mM EDTA and 0.02% NaN3 by a Superdex 75 column . The monomeric Aβ42 concentration was calculated using an extinction coefficient of 1,424 M−1 cm−1 for (A280–A300). For Aβ42 fibrillization kinetics analysis, 80 µL solution containing 10 µM ThT, 3 µM Aβ42 monomer, and different concentrations of rh Bri2 BRICHOS R221E monomer or dimer  at molar ratios 0, 10, 30, 50, 70, and 100% (relative to Aβ42), were added to each well of half-area 96-well microplates with clear bottom , and incubated at 37 °C under quiescent conditions. The ThT fluorescence was recorded using a 440 nm excitation filter and a 480 nm emission filter using a microplate reader . The aggregation kinetics of Aβ42 at different concentrations in the presence of 0.9 µM rh Bri2 BRICHOS R221E monomers and dimers were measured in the same manner. For Aβ42 seeds preparation, 3 µM Aβ42 monomer was incubated for ~20 h at 37 °C, and the fibrils were then sonicated in a water bath for 3 min. For seeding kinetics analysis of Aβ42 fibrillization, 80 µL solution containing 10 µM ThT, 3 µM Aβ42, different concentrations of rh Bri2 BRICHOS R221E monomers or dimers at 0, 10, 30, 50, 70, and 100%, and 0.6 µM seeds  were added at 4 °C to each well in triplicate of 96-well microplates with clear bottom  and immediately incubated at 37 °C under quiescent conditions. The elongation rate constant k+ (see below) in the presence of rh Bri2 BRICHOS R221E monomers or dimers was calculated from the highly seeded experiments. The initial slope of the concave aggregation traces was determined by a linear fit of the first 25–30 min traces. For all the experiments, aggregation traces were normalized and averaged using 3–4 replicates, and data defining one dataset was recorded from the same plate.Recombinant Met-Aβ(1–42), here referred to as Aβ42, was produced in BL21*(DE3) pLysS Rh Bri2 BRICHOS R221E monomer (5 µM), rh wild-type Bri2 BRICHOS oligomer (5 µM), and the mixtures  were incubated at 37 °C overnight. Then the activities  were tested against Aβ42 fibril formation by ThT assay, and for prevention of Aβ42 neurotoxicity by γ-oscillation measurements in hippocampal slices (see below). The samples were also analysed by native PAGE and western blotting using rabbit anti-AU1 antibody .74:τ1/2 is the aggregation half time, and rmax the maximal growth rate, A the amplitude, and F0 the base value. By applying Eq. , is described by the following integrated rate law19λ and κ:kn, k+, and k2 are the primary nucleation, elongation, and secondary nucleation rate constants, respectively, and the parameters nC and n2 are the reaction orders for primary and secondary nucleation, respectively.The aggregation traces of the total fibril mass concentration, k+k2 and knk+ in the absence and presence of BRICHOS required to describe the time evolution of Aβ42 fibrillization.We identified the microscopic events that are inhibited by rh Bri2 BRICHOS R221E by applying Eq.  by rn(t).To illustrate the generation of nucleation units, according to the nucleation rateg, 4 °C). The fibrils were resuspended in 20 µL 1× Tris-buffered saline (TBS), and 2 µL were applied to formvar-coated nickel grids with ~5 min incubation. For blocking, the grids were incubated for 30 min in TBS containing 1% bovine serum albumin, then washed by TBS for 3 × 10 min. The grids were incubated overnight at 4 °C with primary antibody , and washed again with TBS for 3 × 10 min. Finally the grids were incubated with 10 nm gold particles coupled secondary antibody  at room temperature for 2 h, and washed with 1 × TBS for 5 × 10 min. For staining, 2.5% uranyl acetate (2 µL) was added to each grid with 20 s incubation, and excess solution was removed carefully. The grids were dried at room temperature for ~20 sec, and analysed by TEM (Jeol JEM2100F at 200 kV).The Aβ42 monomer (5 µM) was incubated at 37 °C with 50% rh Bri2 BRICHOS R221E monomers or dimers for ~15 h, and the generated fibrils were collected by centrifugation for 1 h . C57BL/6 mice of either sex  were used in the experiments. Before sacrificed, all the mice were anesthetized deeply using isofluorane.3, 25 mM glucose, 1.25 mM NaH2PO4, 1 mM ascorbic acid, 3 mM NaPyruvate, 2.5 mM KCl, 4 mM MgCl2, 0.5 mM CaCl2, and 75 mM sucrose. Horizontal sections (350 µm thick) of the ventral hippocampi from both hemispheres were sliced with a Leica VT1200S vibratome . The sections were immediately transferred to a submerged incubation chamber containing standard ACSF: 124 mM NaCl, 30 mM NaHCO3, 10 mM glucose, 1.25 mM NaH2PO4, 3.5 mM KCl, 1.5 mM MgCl2, and 1.5 mM CaCl2. The chamber was held at 34 °C for at least 20 min after dissection and it was subsequently cooled to room temperature (~22 °C) for a minimum of 40 min. Proteins  were first added to the incubation solution for 15 min, and then the slices were transferred to the interface-style recording chamber for extracellular recordings. During the incubation, slices were supplied continuously with carbogen gas  bubbled into the ACSF.The brain was dissected out and placed in modified ice-cold ACSF . The ACSF contained 80 mM NaCl, 24 mM NaHCO3, 10 mM glucose, 1.25 mM NaH2PO4, 3.5 mM KCl, 1.5 mM MgCl2, and 1.5 mM CaCl2.Recordings were performed with borosilicate glass microelectrodes in hippocampal area CA3, pulled to a resistance of 3–5 MΩ. Local field potentials (LFP) were recorded at 32 °C in an interface-type chamber (perfusion rate 4.5 mL per minute) using microelectrodes filled with ACSF placed in stratum pyramidale. LFP γ-oscillations were elicited by kainic acid . The oscillations were stabilized for 20 min before any recordings. No Aβ42, rh Bri2 BRICHOS R221E species, or combinations thereof were present in the recording chamber either during γ oscillations stabilization, or during electrophysiological recordings. The interface chamber recording solution contained 124 mM NaCl, 30 mM NaHCOInterface chamber LFP recordings were carried out by a 4-channel amplifier/signal conditioner M102 amplifier . The signals were sampled at 10 kHz, conditioned using a Hum Bug 50 Hz noise eliminator , software low-pass filtered at 1 kHz, digitized, and stored using a Digidata 1322 A and Clampex 9.6 programs .Power spectral density plots (from 60 s long LFP recordings) were calculated using Axograph X  in averaged Fourier segments of 8192 points. Oscillation power was from the integration of the power spectral density from 20 to 80 Hz.t-test (unpaired) was used for statistical analysis. All experiments were performed with parallel controls from the same animal/preparation, and the number of biological replicates are given in the figure legends. Significance levels are *p < 0.05; **p < 0.01; ***p < 0.001. The ThT fluorescence data are presented as means ± standard deviation, and the aggregation traces are averaged from 3–4 replicates.The electrophysiology data are presented as means ± standard errors of the means. The Student’s Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting SummaryPeer Review File


Malnutrition, a very common condition in the elderly, is known to increase their vulnerability to adverse health events. This study aimed to estimate the prevalence of malnutrition in the over 75-yr-old community-dwelling population in the “Chiavarese” Local Health Unit district .The short version of the Mini Nutritional Assessment (MNA-SF) was used by General Practitioners (GP) as a screening tool to investigate the nutritional status of elderly people (1039 subjects). The study was conducted in 2012–2013 in the Local Health Unit of Chiavari (Liguria Region), Italy. The malnutrition was subsequently confirmed by means of biochemical parameters. Subjects at risk of malnutrition or malnourished (n=22) received personalized dietary counseling by the GP.P=0.00613).The MNA-SF recognized 21% of the elderly people being at risk of malnutrition and biochemical tests confirmed a malnutrition prevalence of 3.5%. The dietary counseling improved the MNA-SF score and biochemical parameters, but the difference was statistically significant only for the MNA-SF score (Malnutrition can be evaluated with a simple tool such as the MNA-SF, recognized at the earliest stage and successfully treated by the GP. Malnutrition represents “a state of deficient energy or protein intake or absorption, characterized by weight loss and changes in body composition” . It is aPhysiological, social and economic issues, often referred as the “nine d’s”, namely poor dentition, dysgeusia, dysphagia, diarrhea, depression, disease, dementia, dysfunction, and drugs, are the main causes . The so-The prevalence of PEM in the elderly varies considerably, depending on the population studied , the stuManagement of the severe stage of PEM becomes difficult  and earlThe aim of the study was to estimate the prevalence of malnutrition in the population over 75 yr old living at home and resident in the “Chiavarese” Local Health Unit district . The subjects recognized as malnourished or in a condition of risk-received counseling, consisting in a personalized diet recommended by their General Practitioner (GP). The attempt to improve the management of patients with inadequate diet was based on “patient empowerment” in order to change their lifestyle.The study was carried out between Nov 2012 and Dec 2013. The participants were recruited by the general practitioners (GP) of the “Chiavarese” Local Health Unit district  who agreed to take part in the study. Individuals aged 75 yr and older, not institutionalized, that spontaneously attended outpatient clinics for therapeutic management or contacted according to the proactive medicine approach were enrolled.They were informed about the aim of the study and gave written informed consent.Exclusion criteria were: the absence of the above-mentioned requirements, not receiving the information on the study, the presence of a neo-plastic disease diagnosed less than one year before and/or with clinical signs of activity, severe psychiatric symptoms or which in any case did not allow them to provide informed consent, already activated artificial nutrition , chronic renal failure (creatinine > 3 mg/dl), life expectancy shorter than 6 months.Since there are no absolute widely accepted criteria for frailty determination, we focused on socio-demographic attributes and components concerning physical and social domains. Accordingly, individuals presenting at least one of these conditions were considered frail: having more than two chronic diseases or a single complicated chronic disease, living alone or in a condition of social disadvantage, having the prescription charge exemption for low income.Nutritional status was investigated using the Mini Nutritional Assessment Screening Form (MNA-SF), provided with the software Millewin/MilleGPG used by the GPs. The MNA-SF is the short version of MNA, a tool for nutritional screening specifically designed for elderly people. It consists of six questions that consider recent weight and appetite loss, mobility, acute disease or psychological stress and body mass index (BMI). Each question is scored from zero to two or three, for a maximum score of 14. A score of 12 and above indicates satisfactory nutritional status; a score ranging from 8 to 11 suggests a risk of malnutrition and a score of 7 and below shows a malnutrition status . In subjSubjects with an MNA score ≥ 12, not presenting malnutrition, received information on how to maintain an adequate nutritional status and suggestions for the continuation and improvement of a suitable lifestyle. Tailored dietary counseling correlating the degree of malnutrition and the most appropriate dietary indications according to the characteristics of the subject  was recommended by the GP to the patients with an MNA score under 12 and PEM confirmed by biochemical parameters. In the case of impaired chewing ability, a soft diet was suggested. The selection of patients needing dietary advice was made by the GPs with the collaboration of the Nutrition Service of the Local Health Unit that prepared the diets. Two months after the implementation of the suggested dietary regime, a further assessment of nutritional status with the MNA-SF and an additional evaluation of plasma biochemical parameters were performed.The study received the approval of the Local Health Unit Ethics Committee according to current legislation. The collection of data and biological samples was conducted in compliance with the protection of personal data.t-test. The multivariate regression analysis (adjusted for gender and age as confounding factors) was applied to evaluate, before and after treatment, the variations in biochemical parameters and MNA-SF score corrected for the confounding effect due to CRP. Statistical analysis was performed with R  and the significance set at 0.05.MNA-SF score, albumin and prealbumin concentrations expressed as mean ± standard deviation were compared between genders with the Student Eleven GPs participated in the study, involving 1039 over 75-yr-old community-dwelling persons. Complete data are available for 821 of them, representing a participation rate of 79%. The main demographic characteristics are reported in Almost all the participants (95.7%) could be considered frail individuals, mainly due to the presence of illness conditions and the prescription charge exemption for low income. The nutritional evaluation by means of the MNA-SF showed that 21% of elderly people living in the community were in a situation of risk of malnutrition and the prevalence of malnutrition was 3.4%. Both conditions were more prevalent in the females .The determination of biochemical parameters for confirmation of the malnutrition status made it possible to estimate a prevalence of 3.5% . MalnutrA significant proportion (75%) of patients with malnutrition confirmed by blood parameters received tailored dietary counseling. All measured parameters had improved, as showed by median, first, and third quartiles reported in P=0.00613).The regression analysis corrected for the CRP showed that dietary intervention improved all considered parameters but the difference was statistically significant only for the MNA-SF score . The levels of albumin and prealbumin increased and the latter, in particular, exceeded the threshold of 17 mg/dl, indicating a recent recovery of a good nutritional status.The elderly population is particularly vulnerable to malnutrition. Although this condition is more frequent among institutionalized persons, prevalence rates not entirely insignificant and varying according to the degree of independence have also been reported among older people living in the community. Western countries are characterized by a progressive increase in average life expectancy but this implies a higher prevalence of adverse health events. These are strongly associated with malnutrition and include reduced quality of life, increased complications of diseases, higher risk of institutionalization, greater frailty and increased mortality. More attention should be directed at malnutrition, a condition often unrecognized and underestimated.Compared to the Italian population in general, the inhabitants of the “Chiavarese” Local Health Unit district are characterized by a lower number of young people and of the so-called active population, while the elderly component is predominant. With respect to the Italian population in general, there are more than one and a half times more 64-yr-old subjects in this area and more than double over 84-yr-old (4% vs. 2%). The Ligurian population is, in fact, the oldest of Italy and the residents of the “Chiavarese” Local Health Unit district are the eldest of Liguria. Since the demographic transition that already occurred in this area prefigures the transition that the Italian population and those of all economically advanced countries will undergo in the coming decades, this district and its population can represent a test case for assessing and proposing solutions to the health and social problems of an increasingly elderly community.This study made it possible to estimate the prevalence of malnutrition in the over-75-yr-old population cared for by GPs. 3.5% of the enrolled elderly showed a malnutrition status confirmed by biochemical parameters. Due to the variations depending on the method adopted, it is very difficult to compare this finding with those reported by other studies. However, the acquisition of updated epidemiological data on the malnutrition status of the elderly population is a basic requirement for the definition of programs aiming to control and prevent malnutrition. The MNA-SF proved to be a reliable, simple and easy tool to detect the risk of malnutrition in elderly people living in the community. The adoption of a standardized methodology, available to GPs, can contribute to the timely monitoring of the nutritional status of older patients, and the early identification of deficiency situations may allow effective recovery actions to be implemented.Previously, the Nutrition Service of this district had already carried out nutritional surveillance in hospitalized patients both in the hospital setting  and in pAlthough the study involved a limited number of GPs and over 75-yr-old subjects, it made it possible to explore the problem of malnutrition in the elderly living in the community, based on a methodology previously established through two observational studies conducted in hospitals and in protected structures , 21. In The GP, a valuable reference point for elderly patients, can monitor and recognize the malnutrition status or related risk early, thus successfully implementing personalized intervention, in order to achieve regression of the PEM, as shown by the improvement of the MNA-SF score and biochemical parameters. This finding may have been influenced by the fact that the patients received customized dietary indications and were motivated to change their lifestyle by a trusted person like the GP; in addition, the participants in our study presented only a mild PEM status. Given the positive results obtained with a follow up of only two months, we can hypothesize that the extension of the “empowerment” intervention could encourage the patient to continue to follow the dietary recommendations over time, consolidating the result and ensuring a better quality of life.Malnutrition can be assessed with a simple tool such as the MNA-SF, recognized at the earliest stage and successfully treated by GPs, as shown by the increase by two points of the score recorded in patients receiving a personalized diet. This result has important and promising implications in the management of the older population, as most of them live in the community and could benefit considerably from nutritional counseling. The improvement of the nutritional status of community-dwelling people can, in fact, constitute an effective method of prevention of adverse health events such as hospitalizations, complications, readmissions, institutionalization and mortality.Ethical issues  have been completely observed by the authors.