COVID-19 pandemic has created challenges to the ward pharmacy practice. Challenges arose due to new norms in the ward pharmacy practice. Adaptive measures to overcome these challenges were important to sustain the quality of pharmaceutical care. This study aimed to identify the perceived challenges and attitudes towards adaptive measures in the ward pharmacy practice during the COVID-19 pandemic and determined their association with pharmacists’ characteristics.This cross-sectional study was conducted in 14 Perak state hospitals and 12 primary health clinics through an online survey. All ward pharmacists and trainee pharmacists with at least 1 month of ward pharmacy experience and working in government-funded health facilities were included. The validated survey tool consisted of demographic characteristics, pharmacists’ experience towards challenges (22 items), and their attitude towards adaptive measures (9 items). Each item was measured based on a 5-point Likert scale. One-way ANOVA and logistic regression were employed to determine the association of pharmacists’ characteristics against their experience and attitude.Out of 175 respondents, 144 (81.8%) were female, and 84 (47.7%) were Chinese. Most pharmacists served in the medical ward . Commonly reported perceived challenges were difficulties in counselling medication devices (3.63 ± 1.06), difficulties in clerking medication history from family members (3.63 ± 0.99), contacting family members (3.46 ± 0.90), patient’s digital illiteracy in virtual counselling (3.43 ± 1.11) and completeness of the electronic records (3.36 ± 0.99). For attitude towards adaptive measures, improving internet connection (4.62 ± 0.58), ensuring availability of multilingual counselling videos (4.45 ± 0.64), and provision of internet-enabled mobile devices (4.39 ± 0.76) were the most agreeable by the pharmacists. Male  and master’s degree holders  had greater odds of high perceived challenging experience scores. Master’s degree holders  were also more likely to have a positive attitude score towards adaptive measures.Pharmacists faced multiple challenges in the ward pharmacy practice during the COVID-19 pandemic, especially in medication history assessment and patient counselling. Pharmacists, especially those with higher levels of education and longer tenure, exhibited a higher level of agreement towards the adaptive measures. The positive attitudes of pharmacists towards various adaptive measures, such as improvement of internet infrastructure and digital health literacy among patients and family members, warrant immediate action plans from health authorities. The declaration of the COVID-19 pandemic, a global health emergency, on March 11, 2020, by the World Health Organization (WHO), had a tremendous impact on healthcare services, including pharmacy [In Malaysia, pharmacy activities such as medication dispensing to out-patients and pharmacist-led medication therapy adherence clinics, which involved medication knowledge assessment, adherence evaluation, medication-related problem assessment, and therapeutic recommendations, were impacted by the government's quarantine policy and movement control order, leading to disruptions in appointment scheduling . ExtendeThe routine local ward pharmacy activities usually include medication history taking & reconciliation, case clerking, pharmacotherapy rounds with physicians, medication reviewing, medication counselling, and discharge planning for warded patients . During In the local settings, ward pharmacists implemented telepharmacy measures such as maintaining regular communication through phone calls or text messages with the patient care team. This facilitated streamlined drug information provision and remote screening of medication charts . PharmacCurrent literature focused on the overall challenges and the adaptive strategies in pharmacy services during the COVID-19 pandemic –14. The Understanding the perception and attitudes of pharmacists towards challenges and adaptive measures during the COVID-19 pandemic is crucial for maintaining quality patient care. It helps identify system barriers, provides targeted support, and informs training and resource allocation. Additionally, it contributes to understanding healthcare system resilience and preparedness for future crises.Therefore, this study aimed to explore ward pharmacists' experiences in terms of challenges in the ward pharmacy practice according to new norms during the COVID-19 pandemic, their attitude towards possible adaptive measures to overcome these new challenges, and the associated characteristics.This questionnaire-based, cross-sectional survey included all ward pharmacists and pharmacist trainees working in government-funded health facilities  in Perak, a northwest state of peninsula Malaysia.The total number of ward pharmacists was estimated based on internal administrative data provided by the Perak Pharmaceutical Services Division. With an estimated population of 200 ward pharmacists, a confidence interval of 95%, a margin of error of 5%, and a proportion of 50% of pharmacists with positive experiences and attitudes towards challenges and adaptive measures, 132 participants were needed for this study. The projected sample size of respondents was 147 after considering an estimation of a 90% completion rate . All pharmacists who fulfilled the eligibility criteria were sampled through convenience sampling.We included pharmacists who met the following criteria: contract or permanently employed; had experience in the ward pharmacy practice for at least 1 month from January 1, 2021, onward; both trainees and fully registered pharmacists (FRP); served in the government-funded health facilities in Perak State; and consented to participate in the study. We excluded pharmacists who worked at mental health institutions.The self-administered questionnaire was developed based on several studies following extensive literature reviews , 12, 15.The questionnaire was developed into an online, electronic-based survey using Google Forms. With the assistance of the Perak Pharmaceutical Services Division, the link (URL) to the questionnaire was distributed to all chief pharmacists in the state via email and WhatsApp Messenger. The chief pharmacists then distributed the questionnaire to the potential participants in their facilities. Prior to accessing the questionnaire, the participants were directed to an electronic participant information sheet and an informed consent page. The consent form required respondents to choose their intention of participation. A "thank you" note would appear if they chose not to participate. Should they choose to participate, they clicked on “agree to participate” and answered the online questionnaire.The data were managed with SPSS version 20.0. The respondents’ sociodemographic characteristics were analysed descriptively. For perceived challenges scores, 1 point was given to “never”, and 5 points to “always”. The overall mean perceived challenges score was calculated by summing all the items in the perceived challenges domain and dividing by 22. The overall mean perceived challenges scores were then dichotomized, where a mean score equal to or more than 3.00 was categorized as high, and a mean score of less than 3.00 was categorized as low. The cut-off was decided based on the mean perceived challenging experience scores of 3.00 .For attitude scores, 1 point was given for “strongly disagree” and 5 points for “strongly agree”. The overall mean attitude score was calculated by summing all the items in the attitude domain and dividing by 9. The overall mean attitude scores were then dichotomised, with a mean score of 4.00 or higher categorised as a positive attitude and a mean score of less than 4.00 categorised as a negative attitude. The cut-off was decided based on the mean attitude score of 4.00 .One-way ANOVA analysis was performed to generate the mean scores of the perceived challenging experience and attitude domains for each socio-demographic characteristic. Univariate logistic regression was performed, and variables with a P-value less than 0.25 were included to run the multivariate logistic regression. Multicollinearity and interaction terms were checked and not found. The Hosmer–Lemeshow test, classification table and Nagelkerke R square were applied to check model fitness. Histogram and Kolmogorov–Smirnov test were performed to check normality. A backward stepwise multiple logistic regression model was used, and the final regression model included variables with p < 0.05. A Pearson correlation was used to determine the correlation between perceived challenging experience and attitude scores.A total of 175 pharmacists out of 200 invited pharmacists responded. The majority were female (82.3%), between the ages of 21 and 30 (60.0%), Chinese (48.0%), had a bachelor's degree (89.1%), worked in medical wards (70.8%), and in non-COVID-19 wards (86.4%) , difficulties in clerking medication histories from family members (3.63 ± 0.99), difficulties in contacting family members (3.46 ± 0.90), patient’s digital illiteracy in virtual counselling (3.43 ± 1.11) and completeness of the electronic health records (3.36 ± 0.99) Table .Table 2PRegarding attitudes toward adaptive measures, the overall mean score was 4.00 ± 0.38 out of a maximum of 5, ranging from 2.89 to 4.78. The pharmacists were more agreeable to such adaptive measures: improving internet connection (4.62 ± 0.58), ensuring availability of multilingual counselling videos (4.45 ± 0.64), providing internet-enabled mobile devices (4.39 ± 0.76), educating physicians about formulary restrictions (4.25 ± 0.62), and developing local COVID-19 treatment (4.15 ± 0.65) , Chinese (p = 0.010), possessing a master’s degree p = 0.047) and permanent-based FRP (p = 0.009) were associated with higher perceived challenging experience scores. In contrast, pharmacists working in pharmacy-based information systems were associated with positive attitude scores (p = 0.010)  and those with a master’s degree  had higher odds of perceived challenging experience scores. Pharmacists with a master’s degree , contract-based FRP , permanent-based FRP , those who worked in pharmacy-based information system  and those who worked for more than 1 year were more likely to have a positive attitude towards adaptive measures .To the best of our knowledge, this was the first study that assessed the perception of ward pharmacists towards challenges and adaptive measures implemented in the wards. The respondents reported different levels of challenges in carrying out ward pharmacy duties. The highest-ranked perceived challenge was medical device counselling, consistent with the findings of other studies , 12, 17.Malaysia is a multiracial nation, and language barriers could present in patient communication when providing medication counselling . Ward phThe second highest-rated challenge was obtaining a comprehensive medication list from family members. Communication with family members was required when patients had low health literacy and language barriers to providing medication information. Face-to-face communication between pharmacists and the patients' family members was restricted because of the "no visitor policy" during the pandemic. Furthermore, due to home quarantine, the patient's family member might be unable to deliver the patient's previous drugs to the ward . Thus, cThe Pharmacy Information System (PhIS) was an electronic prescribing and medication supply record system widely used in Malaysian government-funded hospitals . TracingDigital health insufficiency might affect the delivery of pharmaceutical care. Similar to our study, internet connectivity issues and a lack of suitable platforms for performing telepharmacy was reported –29. HighIn the early phase of the pandemic, several medications were suggested to be repurposed for the treatment of COVID-19. However, the evidence supporting the use of these agents was inadequate due to a lack of safety and efficacy data , 7. The Insufficient personal protective equipment, shortage of staff and drug shortages were not perceived as the most common challenges by the respondents, but these were the top three pharmacists’ concerns according to a Canadian study . MeanwhiOur study found that male pharmacists and those with master's degrees had higher odds of perceiving challenging experiences. The "reality" of the job environment was influenced by perception, attitude, and opinion. All these factors might differ according to certain characteristics, such as gender  and the This research revealed that fully registered pharmacists, whether on a contract or permanent basis and pharmacists who had worked for more than a year, were more likely to have a positive attitude toward workplace adaptive measures during the pandemic. The pharmacist’s positive attitude towards providing pharmaceutical care was important in gaining patients' confidence in the profession . GeneralOur study was conducted only in the Perak state; hence the findings might not reflect the situation encountered in other states in this country. Furthermore, recall bias might be present among respondents who were no longer working in the ward when answering the survey. In addition, some ward pharmacists were transferred between facilities during the pandemic, causing difficulty in obtaining responses. Nevertheless, the state pharmaceutical service division's involvement maximised the questionnaire's dissemination.During the COVID-19 pandemic, multiple challenges arise in the ward pharmacists’ daily routine, particularly when assessing patients' prescription histories and providing patient counselling. Pharmacists demonstrated a high level of agreement towards the adaptive measures, particularly those with a higher level of education and longer duration of service. Health authorities should develop an early action plan in light of pharmacists' positive attitude towards adaptive measures, such as upgrading internet infrastructure and preparing the health system for digital health and telepharmacy.


Recently, end-to-end deep models for video compression have made steady advancements. However, this resulted in a lengthy and complex pipeline containing numerous redundant parameters. The video compression approaches based on implicit neural representation (INR) allow videos to be directly represented as a function approximated by a neural network, resulting in a more lightweight model, whereas the singularity of the feature extraction pipeline limits the network’s ability to fit the mapping function for video frames. Hence, we propose a neural representation approach for video compression with an implicit multiscale fusion network (NRVC), utilizing normalized residual networks to improve the effectiveness of INR in fitting the target function. We propose the multiscale representations for video compression (MSRVC) network, which effectively extracts features from the input video sequence to enhance the degree of overfitting in the mapping function. Additionally, we propose the feature extraction channel attention (FECA) block to capture interaction information between different feature extraction channels, further improving the effectiveness of feature extraction. The results show that compared to the NeRV method with similar bits per pixel (BPP), NRVC has a 2.16% increase in the decoded peak signal-to-noise ratio (PSNR). Moreover, NRVC outperforms the conventional HEVC in terms of PSNR. In recent years, due to the powerful feature learning ability of deep networks and the robustness defects of manually designed traditional video compression technology modules ,2,3, morAs a novel video compression approach based on implicit neural representation (INR), NeRV  transforHowever, the NeRV network is limited by its single-scale feature pipeline and reliance solely on the MLP network for feature extraction. These restrictions hamper NeRV from accurately fitting the input, making it inadequate for video frame reconstruction and representation. Meanwhile, those also make it challenging for NeRV to handle more complex video frame data. Therefore, we propose a new model of a neural representation approach for the video compression with implicit multiscale fusion network (NRVC) which effectively utilizes features of different scales in the model for video frame representation. By fitting each video frame index on the video timeline with the network model, NRVC allows us to obtain of a highly effective function for representing input videos. The decoder can output any RGB video frame by a simple feedforward operation.The main difference between the proposed NRVC and NeRV networks lies in the structure after the MLP layer. In NeRV, after completing feature extraction, five NeRV blocks are added in sequence, performing simple upsampling operations to restore the original video resolution and complete the video frame reconstruction. In contrast, after the MLP layer, NRVC achieves not only the goal of restoring the original video frame resolution, but also utilizes the MSRVC network, constructed with the channel attention mechanism and the multi-scale normalization network, to achieve more refined feature extraction. This improvement leads to enhanced accuracy in the video frame reconstruction process.The proposed MSRVC network contains two main blocks: normalized residual for video compression (NoRVC) and feature extraction channel attention (FECA).Firstly, utilizing the normalized network, the proposed NoRVC model incorporates feature inputs from other layers during video frame reconstruction, thereby expanding the information content of the features. Normalization networks have shown outstanding capabilities in extracting multi-level style features in style transfer research , allowinSecondly, we utilize the FECA attention mechanism to enhance the attention of feature channels and capture interaction information among feature extraction channels. Moreover, in order to improve the effectiveness of the final feature information, we weigh the corresponding feature map channels based on their effectiveness ,14.In NRVC, all video frame images are transformed into 80-dimensional normalized position vectors with the position encoding layer, which serve as inputs to the MLP. The hidden layer of the MLP has a size of 512, and the output layer’s feature size depends on the target video resolution, with common sizes being 16 × 9 and 4 × 3, and the number of channels is 48. The entire MSRVC network consists of one FECA block and five NoRVC blocks. After the FECA module adaptively extracts features, the outputs are used as inputs to the first NoRVC layer and as additional features for the subsequent layers, facilitating video frame reconstruction. The change in video frame size after upsampling in each NoRVC layer is determined by the upsampling parameters of each layer; specific values can be found in We propose a novel model of multiscale representations for video compression (MSRVC) for video compression, which contains multiple NoRVC blocks that effectively extends feature information to improve the quality of reconstructed images.We discover that inserting the FECA attention module in the MSRVC network can significantly improve the feature extraction performance of the model. This effectively enhances the network’s performance without significantly increasing its complexity.We demonstrate that NRVC has a 2.16% increase in decoded PSNR compared to the NeRV method at similar bits per pixel (BPP). Meanwhile, NRVC also outperforms the conventional HEVC  in termsThe contributions of this paper can be summarized as follows:As fundamental research in computer vision and image processing, video compression has been studied for decades. Prior to the emergence of deep neural networks, manually crafted image compression approaches such as JPEG  and JPEGSince the limitations of deepening individual modules, an increasing number of researchers have been devoted to constructing end-to-end deep video compression schemes, where all modules are implemented based on deep neural networks. Lu et al.  proposedImplicit neural representation is an innovative approach to parameterizing a variety of input information. Due to the powerful modeling plasticity of its treatment of representation information, INR has the capability of parameterizing the information represented through a neural network into a convergent function that maps a coordinate to corresponding values, for example, the pixel coordinates of an image and the RGB values of a pixel. Typically, INR approximates this function by the MLP combined with high-frequency sinusoidal activations  or GaussThe overall framework of NRVC is shown in t leads to a suboptimal performance of capturing high-frequency variations in color details and geometry details of the original video frames. This observation aligns with the conclusions of studies by [t to the high embedding space can obtain high-frequency data information and enhance the fitting effect of the neural network.Despite the fact that neural networks are universal function approximators, we have observed that training the entire network udies by ,34 that b and l, we can control the frequency of the timestamp input, thereby regulating the learning capacity of the MLP network. Formally, we utilize the following encoding function as the input embedding for NRVC:b and l are the hyperparameters of the network, b is used to set the va of input embedded data, and l is used to set the corresponding dimension of embedded data.In previous studies , FourierAfter extracting video sequence features from multi-frequency input in MLP layers, this feature information is fed to the MSRVC network. MSRVC contains FECA, five NoRVC blocks, and four upsampling blocks. The primary function of MSRVC is to reconstruct output video frames, which have the same size and comparable visual performance as the input frames.Due to the lack of sensitivity to local features in MLP, the extraction of local features is insufficient. Therefore, to further extract local features, we proposed the FECA attention mechanism as the first layer of the MSRVC. FECA avoids feature dimension reduction while capturing inter-channel interaction information, and divides the weights to improve feature extraction performance.T obtained from the upper MLP, FECA performs a fast one-dimensional convolution with a size of n to recompute channel weights. The value of n is adaptively determined based on the mapping of the channel dimension C that transmits the feature, representing the number of neighboring channels considered when capturing local cross-channel interaction information for each channel.Our FECA block is presented in n, which represents the extent of inter-channel interaction, is directly proportional to the channel dimension C. The input data dimension of the positional embedding increases together with the value of the positional embedding dimension parameter l. Consequently, more neighboring channels must be taken into account by each channel for interaction. This relationship implies that the positional embedding dimension parameter l is directly proportional to n. Furthermore, the channel dimension C is typically set as a power of 2, and l is usually set as a multiple of 10. Finally, The final calculation formula for the fast one-dimensional convolution is as follows:a. In our all experiments, we set The size of w for regenerating each channel can be calculated through a fast one-dimensional convolution employing a kernel size of n:x represents the original channel weight parameters.The weights The feature maps extracted by the feature extraction module are resized to the original video resolution through multiple NoRVC blocks. The structure of the proposed NoRVC module is illustrated in In the NoRVC block, N is the number of frames of the training video sequence, In the training of NRVC, we adopt the loss function of NeRV , which cL1  and SSIML1 [SSIM  loss forScikit-video is a Python library for processing video data that provides four sample datasets, all with a resolution of 1280 × 720. Among them, the “Big Buck Bunny” sequence, with a total duration of 5 s, is used for general video processing tasks. The “Bikes” sequence, with a total duration of 10 s, is used for scene detection tasks. The “Carphone” pristine and distorted sequences, each with a total duration of 4 s, are used for full-reference quality algorithm testing.The Ultra Video Group (UVG)  dataset We performed quantitative and qualitative experiments on eight different video sequences collected from scikit-video and UVG datasets to compare our NRVC with other video compression approaches. The video sequence of scikit-video is the “Big Buck Bunny” video sequence with a resolution of 1280 × 720 and a total number of 132 frames, and the UVG dataset has 7 videos with a resolution of 1920 × 1080 and 200 frames per video, for a total of 1400 frames.We use the Adam  optimizete table  and set  MS-SSIM .Once completing the video fitting, then the model compression is used to accomplish the purpose of video compression. For fairness of the experiment, we perform video compression with the same method as NeRV. The model compression process consists of three steps: model pruning, weight quantization, and weight encoding. We achieve model size reduction by global unstructured pruning. In our experiments, model quantization is performed after model training.We built the model using PyTorch and trained it at full precision (FP32). All experiments are based on NVIDIA RTX3090.b is set to 7. NRVC still demonstrated superior video reconstruction capabilities to NeRV.Firstly, we compare our method with NeRV . The traThen, we compare the compression performance of NRVC with other compression approaches on the UVG dataset. The results for different pruning ratios in model pruning are shown in Finally, we conducted ablation investigations on the “Big Buck Bunny” dataset, where PSNR and MS-SSIM are utilized to evaluate the rebuilt video. The ablation comparison experiments of NeRV , NeRV onNRVC can maintain the same resolution of the reconstructed video frames as the input video by adjusting the dimension parameters of the MLP’s final output layer and the upsampling parameters of each NoRVC layer. In this section, we select four video sequences from the UVG dataset and modify the video frame resolutions using the ffmpeg tool. Subsequently, NeRV was trained on all frames of these videos with different resolutions to evaluate the compression capability for videos with varying resolutions of NRVC. As shown in In this paper, we explore a novel neural implicit representation for video compression. Compared to end-to-end deep network video compression, we discover that INR-based compression methods demonstrate faster encoding and decoding speeds. Meanwhile, by expanding the feature information during feature fusion, we can accelerate the network’s fitting speed. At high compression rates, reconstructed video frames show improved image performance. In addition, approaches with superior compression performance than conventional video compression representations will be presented based on this method in the future, taking into account the enormous potential of neural implicit methods.


We examine whether associations between prenatal exposure to hazardous maternal alcohol consumption during the first trimester of pregnancy and sleep problems in young children represent a causal association.The population‐based sample consists of 15,911 mothers with 30,395 offspring from the Norwegian Mother, Father, and Child Cohort Study (MoBa) and the Medical Birth Registry of Norway (MBRN). Women self‐reported pre‐pregnancy alcohol consumption and consumption during the first trimester of pregnancy twice: at gestational weeks 17 and 30. Mothers reported their children's sleep problems, when they were 1.5 and 3 years . We tested models adjusting for (1) measured confounders, (2) unmeasured familial risk factors by sibling design, and (3) maternal hazardous drinking in the 3 months prior to pregnancy as an instrumental variable within the sibling design.β = 1.14, 95%CI 0.04–2.25) and 3  years of age. These associations were reduced to close to zero and non‐significant at 1.5  and 3  years when controlling for both familial and measured environmental risk factors.Children of mothers with hazardous drinking during the first trimester were at increased risk of sleep problems at 1.5 (There is a moderate association between maternal hazardous drinking during pregnancy and offspring sleep problems up to age three. This association is explained by risk factors differing between families and does not reflect a cause‐effect relationship. We examined whether associations between prenatal exposure to hazardous maternal alcohol consumption during the first trimester of pregnancy and sleep problems in young children represent a causal association. Using a population‐based sample of 15,911 mothers with 30,395 offspring, we found that the association between maternal hazardous drinking during pregnancy and offspring sleep problems up to age three does not reflect a cause‐effect relationship. The findings help to advance the understanding of associations between maternal hazardous drinking during pregnancy and child sleep problems and inform future intervention and prevention research. Maternal drinking during pregnancy is associated with child sleep problems, but the collective research findings show conflicting results. It remains challenging to disentangle the true association, information crucial to inform intervention and prevention research.Using a population‐based sample of 15,911 mothers with 30,395 offspring we found that the association between maternal hazardous drinking during pregnancy and offspring sleep problems up to age 3 does not reflect a cause‐effect relationship.The findings help to advance the understanding of associations between maternal hazardous drinking during pregnancy and child sleep problems and inform future intervention and prevention research.Audit‐Cthe alcohol use disorder identification test consumptionCBCLchild behavior checklist for preschool childrenCIConfidence IntervalFASfetal alcohol syndromeFASDfetal alcohol spectrum disordersGRM‐IRTgraded response item response theory modelMoBathe Norwegian mother, father, and child cohort studyAbout 10% of young children have sleep problems , a prospective, ongoing pregnancy cohort study described in detail elsewhere  to assess child sleep problems at ages 1.5 and 3 years (29). CBCL/1.5–5 consists of 99 items that describe child behavior in the preceding 2 months, including a sleep problems subscale. The items used were: “Doesn't want to sleep alone” and “Resists going to bed at night”. Mothers responded to statements about child sleep problems on a 3‐point scale ranging from not true (1), somewhat or sometimes true (2), and very true or often true (3). The standardized factor loadings for the items were 0.77 and 0.62, respectively. The graded response item response theory (IRT) modeling of child sleep problems allows for estimating random measurement error in the outcome. In sibling comparison studies random measurement error is located at the lowest level of analysis, making measurement models particularly beneficial in within family studies.We used sibling comparison and maternal hazardous drinking during the last 3 months prior to pregnancy as an instrumental variable to adjust for variables that were stable across pregnancies. This approach has been described in previous research  to model the sleep problem scale of the CBCL; this is a confirmatory factor analysis for ordinal items using a logit link function. Hence, each response category of each item has a separate threshold, and each item has a single slope . To ensure interpretability, we used the CBCL standard of the T‐score (SD of 10) and fixed the variance at the first time point to this value. We used a latent growth curve to model sleep problems across time. The growth curve had its random intercept set to 1.5 years and a fixed slope (30). To model changes in latent means and variances across time, we set the thresholds for each response category and factor loadings for each item to equal across time.In three steps, we regressed the latent intercept and slopes of the growth curves on the hazardous drinking measures and the covariates. First, we adjusted for the covariates. Second, we adjusted for the group mean‐centered hazardous drinking measures to perform sibling control analyses. Third, we exchanged the exposure to hazardous drinking 3 months before pregnancy and adjusted for the group mean‐centered hazardous drinking 3 months before pregnancy as an instrumental variable by regressing hazardous drinking during pregnancy on hazardous drinking before pregnancy in a structural equation model. We used the default Sandwich estimator of Mplus 7.31 to correct for dependent observations in the sibling data. We used Full Information Maximum Likelihood (FIML), thus all available information was used to estimate the model. Using FIML to handle missing data, all cases where at least one outcome time‐point is included under the Missing At Random assumption. Our choice to model both time points in a single model renders the advantage of (FIML) estimation of potential bias from non‐response at either time point correlated with any observed covariate included in the model or unobserved time‐invariant maternal factor correlated with such non‐response.Table r = 0.47.Hazardous drinking during the first pregnancy (4.7%) was more common than in following pregnancies (2.4%). Among women that did not report hazardous drinking in the second pregnancy, 4.2% did so in the previous pregnancy. Conversely, among women who did not report hazardous drinking in the first pregnancy, 1.8% did so in the following pregnancy. The relative risk for a woman with hazardous drinking during pregnancy to also report hazardous drinking in the following pregnancy was estimated to 7.41 (95%CI 5.89–9.33). This estimate was not altered after adjusting for parity and maternal age . The tetrachoric correlation between hazardous drinking over two sequential pregnancies were estimated to β = 1.14, 95%CI 0.04–2.25) and 3  years. In analyses adjusted for measured confounders, hazardous drinking during the first trimester was still associated with sleep problems at 3 years . We adjusted for maternal risk factors stable across pregnancies by sibling control, which markedly reduced the associations between maternal hazardous drinking and child sleep problems. When pre‐pregnancy drinking was included as an instrumental variable, the effects of hazardous drinking during pregnancy on sleep problems were non‐significant at 1.5  and 3 years .Table To disentangle the effect of prenatal exposure to alcohol on sleep outcomes in young children, we compared sleep outcomes in siblings raised in the same family that was differently exposed to alcohol during pregnancy, and we used hazardous drinking before pregnancy as an instrumental variable. Initially, in the unadjusted model, we found that hazardous drinking during pregnancy was associated with offspring sleep outcomes at 1.5 and 3 years. In the adjusted model, there was an association between maternal hazardous drinking and sleep problems when children were 3 years old. However, with sibling control and instrumental variable, the effects were greatly reduced and insignificant. The results did not show a putative causal effect of hazardous drinking during the first trimester on child sleep problems at ages 1.5 and 3 years.To our knowledge, this is the first study that has used sibling control when examining the effect of prenatal exposure to alcohol on offspring sleep problems. Therefore, our findings cannot be discussed in the context of studies using an equally robust research design. However, comparing our findings with previous studies, utilizing a non‐causal design, findings from our initial adjusted model, are in line with findings from several studies that showed an association between prenatal exposure to alcohol and child sleep problems (Alvik et al., The findings from our more comprehensive models, that utilized sibling control and instrumental variable, were not in line with the abovementioned studies; our final models did not suggest a causal effect between maternal hazardous drinking during the first trimester of pregnancy and child sleep problems at ages 1.5 and 3 years. These findings were more in line with a Finnish cohort study that did not show an association between prenatal alcohol exposure and sleep problems infants (Morales‐Muñoz et al., Maternal alcohol use during pregnancy is associated with several environmental risks, including maternal psychiatric and substance use problems, which are also associated with offspring psychiatric problems (Kendler et al., Key strengths of the study include that it is based on a prospective population cohort study with a large sample size. It utilizes several quasi‐experimental approaches to control for unmeasured confounding. Further, we used a validated measure for assessing maternal drinking (Bradley et al., Our findings suggest no causal effect of maternal hazardous drinking during the first trimester on child sleep problems at ages 1.5 and 3 years. The findings underscore the importance of utilizing methods accounting for familial confounding in studies on associations between parental exposures and child outcomes and that failing to do so may lead to erroneous conclusions. The findings should not be interpreted as implying that maternal alcohol use during pregnancy is not harmful; It is well established that maternal drinking during pregnancy is associated with other harmful outcomes for children, including fetal alcohol syndrome and fetal alcohol spectrum disorders (Lange et al., Dr. Ingunn Olea Lund assisted in study conceptualization and study design, assisted in data analysis aspects, interpreted results, drafted the initial manuscript, and reviewed and revised the manuscript. Dr. Eivind Ystrom conceptualized and designed the study, designed the data analysis strategy, conducted the data analysis, assisted in drafting the initial manuscript, and reviewed and revised the manuscript. Both authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.Eivind Ystrøm is a Joint Editor for JCPP Advances. Ingunn Olea Lund has declared no competing or potential conflicts of interest.All study participants provided written informed consent. MoBa has a license from the Norwegian Data Inspectorate, and this study was approved by the Regional Committee for Medical Research Ethics.


The Directive 2010/63/EU has been in force for more than a decade and has brought about significant improvements and novel requirements for the welfare of animals used for scientific purposes, while safeguarding research integrity. The Directive sets clear provisions for its scope, the authorization procedures, animal welfare bodies, national committees, cost/benefit analysis, severity classification, retrospective assessment, and statistical reporting, among many others. From this perspective, indicative areas in the existing legislative texts and guidance documents, which may influence animal welfare and scientific integrity, have been identified by the authors. Suggested solutions to address these areas by potential future revisions in the legislation are discussed, with the aim to clarify and simplify them for all actors involved.The Directive 2010/63/EU of the European Parliament and of the Council has been in force for more than a decade and has brought about significant improvements and novel requirements for the welfare of animals used for scientific purposes, while safeguarding research integrity. The Directive sets clear provisions for its scope, the authorization procedures, animal welfare bodies, national committees, cost/benefit analysis, severity classification, retrospective assessment, and statistical reporting, among many others. From this perspective, indicative areas in the existing legislative texts and guidance documents, which may influence animal welfare and scientific integrity, have been identified by the authors after years of working in this field. Suggested solutions to address these areas by potential future revisions in the legislation or other actions to benefit animal welfare are discussed, with the aim to clarify and simplify them for all stakeholders involved. The Directive 2010/63/EU is the outcome of a decade of consultation among stakeholders in order to uphold animal welfare, while concomitantly enabling scientists to conduct research under specific regulations [The revision, which led to the Directive 2010/63/EU, was based on four main sectors, including scope, authorization procedures, ethical review process, and cost/benefit analysis. Furthermore, significant issues such as education and training of staff, national committees for the protection of animals used for scientific purposes, alternative approaches, new requirements for social housing, and provision of enrichment to animal enclosures were included. Regulated procedures were defined as those that are at least equivalent to the insertion of a needle in accordance with good veterinary practice.The Directive 2010/63/EU currently provides a high welfare standard for animals used for scientific purposes. Furthermore, the amendment of the Directive with Regulation 2019/1010/EU contributed significantly to the increase in the transparency of information for the public, since the latter imposes horizontal provisions on the alignment of reporting obligations in the field of environmental legislation “and increasing transparency for the benefit of the public, for instance with the use of harmonized templates for the publication on non-technical summaries, related retrospective assessments as well as implementation and statistics” , as wellThe aim of the present perspective is to highlight areas that can be seen as grey zones that may affect animal welfare and scientific integrity in the existing legislative framework and guidance documents, as well as to indicate some gaps and issues identified in practice during recent years in Greece that could be addressed by potential revisions. This perspective is not intended to come across as overly critical, but aims very much at trying to be beneficial and to have a positive impact on the application of the Directive and animal welfare.In the following sections, indicative areas in the legislative or guidance documents, identified as having a potential effect on animal welfare with possibilities for improvement, have been selected based on our experience with the issues that have arisen during the implementation of Directive 2010/63/EU in Greece, without this selection being exhaustive. While we acknowledge that our experience with the Directive derives from working in one Member State, several of these issues may also potentially be applicable to other Member States to a certain degree. The areas of concern are briefly described and discussed, in the sequence that they are presented in the Directive, and future solutions or actions to be taken are suggested.The Education and Training (E&T) Framework document developed by the European Commission, as guidance to assist the Member States on how the requirements of the Directive may be met, and to facilitate its implementation, sets out the knowledge and skills required to ensure competence and facilitate free movement of personnel . It is rMember States may of course set strict educational requirements in their transposition of the Directive to safeguard animal welfare.Additionally, it may be beneficial to animal welfare if there were many available courses to train official inspectors in their duties, who work for the Competent Authorities and conduct audits of establishments, i.e., official controls for the implementation of the Directive 2010/63/EU in breeder, supplier and user establishments. A possible solution could be for the European Commission to organize training courses similar to those of DG SANTE “Better Training for Safer Food” (BTSF), which is a training initiative for Competent Authorities and stakeholders in Member States and non-EU countries, for various topics including animal welfare official controls [This is an important topic regarding the purposes for which procedures on live animals may be carried out, as mentioned in element (f) of Article 5 of the Directive 2010/63/EU  manages In addition to veterinary students, scientists using animals for scientific purposes also need to be trained using healthy live animals for some procedures in order to acquire the necessary skills and become competent in the conduct of these procedures. As a general conclusion, education and training needs, particularly of some scientific professions whose hands-on learning components on live animals are basic for the acquisition of competence and expertise, should be better defined, indicating, where appropriate, a tier-level approach, e.g., from dummy alternatives to dead animals to anesthetized animals to awake animals ,9. Addithttps://courses.etplas.eu/en, accessed on 30 June 2023) [https://felasa.eu/education-training/course-listings, accessed on 30 June 2023) [The process of project evaluation as described in article 38 of the Directive 2010/63/EU requests the participation of experts in the required fields, but also in the process itself, e.g., the evaluation of application forms according to legal provisions, the application of an ethical evaluation, and a harm/benefit analysis of the use of animals for the approval of a project. In most Member States, there are courses that are developed for Functions A, B, C, and D, designed according to their description in article 23 of Directive 2010/63/EU and in the E&T Framework document . The E&Tne 2023) . The proThe composition of the Animal Welfare Bodies (AWBs) in most Member States is defined by the Directive and its transposition to national legislation. In practice, the diversity of AWB’s membership depends on the availability of specialties within each establishment. The input of the designated Veterinarian is required by the Directive, without defining a mandatory participation amongst the AWB’s members. Many Member States have incorporated this designated Veterinarian membership requirement into their national legislation , which wNon-technical summaries of projects with severe procedures, the use of non-human primates, or for other reasons justified by the evaluating Competent Authority  need to be updated by the latter with the results of a retrospective assessment report according to article 43 (2) of the Directive 2010/63/EU as amended by Regulation 2019/1010/EU . To dateNTSs that are produced in EU Member States have been published in the NTS European Commission open webpage  since 1/National Reference Laboratories (NRLs) for the development and validation of alternative methods as suggested in Article 47 (2) of the Directive 2010/63/EU [There is guidance developed by the European Commission for all those involved in the oversight of and in the care and use of animals used in scientific procedures, to meet the requirements of the Directive with regard to the composition and duties of establishment Animal Welfare Bodies and the National Committee, which leave flexibility to the Member States . HoweverThe European Commission has been publishing statistical reports regarding the use of animals for scientific purposes in Member States since 1991. The reports were published every five years and then every three years from Member States . These rNarratives are drafted by the Member States’ Central Competent Authorities and are sent annually to the European Commission, which has published them separately for each Member State since 2018, in Part C of the Commission Staff Working Documents on Summary Report on the statistics on the use of animals for scientific purposes . The narFor instance, in many cases, significant increases or decreases in the use of animals are not sufficiently justified as remarkable trends by the Central Competent Authorities of Member States. Exact numbers of the species used, the re-use of some animals, and the use of Genetically Altered (GA) animals are presented repetitively by most Member States, although these are already presented in EU statistical reports on the use of animals for scientific purposes . The treAnother reason for an increase in project authorizations and, relatively, the use of animals, could be large-scale scientific project funding, such as that of Horizon 2020. In certain occasions, this is not often explained by the Competent Authorities of Member States with low numbers of animals used, despite the fact that this declaration is not required by legislation. We suggest that these Competent Authorities could try to offer a general overview of this fact, always taking into account EU data protection and national legislation, plus sensitive data on individual funding of scientists. The aim of this observation is to try to show trends in general funding that significantly increases the number of animals used during some particular years, and not presentation of specific examples that would add unnecessary administrative burden.Annex III  amendments of the Directive 2010/63/EU [The severity classification of procedures is very well described in the Directive (Annex VIII)  and inclThere was no clear provision in the Directive 2010/63/EU for GA animals. The European Commission has tried to address the above issue, plus other important issues regarding GA animals, with updated detailed guidelines, which have indeed proven to be very helpful to users and Competent Authorities . ReportiDuring these years of the implementation of Directive 2010/63/EU, Central Competent Authorities of Member States have addressed questions originating from tentative project leaders or user establishments’ supervisors about scientific ideas that need to be defined in terms of whether they fall into the scope of the Directive. Some specific examples to illustrate these issues are presented below.Projects using farmed fish , both in user establishments and commercial fish farms that have been authorized as such, have a nutritional aim and might not always be considered as projects under the scope of the Directive. It remains unclear if, for instance, changing the diet of fish is considered a procedure under the scope of the Directive 2010/63/EU, and thus needs to undergo a project evaluation and authorization. Several of these nutritional studies may be considered to belong to a grey zone regarding their licensing and reporting. These cases need to be clarified in a potential Revision of the Directive. It is noteworthy that some peer-reviewed journals require a project authorization license to have been issued for these studies, in order to be considered for publication, whereas this requirement is not clear in the Directive, despite the fact that these cases could be considered as procedures according to article 5.b.(ii) [Central Competent Authorities of Member States often receive queries about whether a research idea is considered a clinical trial or a basic research project under the scope of Directive 2010/63/EU. Clinical trials do not fall under the scope of this Directive. It often remains unclear which studies should be considered as projects and therefore need an authorization, and which do not. For example, there is a dilemma if the application of an authorized medicine  to farm animals is considered a procedure under the Directive or a veterinary clinical trial. The latter usually requires authorization by the respective national medicines’ agencies of Member States. However, no relevant clear rules are presented in the current text of the Directive 2010/63/EU [The European legislative bodies and the scientific community, as years pass, have acknowledged the need for additional guidance to be issued on topics that are not clearly addressed in the available legislative text. The aforementioned issues are only examples that have been identified by the authors that present a need of specific legislative guidance and clarifications, at least in some Member States, in order to maintain animal welfare to the highest standards and provide valuable advice to the user community for the conduct of their duties. One of these is the reporting of non-Technical Project Summaries and their related retrospective assessments, which needs to be conducted in a uniform manner throughout the Member States, including substantial information about the overall animal experience within each project.Another issue noted is the reporting of surplus animals that are euthanized without having undergone a procedure according to the Directive, which is an important matter regarding the Reduction principle applied by the management of breeder and user establishments. The experience of Central Competent Authorities of the Member States of the need for project authorizations of various scientific ideas that might not necessarily fall within the scope of the Directive 2010/63/EU but remain vague in the current text, should be effectively used by European legislative bodies, in order to further contribute to the justified use of animals for scientific purposes in the EU.It is to be hoped that all actors involved in the protection of animals used for scientific purposes will contribute with their expertise to promote new legislative and guidance documents that will address these and other issues, which will enhance animal welfare.


This study updates a 2009 study which examined uniform resource locator (URL) decay in health care management journals and seeks to determine whether continued URL availability relates to publication date, resource type, or top-level domain. The authors also provide an analysis of differences in findings between the two study periods.The authors collected the URLs of web-based cited references in articles published in five health care management source journals from 2016 to 2018. The URLs were checked to see if they were still active and then analyzed to determine if continued availability was related to publication date, resource type, or top-level domain. Chi-square analysis was conducted to determine associations between resource type and URL availability, and top-level domain and URL availability. A Pearson's correlation was conducted to determine the relationship between publication date and URL availability.There were statistically significant differences in URL availability across publication date, resource type, and top-level domain. Domains with the highest percentage of unavailable URLs were .com and .net, and the lowest were .edu and .gov. As expected, the older the citation, the more likely it was unavailable. The overall percentage of unavailable URLs decreased from 49.3% to 36.1% between studies.URL decay in health care management journals has decreased in the last 13 years. Still, URL decay does continue to be a problem. Authors, publishers, and librarians should continue to promote the use of digital object identifiers, web archiving, and perhaps study and replicate efforts used by health services policy research journals to increase continued URL availability rates. This study is an update of a study published in 2009 which examined uniform resource locator (URL) decay in health care management journals . The oriThe original study found that URL decay was a serious problem in health care management journals. Half of the web resources in that study could not be located at the cited URL, with older articles being more likely to have inactive URLs. Whether a URL was active varied by top-level domain but not resource type . The domain extensions with the largest percentage of inactive URLs were the .com (53%), .gov 51.6%), and .org (47.5%) extensions. The authors of that study were able to find 59.8% of all inactive URLs using the Wayback Machine of the Internet Archive, 48.8% using Google, and 39% using the websites' search functions [.6%, and American Political Science Review, Journal of the Medical Library Association) [This updated study is important because URL decay continues to be an issue of concern to the scholarly community. Numerous calls to remedy the issue have been made -16, inclciation) -11. A smciation) -16, but Health Affairs, Health Services Research, Health Care Management Review, Journal of Healthcare Management, and Medical Care Research and Review. These journals cover the health care management field comprehensively, as confirmed by surveys of health care management researchers as well as the authoritative Medical Library Association Public Health/Health Administration Core Public Health Journal Project list [The URLs of web-based resources cited in articles published in 5 health care management source journals from 2016 to 2018 were compiled in a Microsoft Access database. The citations were gathered from 5 source journals: ect list . The URLect list ; thus thEach web-based resource was manually searched using the cited URL to determine if the link was active or inactive. The URL was recorded as active if it brought the authors to the resource cited in the journal reference or if the URL automatically redirected, thereby connecting the authors with the resource. To match the protocol of the previous study the resource had to match the cited date to be considered active. If the resource was revised or was an updated edition it was considered inactive. It is important to locate the originally cited article because researchers rely on references to find original sources and additional information . Any URLIf the URL was inactive, further checking was done to see if the cited resource could be retrieved by various other searching strategies. These strategies were chosen to match the methodology of the previous study. The title as well as analysis of the URL were used in attempts to locate the resource. First the title was entered into the search box of the corresponding homepage of the cited resource. Next, the URL was searched by shaving extensions working from right to left with each backslash. Additionally, the title of the resource was searched using Google as the search engine. Finally, the Wayback Machine of the Internet Archive was used to locate the resource. The Wayback Machine crawls the Internet daily to capture and then archive versions of web pages . When a Statistical analyses were performed using Excel. The authors analyzed the differences across journal article publication date, resource type, and top-level domain with URL availability. Additionally, data from the original study was compared to the data collected in this study. The level of significance was set at .05 for all statistical analyses done in this study. Throughout this study, URL decay is synonymous with the percentage of inactive URLs and was calculated by dividing the number of inactive URLs by the number of total URLs. The percentage increase or decrease between two percentages was calculated by dividing the difference between the two percentages by the original number.A Pearson's correlation coefficient was calculated to determine if there was a relationship between the number of URLs and the percentage of URL inactivity. A Pearson's correlation was also used to check if the age of a source journal article's publication date and URL availability were related.A Chi-square analysis relating resource type to URL availability was performed. Each URL was recorded as one of four resource types . These categories correspond to the categories used in the authors' earlier study . JournalA Chi-square analysis was used to examine the relationship between top-level domain and URL availability. The top-level domain for each resource was recorded as .com, .edu, .gov, .net, or .org. If the URL did not use one of those extensions, the researcher used the cited URL to connect to the resource and determined which one of these categories would be the closest match. Most often, the “About Us” information was used. Corporate entities were considered .com, universities and educational institutions were considered .edu, government-sponsored sites were considered .gov, and nonprofit organizations were considered .org.The original 2009 study included citations from articles published from 2002 to 2004 and the URLs were checked twice in 2007, once in March and once in August. Similarly, data from the current study were also from articles published during a three-year period (2016 to 2018) although URLs were checked one year apart in August 2020 and in August 2021. This methodology was chosen to replicate the previous study's final check at 3 years post-publication, and also to observe the change in URL decay within the time span of 1 year. Additionally, URLs could have been temporarily unavailable the first time they were checked, and the authors wanted to account for that possibility.Paired sample t-tests were performed using Excel to compare the percentage of active and inactive URLs by year of publication and by domain extension between the two studies.The data from this study consisted of 10,319 URLs extracted from the cited references of 5 source journals which included 51,758 total citations. The initial check for URL availability found that 27.7%  of the citations could not be located with the cited URL. When the URLs were checked again one year later, 36.1%  of the citations could not be found at the cited URL. Within the time span of 1 year, there was a 30.3% (866) increase in the percentage of inactive URLs. Only 48 URLs (0.47%) were inactive the first time they were checked and then found active on the second check 1 year later. This may have been because the servers were temporarily unavailable, or the web sites were down when the initial check was done. The data from the 2021 final check were used in the tables and analysis to follow.Health Affairs, which had the greatest number of total URLs, had the lowest percent of inactive URLs . Health Care Management Review had the highest percent of URL decay. Health Affairs, Health Services Research, and Medical Care Research and Review  are health services policy research oriented, while Health Care Management Review and Journal of Healthcare Management  are practitioner oriented. The health services policy research journals in this study had lower overall URL decay than practitioner-oriented journals . No significant difference was found between the total number of URLs and the percentage of inactive URLs by journal (r(3)=0.78, p = 0.124). For that reason, the number of total URLs for a specific journal does not account for the difference between the percentage of inactive URLs in health services policy research journals and practitioner-oriented journals.r=0.054, p = .00001). The highest percentage of unavailable URLs were in 2016 and the lowest were in 2018.2=20.13, df=3, p < .0002, n=10,319). The resource type with the highest percentage of unavailable URLs was websites, followed by government resources.2=80.46, df=4, p < .00001, n=10,319). The domains with the highest percentage of unavailable URLs were .net and .com, and the domains with the lowest were .edu and .gov.Of the 4 recovery methods used to retrieve the cited resources with inactive URLs, the Wayback Machine was the most effective method, recovering 2,143 (76.2%) of the 2,812 URLs that were inactive in both checks. Searching the title in Google was the second most effective method, recovering 2,044 (72.7%) URLs. The homepage search recovered 828 (29.4%) of the URLs. The truncated or shaved search was the least effective, recovering 285 (10.1%) URLs. There were still 192 (6.8%) that the authors were unable to locate by any of the recovery methods chosen in this study.t(4)=2.776, p = 0.018). Therefore, this study concludes that URL decay decreased significantly over the 13 years between the two studies. Health Affairs continued to have the lowest percentage of unavailable URLs, and Medical Care Research & Review showed the greatest improvement with a 36.5% decrease in the percentage of inactive URLs.The number of URLs collected and analyzed for this study  was 5.1 times greater than the original data collected in 2009 , even though both studies examined a sample of 3 years of URLs from the same 5 journals. In this same time period the number of articles also increased, though to a lesser degree from 989 to 1,410. t(2)=2.924, p = 0.1). However, the trend in the rate of URL decay shows the percentage of inactive URLs decreasing at a slower rate in the current study than in the first study. Between 2016 to 2018, there was a 15.6% decrease in the percentage of URL inactivity (from 40.3% to 34.0%). Comparing 2002 and 2004, there was a 35.8% decrease in the percentage of URL inactivity (from 61.1% to 39.2%).t(5)=0.921, p = 0.4). However, when looking at the domains individually, .edu is the only domain that remained stable. The domain with the greatest percentage decrease for inactive URLs was .gov (34.3%). There was a 12.6% decrease for the .com domain and a 25.7% decrease for the .org domain. The domain with the greatest percent increase for inactive URLs was .net (31.95%).As expected, the older the URL, the higher the percentage of decay. The total number of URLs in this sample was 5.1 times greater than the original study even though both studies examined a sample of 3 years of the same 5 journals. This large increase in the total number of URLs cited in the scholarly literature has also been reported by numerous other studies, including a recent paper analyzing URL decay in the biomedical literature , and papThere were statistically significant differences in URL availability across resource type and top-level domain in this study. Websites were the resource type with the most URL decay. Domains with the most decay were .com and .net and the domains with the least decay were .edu and .gov.Overall, URL decay decreased from 49.3% to 36.1% between the two study periods. Even though this is a statistically significant improvement from the previous study, this still means that over one-third of all citations to URLs in the latest study became irretrievable over a relatively short period of time.The trend in URL decay in this study appears to be more stable overall than the original study, with only a 15.6.% decrease in the percentage of inactive URLs from 2016 (40.3%) to 2018 (34.0%) compared to a 35.8% decrease during a similar time span in the original study. Furthermore, even though there was a longer time between rechecking the links in this study (6 months vs. 1 year), there was still a 30.3% increase in the percentage of inactive links between checks.Both studies showed significant differences in URL decay between top-level domains, but there was no significant difference between the two studies. URL decay for the .edu domain extension remained stable, standing out as the domain with the lowest percentage of decay across both studies. This is not surprising considering the relative stability of educational institutions when compared to other sources. Interestingly, the .gov domain extension saw the largest decrease in the URL decay, perhaps due to web archiving and permanent URL initiatives by government agencies . The .neTo decrease URL decay of citations to web resources, it has already been suggested that publishers, editors, and authors should work together to require authors to retain a digital backup or printed copies of cited web resources, advocate for the inclusion of cited web resources in online archives, and check URLs before publication for typos . InclusiThe findings of this study are relevant to health care managers who need to make evidence-based decisions regularly. Citations are the backbone of a journal article. If citations are irretrievable, practitioners may be unable to apply research findings to their own specific health care setting. The findings are also applicable to health services researchers who are increasingly following open science principles, which promote best practices around reproducibility, transparency, and research data management .The original 2009 study provided an extensive list of possible resources for combating the problem of URL decay, including digital object identifiers (DOIs), uniform resource names (URNs), persistent uniform resource locators (PURLs), robust hyperlinks, institutional repositories, and personal web archiving tools like WebCite and the Wayback Machine of the Internet Archive . The autOf relevance to reference librarians, the Wayback Machine of the Internet Archive was highly effective in recovering inactive URLs in this study. The Wayback Machine archives previous versions of web pages, so it has an edge over the other methods, including Google, in locating the cited version of a resource. Librarians should promote the use of the Wayback Machine for researchers attempting to track down web citations. It also continues to be the most widely used and longstanding web archiving service, and so the authors recommend it as the most sustainable solution to the problem of URL decay. Another available option that has emerged in recent years for web archiving is perma.cc. It was developed by Harvard Library Innovation Lab and allows authors to archive their cited web resources. This is a free service for up to ten URLs per month for individual authors and unlimited access for academic institutions and courts. Other organizations can purchase subscriptions . Perma.cURL decay in health care management journals remains high at 36.1%, although not as high as the previous study where 49.3% of all URLs were not available 3 to 5 years after the date of citation. This 26.8% overall decrease in URL decay in the 13 years between studies is promising, though it can still be concluded that URL decay in health care management journals is still an issue since it is not negligible. Other studies examining URL decay in various other fields have found a similar pattern of decreasing URL decay over a similar time period, so this study supports that overall trend. It should be noted, however, that these studies did not use the same set of journals as each other, nor did they follow the same methodology of this study. For instance, independent studies looking at URL decay in Library and Information Science journals found 45.4% of URLs to be inaccessible in 2003 , 31% in This study is unique because no other study known to the authors has looked at the change in URL decay rate of a specific set of journals over time. Further research should look at changes in publisher policies mandating web archiving or DOIs, especially in the last two decades, to determine if there is any correlation between changes in publisher policies and changes in URL decay. This study did not investigate whether there was a difference in accessibility due to content drift or failure of the link itself. This would also be an area for further research.This study analyzed the citation data from five health care management journals over a period of three years. The results should not be generalized to cover all journals over an extended period. The authors' choice of using Google as a search engine may have affected the recovery rate as no one single search engine indexes every resource.


However, the mechanism by which Mrs2 permeates Mg2+ remains unclear. Here, we report four cryo-electron microscopy (cryo-EM) reconstructions of Homo sapiens Mrs2 (hMrs2) under various conditions. All of these hMrs2 structures form symmetrical pentamers with very similar pentamer and protomer conformations. A special structural feature of Cl−-bound R-ring, which consists of five Arg332 residues, was found in the hMrs2 structure. Molecular dynamics simulations and mitochondrial Mg2+ uptake assays show that the R-ring may function as a charge repulsion barrier, and Cl− may function as a ferry to jointly gate Mg2+ permeation in hMrs2. In addition, the membrane potential is likely to be the driving force for Mg2+ permeation. Our results provide insights into the channel assembly and Mg2+ permeation of hMrs2.Mitochondrial RNA splicing 2 (Mrs2), a eukaryotic CorA ortholog, enables Mg 2+.Mrs2 is a mitochondrial Mg2+ channel that is essential for metabolic function. Here, the authors present cryo-EM structures of human Mrs2 revealing symmetrical pentameric assembly and how Mrs2 permeates Mg It often functions as a cofactor and activator of ATP molecules to facilitate enzymatic reactions2. Recently, Mg2+ has been shown to be a second messenger that regulates CD8+ T-cell activity in the immune system3 and transduces signals through metabolic circuits4. Therefore, maintaining Mg2+ homeostasis is important to living organisms5. Mg2+ imbalance can lead to serious diseases, including cardiovascular disease, hypertension, obesity, Parkinson’s disease and cancer, etc7.The magnesium ion , a homolog of prokaryotic CorA, anchors to the inner mitochondrial membrane and mediates the influx of Mg2+ into the mitochondrial matrix. It is essential for mitochondrial Mg2+ homeostasis11. In human cells, Mrs2 deficiency leads to marked changes in mitochondrial morphology and severely dysregulates mitochondrial physiology13. On the other hand, cells overexpressing Mrs2 show increased mitochondrial and whole-cell uptake of magnesium ion14.MgThermotoga maritima (TmCorA)18 and one archaea CorA structure in Methanocaldococcus jannaschii (MjCorA)19, have been reported to date. CorA proteins typically assemble as homo-pentamers with large funnel-shaped intracellular domains (ICDs) and transmembrane domains (TMDs). Based on structural models and functional studies, four main models of CorA gating mechanisms have been proposed: an iris-like opening21, stem-helix rotation22, considerable helical torque23 and three distinct motions24. In 2016, the cryo-EM structure of TmCorA showed that the absence of Mg2+ results in the disruption of intracellular subunit symmetry in CorA and expansion of the ion-conducting pore18. Subsequently, molecular dynamics (MD) simulations showed that the asymmetric motion of the monomer enables the rotation of helix α7 and the cytoplasmic subdomain to open the gate25. Recent studies have shown that CorA is in a dynamic equilibrium between different states of symmetry and asymmetry, and that the conducting state increases upon activation26.Several prokaryotic CorA structures, such as those in 2+ transport via the CorA family in eukaryotic systems remains unclear. To better elucidate the mechanism through which Mg2+ moves through the eukaryotic CorA homolog Mrs2, we report on several cryo-EM reconstructions of human Mrs2 (hMrs2) under different conditions. Structural analysis, MD simulations and mitochondrial Mg2+ uptake assays showed that hMrs2 may function as a membrane potential-driven Mg2+ channel enabling permeates Mg2+ facilitated by Cl− ions.However, the mechanism of Mg2+ or at different pH values using the detergent glycol–diosgenin (GDN) and then concentrated for cryo-EM sample preparation. All datasets were collected using a Titan Krios electron microscope operated at an accelerating voltage 300-kV with a Gatan K2 Summit or a Thermo Falcon 4 direct-electron-counting detector. Details about the data collection and data processing are shown in Supplementary Figs. 2+, pH 8.0 (hereinafter referred to as hMrs2-Mg); from the dataset without the addition of external Mg2+ or EDTA, pH 8.0 (named hMrs2-rest); from the dataset in the presence of 1 mM EDTA, pH 8.0 (named hMrs2-lowEDTA); or from the dataset in the presence of 5 mM EDTA, pH 6.8 (named hMrs2-highEDTA). The final 3D reconstructions of hMrs2-Mg, hMrs2-rest, hMrs2-lowEDTA and hMrs2-highEDTA were determined at overall resolutions of 2.6 Å, 2.9 Å, 2.5 Å and 2.7 Å, respectively , the hMrs2 channel pentameric architecture is similar28, albeit that its sequence homology with them is low  flanked on one side by a short helix (H1) and on the other side by one short helix (H2) and two long helices (H3 and H4) Fig. . The twoThe hMrs2 channel exhibits extensive intersubunit interactions, and most of these involve in the extra-long helix H8. From the intermembrane space side down to the matrix side, representative interactions between two neighboring protomers can be classified into seven major interfaces, which are shown in Supplementary Fig. 2+-binding sites and one Cl−-binding site were identified  is located inside the pore mouth on the intermembrane side  is located in the pentameric center of the matrix part of the hMrs2 channel, positioned immediately below the inner leaflet of the membrane  is located in the middle high region of the hMrs2 matrix part within the interface between two adjacent protomers  in one protomer and one carboxylic acid group of residue E312 in another protomer. The fourth Mg2+-binding site  is located in the near bottom region of hMrs2 within the interface between two protomers  in one protomer and one carboxylic acid group of residue E261 in another protomer. The Cl−-binding site  is located in the pentameric center of the transmembrane part of the hMrs2 channel, very close to the inner leaflet of the membrane . The distance between the nitrogen atom and Cl− is approximately 3.2 Å. Of the five ligand-binding sites, three sites  but not the other two binding sites (Mg-3 and Mg-4) are located in the three other aforementioned hMrs2 structures . Furthermore, Mg-1, Mg-2 and Cl-1 are unlikely to be misidentified due to artificial densities caused by the fivefold symmetry operation, as the density of each of the three sites is clearly visible in cryo-EM reconstruction of hMrs2-highEDTA without posing any symmetry , Mg-1, Mg-2, Mg-3 and Mg-4 are in similar positions to the ion-binding sites in the MjCorA structure2+ conduction. The pore size distribution plot calculated with the HOLE software (2.2.005)30 shows that the main contributor to this positively charged repulsion barrier is the R-ring, which forms the narrowest region (radius of approximately 1.1 Å) in the entire pore , lysine residue (R332K) or glutamate residue (R332E) and then measured mitochondrial Mg2+ uptake. The amount of Mg2+ permeating the mitochondria in cells overexpressing any of these three mutants was largely unchanged  phenylhydrazone (FCCP) or valinomycin reduced the amount of Mg2+ taken up 10.To address the molecular mechanism of Mgrix Fig. . Overexp up Fig. . To inveged Fig. , suggest up Fig. . In part2+ membrane permeation, we used steered-MD simulation to employ water coordination during Mg2+ permeation. In bulk solution, the first hydration shell of Mg2+ coordinated 6 water molecules, which was consistent with previous studies32. Throughout the pore, the number of water molecules coordinated by Mg2+ was between 4 and 6, indicating that Mg2+ is partially dehydrated during permeation , was identified in hMrs2. Sequence alignment analysis indicated that the residue Arg332 was not conserved among members of the CorA family  to a pronounced asymmetric structure (open state)33. However, at different pH values and after EDTA treatments at different concentrations, all four hMrs2 structures formed symmetric holo-pentamers. Similar results have been previously reported for EcZntB, which maintained a symmetric pentameric state even after EDTA treatment27. Moreover, the four hMrs2 structures were very similar in terms of the pentamer and protomer structures. These findings suggest that no significant structural rearrangement occurs when the hMrs2 loses Mg2+ binding (hMrs2-Mg versus hMrs2-highEDTA) in the matrix domain. In MD simulations after Cl− was manually removed from the pore, the continuous distribution of water channels was observed in the ion-conduction pore without the need for the conformational change in the main chain of the hMrs2 structure. In addition, when the mitochondrial matrix was maintained at physiological Mg2+ concentrations (~0.8 mM), purified mitochondria took up large amounts of Mg2+ when they overexpressed the hMrs2 protein. Taken together, the matrix Mg2+ concentration appears to be dispensable for Mg2+ permeation via hMrs2, reinforcing the argument that hMrs2 and TmCorA may employ different Mg2+ permeation mechanisms.Previous studies have suggested that Mg2+ uptake10. For TmCorA, the membrane potential facilitates the uptake of magnesium ions, as shown via liposome reconstitution and cellular uptake assays34. Our results showed that FCCP or valinomycin treatment greatly reduced the amount of Mg2+ taken up by mitochondria overexpressing hMrs2, suggesting that the membrane potential is the main driver of Mg2+ permeation via hMrs2. We also observed that mitochondria with endogenous hMrs2 took up Mg2+ after treatment with FCCP or valinomycin. We suspect that another unidentified mitochondrial Mg2+ transporter may function independent of the membrane potential.Previous reports showed that purified yeast mitochondria overexpressing Mrs2p after treatment with valinomycin showed reduced Mg2+ permeation in the hMrs2 channel. First, an increase in cytosolic Mg2+ concentration causes Mg2+ to enter the selection filter, GMN motif. Next, the membrane potential drives Mg2+ flow to the matrix. The R-ring is then used as a charge repulsion gate to impede the forward movement of Mg2+. Finally, Cl− may play a ferry-like role in facilitating Mg2+ transport into the matrix through the R-ring. The principle of “anion-assisted cation permeation” has also been observed in the calcium channel Orai protein located in the plasma membrane36.Considering the results of our structural and MD simulations and functional studies, we propose a model for Mg28. Consequently, the possibility of chloride-assisted Mg2+ transport cannot be entirely disregarded for other members of the CorA family, at least for the current time being.It is worth noting that while the R-ring in the transmembrane pore region of the hMrs2 channel is not present in other members of the CorA family, wider arginine rings in the matrix domain have been observed in the structures of TmCorA, MjCorA, EcZntB, and PaZntBHomo sapiens Mrs2 (UniProt ID: Q9HD23) was synthesized with a C-terminal FLAG tag  and cloned into a pcDNA4/Myc-His A vector (Thermo Fisher). The hMrs2 mutants  were generated through standard PCR-based mutagenesis methods and verified by DNA sequencing (GENEWIZ). Primers sequences were listed in Supplementary Data E. coli and purified with a GoldHi EndoFree Plasmid Maxi Kit (CWBio).The gene encoding 2 at 37 °C. Prior to transfection, the cells were diluted to a density of 0.7 × 106 cells mL−1 and cultured for 24 h. Transient transfection was performed using PEI 25k . For 1.0 × 109 cells, 1.0 mg of plasmid and 3 mg of PEI were preincubated in 90 mL of fresh medium for 20 min. Then, the DNA/PEI mixture was added to the cell cultures. After another 24 h, SMS 293-SUPI cell culture supplement  was added to the cultures at a ratio of 3.4% (v v−1). The cells were harvested 60 h after transfection and stored at −80 °C.HEK-293F cells were cultured in SMM293-TII expression medium , supplemented with 5% CO−1) n-dodecyl-ß-D-maltoside  and 1 × cocktail (TargetMol). Cell lysates were incubated at 4 °C for 2 h, followed by ultracentrifugation at 120,000 × g for 45 min. The resulting supernatant was then incubated with Anti-DYKDDDDK Tag (L5) Affinity Gel (BioLegend) at 4 °C for 4 h. The resin was washed sequentially with buffer A  DDM), buffer B , and buffer C  GDN (Anatrace)). The target protein was eluted with buffer C containing 0.4 mg mL−1 DYKDDDDK peptide. The eluent was concentrated to a 1 mL volume using a Microsep Advance ultrafiltration device with a 100-kDa cutoff . Subsequently, the concentrated eluent was loaded onto a Superose 6 increase 10/300 GL column (Cytiva), which had been preequilibrated with buffer D  GDN). For the hMrs2-Mg structure, 20 mM MgCl2 was added to buffer D. For the hMrs2-lowEDTA structure, 1 mM EDTA was added to buffer D. For the hMrs2-highEDTA structure, buffer E  GDN) was used for gel filtration. The peak fractions were pooled and concentrated to a final concentration of 5–15 mg mL−1 for cryo-EM.For protein purification, each liter of cells was resuspended in 100 mL of buffer consisting of 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1% . The grids were blotted for 4 s with blot force 7–8 at 8 °C and 100% humidity. Subsequently, the grids were rapidly plunge-frozen in liquid ethane cooled by liquid nitrogen using the FEI Vitrobot Mark IV.37 in counting mode with the pixel size of 1.014 Å and defocus range of −0.8 to −1.6 μm. Each movie stack was dose-fractionated over 40 frames by 8 s exposure with a dose rate of 1.35 e− per Å2 per frame. hMrs2-Mg, hMrs2-lowEDTA and hMrs2-highEDTA samples were automatically imaged with EPU software (2.14) in a 300 kV Titan Krios G3 cryo-electron microscope (FEI) equipped with Falcon 4 direct-electron detector and Selectris energy filter (ThermoFisher). Image stacks were recorded in EER mode, with a pixel size of 0.93 Å and a defocus range of −0.8 to −1.6 μm. The image stacks in EER format were converted to TIFF format using the relion_convert_to_tiff command in RELION software (4)38. For hMrs2-Mg samples, a total of 3329 image stacks were collected with a total dose of 51.39 e− Å−2, each compressed image stack containing 45 frames with dose rate of 1.13 e− per Å2 per frame. For hMrs2-lowEDTA samples, a total of 3217 image stacks were collected with a total dose of 51.39 e− Å−2, each compressed image stack containing 45 frames with dose rate of 1.13 e− Å−2 per frame. For hMrs2-highEDTA samples, a total of 1708 image stacks were collected with a total dose of 59.97 e− Å−2, each compressed image stack containing 45 frames with dose rate of 1.33 e− Å−2 per frame.A total of 690 image stacks were collected for hMrs2-rest sample by a 300 kV Titan Krios G3 cryo-electron microscope (FEI) equipped with K2 Summit direct-electron detector (Gatan) using SerialEM software (3.8)39 for 5 × 5 patch drift correction with dose weighting. Non-dose-weighted images were used for contrast transfer function (CTF) estimation by CTFFIND software (4.0.8)40. Poor quality images were manually removed before the particles were picked. The particles were picked up semi-automatically by Gautomatch software (0.56) (www.mrc-lmb.cam. ac.uk/kzhang/) and extracted by RELION software (3.1)41 with a 260-pixel box size.Image stacks were processed by MotionCor2 software (1.3.2)42. The initial model was pass-filtered to 30 Å resolution and applied for 3D classification in RELION software (4). After two rounds of 3D classification, the selected subset containing 60,865 particles was used for 3D auto-refine and Bayesian polishing with C5 symmetry. The polished particles were imported into CryoSPARC software (3.3.2), followed by non-uniform refinement, CTF refinement, and symmetry expansion according to C5 symmetry. The mask output at the non-uniform refinement step was used as the initial mask for the subsequent local refinement step. The local refinement procedure, integrating a dynamically generated mask in each refinement iteration, was executed in conjunction with non-uniform adaptive regulation, leading to the production of a final map with a resolution of 2.6 Å with C1 symmetry. The automatically sharpened map derived from the local refinement step was subsequently employed for subsequent model building and refinement.For the hMrs2-Mg dataset, 473,653 particles picked from 2,375 selected images were subjected to 2 rounds of 2D classification in RELION software (4) to remove bad particles, yielding 147,386 particles. The selected particles were then used to generate an initial model by the Ab initio program in CryoSPARC software (3.3.2)For the hMrs2-rest dataset, 170,290 particles picked from 635 selected images were subjected to 2 rounds of 2D classification in RELION software (3.1) to remove bad particles, yielding 36,283 particles. The selected particles were then used to generate an initial model by the Ab initio program in CryoSPARC software (3.3.2). The initial model was pass-filtered to 30 Å resolution and applied for 3D classification in RELION software (3.1). After two rounds of 3D classification, the selected subset containing 20,800 particles was used for 3D auto-refine and Bayesian polishing with C5 symmetry. The polished particles were then imported into CryoSPARC software (3.3.2) for homogeneous refinement and then symmetry expansion according to C5 symmetry. The mask output at the homogeneous refinement step was used as the initial mask for the subsequent Local Refinement step. The Local Refinement procedure, integrating a dynamically generated mask in each refinement iteration, was executed in conjunction with non-uniform adaptive regulation, leading to the production of a final map with a resolution of 2.9 Å with C1 symmetry. The automatically sharpened map derived from the Local Refinement step was subsequently employed for the subsequent model building and refinement.41 to remove bad particles, yielding 58,010 particles. The selected particles were then used to generate an initial model by the Ab initio program in CryoSPARC software (3.3.2). The initial model was pass-filtered to 30 Å resolution and applied for 3D classification in RELION software (4). After a round of 3D classification, the selected subset containing 42,527 was used for 3D auto-refine and Bayesian polishing with C5 symmetry and then imported into CryoSPARC software (3.3.2). After a further round of 2D classification, 34,277 particles were selected and subsequently subjected to non-uniform refinement, CTF refinement, and then symmetry expansion according to C5 symmetry. The mask output at the non-uniform refinement step was used as the initial mask for the subsequent Local Refinement step. The Local Refinement procedure, integrating a dynamically generated mask in each refinement iteration, was executed in conjunction with non-uniform adaptive regulation, leading to the production of a final map with a resolution of 2.7 Å with C1 symmetry. The automatically sharpened map derived from the Local Refinement step was subsequently employed for the subsequent model building and refinement.For the hMrs2-highEDTA dataset, 374,846 particles picked from 1244 selected images were subjected to 2 rounds of 2D classification in RELION software (4)For the hMrs2-lowEDTA dataset, 631,480 particles picked from 2324 selected images were subjected to 2 rounds of 2D classification in RELION software (4) to remove bad particles, yielding 203,681 particles. The selected particles were then used to generate an initial model by the Ab initio program in CryoSPARC software (3.3.2). The initial model was pass-filtered to 30 Å resolution and applied for 3D classification in RELION software (4). After two rounds of 3D classification, the selected subset containing 37,000 particles was used for 3D auto-refine and Bayesian polishing with C5 symmetry. The polished particles were imported into CryoSPARC software (3.3.2) and subsequently subjected to non-uniform refinement, CTF refinement, and then symmetry expansion according to C5 symmetry. The mask output at the non-uniform refinement step was used as the initial mask for the subsequent Local Refinement step. The Local Refinement procedure, integrating a dynamically generated mask in each refinement iteration, was executed in conjunction with non-uniform adaptive regulation, leading to the production of a final map with a resolution of 2.5 Å with C1 symmetry. The automatically sharpened map derived from the Local Refinement step was subsequently employed for the subsequent model building and refinement.43. The workflows for the data collection and reconstruction are shown in Supplementary Figs. The resolution of the maps was evaluated using the “gold-standard” FSC = 0.143 criterion. The local resolution was calculated using ResMap software (1.1.4)44, and pre-fitted single-chain model was refined by PHENIX software (1.19)45 in the real_space_refinement with model morphing and secondary structure and geometry restraints. After the real space refinement, the regions with poor density fitting and side-chain orientation were manually built and refined according to the density map in COOT software (0.8.9.3)46. After five chains of the model were manually fitted and refined, the entire model was refined by PHENIX software (1.19) real_space_refinement with secondary structure and geometry restraints. Based on refined hMRS2-rest model, hMRS2-Mg, hMRS2-lowEDTA and hMRS2-highEDTA structures were built and refined by COOT software (0.8.9.3) and PHENIX software (1.19). The quality of the models was assessed by MolProbity module of PHENIX software (1.19). Refinement statistics and validation statistics for hMRS2-rest, hMRS2-Mg, hMRS2-lowEDTA and hMRS2-highEDTA models are shown in Supplementary Table The hMRS2-rest model building used a predicted human Mrs2 (UniProt ID: Q9HD23) model from the Alphafold2 database. The predicted coordinates were fitted into hMRS2-rest map in UCSF ChimeraX 1.5).544, and14. HEK-293F cells were harvested by centrifugation . The cells were then washed with ice-cold PBS and resuspended in cold isolation buffer  at a ratio of 4 mL per gram of cells. BSA was added to the solution so that the final concentration was 0.25%. The resuspension was added to a glass homogenizer, and the pestle was pumped up and down 10~15 times. An additional threefold volume of buffer IB was added to the homogenate, and the pellet was removed after two rounds of centrifugation . The supernatant was then centrifuged  to obtain mitochondria. Purified mitochondria were quantified by Bradford’s method.Mitochondrial isolation experiments were performed in accordance with the literature2+ uptake assay buffer (AB) consisted of IB supplemented with 0.5 mM ATP, 0.2% succinate, and 0.01% pyruvate. Mitochondria were resuspended at 0.5 mg mL−1 in AB and loaded with 2 μM Mag-fluo-4 (Invitrogen) and 0.02% Pluronic F-127 (Thermo Fisher) for 25 min at room temperature (RT). Mitochondria were washed twice with AB and then incubated for 35 min. The mitochondria were then washed twice more with AB and dispensed into a 96-well plate at a ratio of 96 μL per well. Fluorescence was monitored at RT with Spark multimode microplate reader (Tecan) using filter sets  before and after Mg2+ injection. The average fluorescence value before and after Mg2+ addition was subtracted and plotted in a bar graph.MgThe primary antibodies: rabbit anti-MRS2 antibody  and mouse anti-cytochrome C , and the secondary antibodies: rabbit anti-mouse IgG  and goat anti-rabbit antibody  were diluted in TBST. Samples were loaded on 12.5% SDS‒PAGE gels, and the proteins were transferred to PVDF membranes. The membranes were incubated with 5% nonfat dry milk for 1 h at RT. After washing 3 times with TBST buffer, primary antibodies were incubated with the membranes for 1 h at RT. After washing another 3 times with TBST, the secondary antibodies were incubated with the membrane for 1 h at RT. Finally, after washing 3 times with TBST, the membranes were soaked in a chemiluminescent HRP substrate (Millipore) and imaged on a Tanon 5200 Multi Chemiluminescent Imaging System (Tanon).−) and magnesium ion (Mg2+) were retained. The protonation state for titratable residues was determined using the H++ program47. All proteins were capped at the N- and C-termini with acetyl and methyl amide groups, respectively. All missing side-chain non-hydrogen atoms and hydrogen atoms were added automatically using the LEaP module in AMBER-2020 (http://ambermd.org/AmberMD.php). The hMrs2 protein was inserted into 135 Å × 135 Å bilayers of palmitoyl oleoyl phosphatidyl choline (POPC) with the pore axis aligned parallel to the z axis by using the VMD software (1.9.3) Membrane Builder plugin48. The protein-POPC system was then solvated in SPC/E water boxes49 and neutralized by 0.15 M NaCl and 0.02 M MgCl2. The size of the system Mrs2 with Cl− (Mrs2-withCl) was initially measured as 135 Å× 135 Å × 180 Å. The Cl− from the Mrs2-withCl system was omitted to build the Mrs2 without Cl− (Mrs2-noCl) system. One sodium ion was removed randomly to neutralized the system. The systems were consisted of 316,626 and 316,624 atoms, respectively (Supplementary Table The hMrs2-Mg structure was prepared for the MD simulations. The bound chlorine ion (Clhttp://ambermd.org/AmberMD.php) with the AMBER force field FF19SB50, LIPID2151, and SPC/E water model52. The Na+ and Mg2+ ions parameters were provided by the in-silico studies  algorithm57. The 10,000 steps minimization was carried out for each system, followed by the thermalization of each system heating from 0 K to 310 K in 500 ps using the Langevin thermostat58. The lipids were equilibrated for 30 ns with the proteins constrained , followed by the 20-ns optimization for missing atoms with the other protein atoms constrained with the gradually reduced harmonic restraints . After that, the 20-ns equilibration phase was carried out, without any constraint applied for the entire system. Finally, three independent 500-ns production phase was performed for each system. The NPT ensemble was utilized at a constant pressure of 1 bar by the Berendsen barostat59. The time step was set to 2 fs. The frames were saved every 5000 steps for analysis.All MD simulations were performed with AMBER-2020 with the PMEMD engine  was set to the reaction coordinate (RC) after placing the center of the lipids into the origin and a harmonic restraint  was used to steer the Mg2+ permeation through the Mrs2 from intermembrane space to the matrix. The profiles of the coordinated water number were calculated based on three independent steered-MD simulations.Due to the size (<2 Å) of two narrow regions (R332 and N362) of the hMrs2 pore is smaller than the size (approximately 2.4 Å) of the first hydration shell of magnesium ion62 was used in 51 windows which starts from the extracted frames from steered-MD trajectory and dispersed uniformly along the RC where the distance ranges from −70 Å to +30 Å, at 2 Å intervals. In each simulation, the ion position along the Z direction was restrained using a harmonic restraint with a force constant 2.5 kcal mol−1 Å−2. The PMF was generated by fully sampling the configuration space in each window and then continued until the convergence of PMF was achieved. The total accumulated simulation time of trajectories used for the PMF calculations is 1.02 μs for Mrs2-withCl and Mrs2-noCl system, respectively. The PMF profiles utilized the WHAM software (2.0.11) (http://membrane.urmc.rochester.edu/wordpress/?page_id=126) and used the bin width of 0.1 Å.An umbrella sampling proceduret-test was performed to determine statistical significance of effects. Differences with a P-value of <0.05 were considered significant: *P < 0.05 and **P < 0.01.The number of independent experiments (N) and the relevant statistical parameters for each experiment (such as mean or standard deviation) are described in the pertinent figure legends. No statistical methods were used to pre-determine sample sizes. An unpaired two tailed Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummarySource Data