In in-silico prediction for molecular binding of human genomes, promising results have been demonstrated by deep neural multi-task learning due to its strength in training tasks with imbalanced data and its ability to avoid over-fitting. Although the interrelation between tasks is known to be important for successful multi-task learning, its adverse effect has been underestimated. In this study, we used molecular interaction data of human targets from ChEMBL to train and test various multi-task and single-task networks and examined the effectiveness of multi-task learning for different compositions of targets. Targets were clustered based on sequence similarity in their binding domains and various target sets from clusters were chosen. By comparing the performance of deep neural architectures for each target set, we found that similarity within a target set is highly important for reliable multi-task learning. For a diverse target set or overall human targets, the performance of multi-task learning was lower than single-task learning, but outperformed single-task for the target set containing similar targets. From this insight, we developed Multiple Partial Multi-Task learning, which is suitable for binding prediction for human drug targets. Discovering novel compounds that bind to human proteins for use as drugs is gaining increased interest in clinical research. As metabolism within the human body is controlled by the interaction between molecules, predicting and validating potential molecular binding is essential for novel drug development . In factIn this context, various in-silico-based approaches in pharmaceutical research have been proposed to overcome the low success rate of novel drugs. Computer aided drug design (CADD) assists in the retrieval of viable drugs from a large-scale compound database, thereby substantially reducing the time and cost for clinical approval . PredictIn the early stage of a machine learning approach for drug-target binding prediction, Quantitative Structure-Activity Relationship (QSAR) tasks based on linear regression have had considerable success . SubsequPrevious works applying DNN for QSAR binding prediction commonly insist that multi-task learning helps to improve prediction performance. Multi-task learning refers to an approach that simultaneously trains a neural network that is shared by multiple tasks with output neurons for individual tasks . In contNevertheless, for the practical purpose of predicting drug-candidates for human drug targets, the effectiveness of multi-task learning requires more precise examination. Firstly, the exact deep learning architecture that is used for multi-task learning is quite different for each study and no fair comparison has been made among them. Secondly, the target sets that are used in previous studies do not focus on the large-scale human genome, which obscures whether the performance is still valid for general human drug targets. Among around 30,000 human genomes, an estimated 600–1500 genes are sorted as “drug targets” . In thisIn this study, we compared various types of multi-task learning methods from previous researches using a normalized validation method. According to previous studies, the output neurons for multi-task learning architecture, which are task-specific layers, can be constituted as a multi-label classifier ,20,22 orThe ChEMBL ,27 databThe SMILES format of compound structures is a text-based representation of molecules and does not represent scalable features that are required for training. Thus, further modification for compound data must be facilitated for machine learning. First, the ChemAxon standardizer  standardAs mentioned previously, multi-task architecture in deep learning can adapt a multi-label classifier or multiple binary classifiers as its task-specific output layer a. BecausLoss function for multi-label classifier using masking:i,t stands for the predicted value for the binding activity between a query compound i and target t. yi,t indicates the true activity corresponding to xi,t with 1 for positive and 0 for negative. mi,t is an element representing a mask with 1 for a known interaction and 0 for an unknown. wt is the weight of a target t which is the reversal of the number of known activities of the target.Loss function for multi-label classifier using masking and task-weighting:Meanwhile, the multi-task architecture using multiple binary classifiers (MT-binary) is free from many issues derived from batch generation. This is because they can build independent mini-batches for each task ,23. Nonei,0 and xi,1 represents the predicted value for binding and non-binding activities of a compound i, respectively. yi indicates the true activity between compound i and the target with 1 for positive and 0 for negative. N indicates the number of compounds in a batch.Loss function for multiple binary classifier defined for each target:TensorFlow (version 1.10.1)  was usedAccording to a study by Mayr et al., machine learning prediction for the chemical database has a variety of potential biases . FirstlyROC-AUC is one of the most common metrics to evaluate the performance of binary classification (active or inactive). However, based on the purpose of multi-task learning, validating the ROC curve across all targets cannot represent the prediction accuracy for individual targets. Some of the tasks may suffer from low prediction power while other tasks may display overwhelming accuracy. To check such skewed performance, we used target-AUC, which indicates the average ROC-AUC over validation fold for each task . ValidatAs high inter-task correlation is known to improve the effectiveness of multi-task learning , we specNormalized BLOSUM62 distance between two protein sequences:https://github.com/KyoungYeulLee/MPMT.We developed a novel multi-task learning strategy that is effective for molecular binding prediction of human targets in ChEMBL. This was derived with reference to the results from various validations and comparisons of DNNs. Instead of performing multi-task learning for all human targets at once, we clustered human targets based on their sequence similarities in their binding domain sequences. For each cluster, targets in a cluster were trained using multi-task learning c. TargetBioactivity data from ChEMBL (version 23) was divided into two types of python array data. Feature space for binding compounds was generated for Semi-sparse, ECFP4, and ECFP6 fingerprints. Target space representing activities between compounds and protein targets was also generated. All possible activity types including active interactions, inactive interactions, and unknown interactions were distinguished by imposing different values. Targets were also clustered based on their sequence similarities at known binding domains to build data with different target compositions. Various deep neural architectures including multi-task and single-task learning were Constructed by TensorFlow, which were trained and tested with bioactivity data. Each DNN was tested fairly and compared by nested cluster cross validation, which is constructed to avoid overfitting to specific compound scaffolds or hyperparameters. Using this normalized scheme, various multi-task strategies were evaluated to find the best practices for binding prediction of human targets .We collected targets that are only expressed in Homo sapiens (tax id = 9606). Targets with at least one active and one inactive data for each fold in compound clusters see  were gatTarget-AUC, which indicates an average ROC-AUC over validation fold for each target, was used as a standard to quantitatively compare the performance of deep neural architectures. As target-AUC can measure prediction performance for each target, we could precisely verify the strengths and weaknesses of various architectures and find practical strategies for different target compositions. Not only average target-AUC across targets, but also standard deviation, maximum, and minimum of the target-AUC were measured, providing insights on the unique properties of each architecture. In addition, the robustness of multi-task architectures, representing the ability of networks to improve performance across all targets, was examined using single-task learning as a baseline . The proThe result clearly shows that multi-task learning can outperform single-task learning when targets are similar. However, for diverse target sets or overall human targets, no multi-task architecture could outperform single-task learning. Although it is known that inter-task correlation is an important factor for the performance of multi-task learning, little is known about the adverse effects of multi-task learning. This result suggests that multi-task learning may reduce prediction performance when the target list is not cautiously selected. Furthermore, simply applying multi-task architectures across human targets can be problematic. Nevertheless, multi-task architecture demonstrated its effectiveness when applied to targets with high similarity. The details of several meaningful cases were further examined and the exact difference between deep-neural architectures was compared to elucidate their properties and the best practices. The performance using ECFP4 and ECFP6 as compound representation is reported in When testing overall human targets without considering similarity, the prediction performance of single-task learning outperformed that of multi-task learning. Surprisingly, target-AUC for the single-task network generally showed better performance across targets a. More tMulti-task network with binary classifiers (MT-binary) showed the most qualified performance when validating highly similar targets (d ≤ 0.2). Of the targets, 76.9% had better prediction performance using MT-binary than single-task learning b. An intBecause multi-task learning is promising for similar targets but inaccurate for diverse targets, a more sophisticated technique is needed for large-scale human drug targets. We developed a technique that takes advantage of both multi-task learning and single-task learning. Multi-task learning has the strength of using information from targets with qualified data to train targets with low accuracy. Single-task learning can more clearly identify the difference between targets than multi-task learning. In this technique, multi-task learning (MT-binary) is performed multiple times for partial target sets by clustering similar human targets. Through this process, diverse targets did not share common layers, thereby enabling the identification of the differences between dissimilar targets. For similar targets, multi-task learning improved the accuracy, especially for the targets with low performance by single-task learning. We named this technique Multiple Partial Multi-task learning (MPMT).The performance of MPMT must be compared to that of single-task learning. First, we compared target-AUC for targets included in the similar target clusters. As a sufficient number of targets for effective multi-task learning were ambiguous, we selected clusters using different cutoffs for the minimal number of targets in a cluster. For selected clusters, MPMT trained targets by multi-task learning for each cluster. The overall targets in selected clusters were also trained by single-task learning for comparison. Clustering of targets used normalized BLOSUM62 distance with a distance cutoff of 0.2 or 0.3. Mean and minimum target-AUC were compared between MPMT and single-task learning for various cutoffs and clustering distances . The res−3 for the alternative hypothesis that MPMT shows higher target-AUC compared to single-task learning, and p-values are even smaller when compared to other architectures. This result concludes that the deep learning model that is built by MPMT shows the most reliable binding prediction for human targets.We also compared the performance of MPMT to that of other deep neural architectures for 1067 human targets . Human tSome of the human targets cannot be trained properly because of the lack of known active compounds, biased compound structures, or network overtraining. As multi-task learning is known to relieve such bias, MPMT was applied for the targets with low accuracy when trained by single-task learning. Targets with ROC-AUC ranging from 0.5 to 0.6 for single-task learning were trained by MPMT with a distance cutoff of 0.2 and the performances were compared. The results clearly showed that the prediction accuracy of the target models improved when they were trained by MPMT. The rate of improvement was about 84% as the performances of 16 targets out of 19 targets were improved. Some of the targets improved greatly such as Tyrosine-protein kinase Lyn (ChEMBL ID: 3905) of which ROC-AUC increased from 0.574 to 0.758 using MPMT. The performance of two targets were slightly decreased and only one target, Peptidyl-prolyl cis-trans isomerase FKBP5 (ChEMBL ID: 2052031), showed a significant performance decrease from 0.5921 to 0.4878. Such decreases might be caused by selecting the wrong binding domains during target clustering or underestimating the difference of the target from similar targets during multi-task learning.Even though the performance of MPMT seems better than that of single-task learning statistically, we need to check whether it is helpful for finding potential drug candidates. For this purpose, we investigated approved drugs binding to Tyrosine-protein kinase Lyn and assessed the probability of MPMT to identify those drugs as potential candidates. The results compared with single-task learning are represented in Although the importance of inter-task correlation when performing multi-task learning is known, the adverse effect of multi-task learning for dissimilar tasks has not been adequately investigated. In this study, we found that multi-task learning can interfere with proper target prediction if the degree of similarity between the targets is low. By applying multi-task learning to large-scale targets, such as overall human targets, significantly worse performance was achieved compared to that with single-task learning. This suggests that the target set for multi-task learning should be selected more carefully for practical purposes .According to the results of this study, clustering human targets based on their similarity may be effective in improving the accuracy of molecular binding prediction for large-scale human targets. In this study, target similarity was calculated using sequence similarity at binding domains. This improved the efficiency of multi-task learning for similar human targets. In addition, through the strategy termed MPMT, target sets with high similarity were trained by multi-task learning while other targets were trained by single-task learning. The results demonstrate that, compared to previous methods, the prediction performance for overall human targets in ChEMBL was improved. However, a considerable number of targets remain unclustered because of unknown binding domain or low similarity to other targets, thereby functioning as an obstacle of MPMT in achieving significant improvement in prediction accuracy. If we can cluster targets more precisely to ensure most targets have neighboring targets, a further improvement in the performance of MPMT is expected. The novel clustering method may exploit the characteristics of known drugs or the family of targets besides sequence similarity. In addition, the algorithm for partial multi-task learning can be developed beyond a simple divide and conquer method. Therefore, MPMT is expected to be a technique that will more likely improve in the future.Increasing the performance of drug-binding predictions through multi-task learning can increase the efficiency of drug development. Accurate in-silico binding predictions will dramatically reduce the time and cost that is necessary for drug screening . Additio


This cross-sectional was aimed to assess the prevalence of pre-pregnancy care services usage and its determinant factors among women of reproductive age in Kedah, Malaysia.Overall, 1347 respondents who attended 24 government health clinics, were chosen using systematic multistage random sampling. A validated self-administered questionnaire which consisted of sections including socio-demographic characteristics, social support, knowledge on pre-pregnancy care, perception on risk of pregnancy, health status, as well as intention and awareness on pre-pregnancy care services were distributed.P<0.001), high education level (P<0.001), non-working mothers (P<0.001), multipara (P=0.001), awareness on the existence of pre-pregnancy care services in government health facilities (P<0.001), intention to use the services (P=0.0030), having medical illness (P=0.005), having social support (P=0.001), high knowledge (P<0.001), and positive perception (P<0.001).The prevalence of utilization of pre-pregnancy care services was still low i.e. 44.0%. Bivariate and multivariate analysis showed consistent significant level between all factors and pre-pregnancy care usage except for family planning practice. The factors that showed significant difference with the usage of pre-pregnancy care services were age of more than 35 (Low usage of pre-pregnancy care services can be improved through health screening on reproductive-aged women with positive determinant factors at the triage level in integrated clinics. Information and knowledge on pre-pregnancy services should be disseminated among community members through various means including roadshows and pre-wedding workshops. Today, the continued increase in the burden of maternal and infant mortality has become a public health concern globally. Worldwide, over 350000 women of reproductive age die each year from complications of pregnancy and during delivery, while more than 15 million women suffer from long-term illnesses or disability .In addition to the burden of maternal mortality and morbidity rate which showed increasing trend in developing and less developed countries, globally, in which each year about 3.6 million newborns die within the first four weeks after birth . WorldwiIn Malaysia, although the mortality rate among women of reproductive ages has reduced over the years, this reduction has been almost stagnant for the past ten years (27.9 per 100000 live birth in 2005 to 25.2 per 100000 live birth in 2013) .Center of Diseases Control and Prevention (CDC) 2006 defines pre-pregnancy care as “a set of intervention that aims to identify and modify biomedical risk, behavioral and social risks to a women’s health through prevention and treatment” –8. Pre-pAlthough pre-pregnancy care services had been proven to be successful, the level of utilization of these services and the knowledge of Malaysian women in the reproductive ages about pre-pregnancy care and its services remains unclear. Many women in this country including those from the state of Kedah do not use pre-pregnancy care services, which are readily available and affordable. If these diseases were not controlled promptly, it will result in adverse outcome to the mother as well as the baby –13. The We aimed to determine the prevalence of pre-pregnancy care services usage and its determinant factors among women of reproductive ages in Kedah, Malaysia.This cross-sectional study was carried out among married reproductive-aged women between the ages of 18 to 45 year old from all ethnic groups in Kedah in May 2015. It was conducted in 12 districts in Kedah chosen through a multistage random sampling. Eight districts were chosen and three government health clinics were then selected from each district. Overall, 24 government health clinics were finally selected to be included in this study. Selection of the respondents were through systematic random sampling where it was based on registration number listed in the registration book at the integrated counter in the 24 government health clinics involved in this study. Every five women who fulfilled the inclusion criteria were chosen to avoid bias.We used a validated self-administered questionnaire which had satisfactory value of Cronbach’s alpha of 0.927. This questionnaire consisted of 7 domain sections including socio-demographic characteristics, socio-economic background, social support, and knowledge on pre-pregnancy care, perception on risk of pregnancy, health status, as well as intention and awareness on pre-pregnancy care services. Overall, 1347 mothers answered the self-administered questionnaire in the Malay language in the clinics.Six sub-questions, which consisted of 44 fractional responses, were used to assess the knowledge of pre-conception care and its services which includes knowledge of what was pre-pregnancy care, to whom such services should be designated for, the importance and goals of pre-pregnancy care services, types of services provided in the pre-pregnancy care clinics and where to get the service. Twelve items questionnaire related to pre-pregnancy care and its services were used to test the perception of respondents including perception towards risk of pregnancy as well as perception towards outcome of pregnancy and its complication. Meanwhile, in the social support section, there were four questions given to respondents.P-value<0.05) with the uptake of pre-pregnancy care services. Dependent variable was the usage of pre-pregnancy care services while the independent variables were the social-demographic factors, awareness, intention, knowledge, perception, social support and medical history. Data were analyzed using SPSS ver. 20.0 .Descriptive characteristics were summarized and presented as numbers and percentages. Chi-square test was used to examine the relationship between each variable and usage of pre-pregnancy care services. This was followed by multiple logistic regression analysis utilized to examine the associations between the significant variables  due to these services.It is very important to improve the knowledge and perception of pre-conception care and services among women of reproductive age in this country, considering the low utilization of pre-pregnancy care services as shown in this study. With the effort by multiple stakeholders taken to improve the knowledge and perception of reproductive-aged women on such services, especially among women who are younger, less educated and do not have children, may result in the reduction of poor pregnancy outcome. This include the reduction of maternal mortality rate and at the same time, we hope to achieve one of the SDG targets in 2030, i.e. to reduce maternal mortality rate to 8.7 per 100,000 live births.Ethical issues  have been completely observed by the authors.


The first section describes strategies employed to locate target macromolecules within large cellular volumes. Next, we explore methods to study thick specimens by sample thinning. Finally, we review examples of macromolecular structure determination in a cellular context using cryo-ET. The examples outlined serve as powerful demonstrations of how the cellular location, structure, and function of any bacterial macromolecule of interest can be investigated using cryo-ET.Electron cryotomography (cryo-ET) is an imaging technique uniquely suited to the study of bacterial ultrastructure and cell biology. Recent years have seen a surge in structural and cell biology research on bacteria using cryo-ET. This research has driven major technical developments in the field, with applications emerging to address a wide range of biological questions. In this review, we explore the diversity of cryo-ET approaches used for structural and cellular microbiology, with a focus on  Now, the role of macromolecules in driving cellular reactions can be investigated in unprecedented detail using a variety of imaging methods available to the modern cell biologist. These methods can be used to uncover the spatial arrangements of molecules in biological specimens, and reveal how they coordinate fundamental physiological processes. The choice of imaging approach is dependent in the first instance on the biological question, but must also account for the properties of the specimen being investigated. Eukaryotic cells are typically several microns in size, and thus many eukaryotic cellular processes can be readily monitored using light microscopy techniques. On the other hand, bacterial cells are often an order of magnitude smaller than eukaryotic cells and thus present different challenges for cellular imaging. Studying biological processes inside bacterial cells generally requires higher spatial resolution, since processes typically occur over a much smaller scale (<1 μm).An imaging method that is ideally suited to studying bacterial cells is cryo-EM (electron cryomicroscopy or cryo-electron microcopy). Cryo-EM is a unique imaging tool for the visualization of unstained biological material at high spatial resolution . The speThe signal to noise ratio of cryo-EM data is inherently limited by specimen thickness, since the number of inelastic scattering events, or ‘noisy’ electrons increase with increasing thickness. During cryo-ET data collection, the effective thickness of the slab-like specimen increases significantly at high tilt angles. Thin (< 1 μm) bacterial cells are therefore more suited to cryo-ET imaging than eukaryotic cells, because the entire organism can be imaged using this method. Applied in this way, cryo-ET can provide not only structural information, but important contextual information about any macromolecule of interest, in particular its organisation or interactions with other molecules within the cell. Imaging unstained, whole, frozen-hydrated cells provides unique interpretative clarity when compared with methods that require extensive interventions ahead of data collection, such as chemical fixation and heavy metal staining.in situ within cells or on bacterial membranes. Cellular structures derived from cryo-ET represent native conformations of macromolecules found within bacteria, and also shed light on the mechanisms responsible for bacterial evasion of antibiotics, which can inform the rational design of novel strategies to circumvent antimicrobial resistance [As evidenced by the wealth of recent articles on the subject, cryo-ET is rapidly becoming a fundamental technique for macromolecular structure determination, particularly of molecules in their cellular context . Pioneersistance .In this article, we review recent studies that describe spatial mechanisms in bacterial physiology, placing a special focus on strategies used to localize macromolecules inside cells with the end goal of sub-tomogram averaging and structure determination. We include a discussion of supporting techniques such as cryo-fluorescence microscopy, which provide a crucial navigational guide within subcellular volumes for targeted cryo-ET imaging, as well as sample thinning strategies for collecting high-quality cryo-ET data. Once the location of the target macromolecule is ascertained, cryo-ET data can be used for sub-tomogram averaging, where information about the secondary structure of macromolecules may be resolved. Studies using this approach have revealed fascinating details about the functional states of complexes in their cellular context, which are otherwise inaccessible through other biochemical methods. We thus provide a comprehensive overview of modern cryo-ET workflows as they have been applied to the investigation of bacterial cells, assessing the associated challenges and the approaches used to overcome them. The studies described demonstrate how cryo-ET has been and will continue to be a powerful technique for probing the ultrastructure of whole bacterial cells, for locating molecules of interest with high-spatial resolution, and for structure determination, which together provide a unique glimpse into the subcellular world with near atomic resolution.22.1A critical step in the cryo-ET workflow is the identification and localization of macromolecules of interest within tomographic volumes of the specimen. Sub-tomograms of the target macromolecule can then be extracted and averaged for structure determination. The problem is that cryo-EM imaging produces greyscale (black and white) images. As a result, macromolecules of interest are often obscured by, or indistinguishable from their crowded environment. Many cryo-ET studies have therefore focussed either on large proteinaceous assemblies such as sheets or filaments, or on molecules associated with defined cellular locations like membranes or the nucleoid.Caulobacter crescentus mid-cell [C. crescentus and Escherichia coli during cytokinesis [in vitro experiments led to a model of sliding FtsZ filaments in a ring driving membrane constriction at the mid-cell [C. crescentus and Proteus mirabilis showed that short FtsZ filaments can cause asymmetric cell envelope constriction at the beginning of cytokinesis [C. crescentus, E. coli and P. mirabilis). Together, these strategies led to an unambiguous identification of FtsZ inside bacterial cells and provided important insights into the fundamental process of cell division.One area of intense research is the study of protein and membrane dynamics involved in bacterial cytokinesis. Bacterial cytokinesis is mediated by the bacterial homologue of tubulin known as FtsZ . A ring mid-cell . Upon ovmid-cell . Togetheokinesis  . A dramatic example of this strategy is provided by studies on membranous organelles present in Magnetospirillum bacteria called magnetosomes. Magnetosomes are flanked by a network of actin-like proteins called MamK. MamK protein filaments appear to organise magnetosomes into linear chains that provide magnetic orientation to cells [mamK deleted cells using cryo-ET [A related strategy to locate molecules in cells using cryo-ET is to either delete the gene of interest completely, or perturb its copy number in a controlled manner, leading to distinctive changes in the ultrastructure of the cell. By comparison with wild-type cells, the location of the target molecules may be inferred. The success of this strategy depends on the possibility to identify the structures of interest (or lack thereof) after genetic manipulation. Despite this requirement, this approach is extremely useful for macromolecular identification of larger protein assemblies  in cells. In the examples presented above, a common approach used is modification of the copy number of the macromolecule of interest, and comparison of the resulting cellular ultrastructure with that of wild-type cells to understand the location and function of the macromolecule.The examples described in this section illustrate that while cryo-ET is a visual, image-based technique which, when taken in isolation, may seem merely descriptive, it can provide profound mechanistic insights into bacterial cell biology. Comparing different perturbed states of the system under observation allows researchers to glean mechanistic understanding of dynamic biological processes. These studies are particularly suited to study macromolecules that oligomerise into an easily identifiable shape  and final image resolution. Additionally, cryo-LM adds an extra cryotransfer step to the workflow, which increases the risk of ice contamination and/or devitrification. Furthermore, bacterial cells are extremely small, and high-resolution LM images are required for sub-cellular localisation of any fluorescent signal. Super-resolution LM approaches, and their associated benefits, are now being ported into cryo-LM systems . TechniqStreptomyces is a dramatic process involving the formation of long hyphae separated by cross-walls that exclude nucleoids. The distance between cross walls is several micrometers, thus requiring a modest resolution in cryo-LM for visualization and unambiguous correlation. A workflow to investigate this process using cryo-CLEM on Streptomyces cells was developed and applied to probe the underlying ultrastructure of cross walls [Cryo-LM/cryo-ET workflows have been successfully applied to study a variety of bacterial processes. The vegetative growth phase of ss walls . A fluorss walls . The memMyxococcus xanthus cells [M. xanthus cells, which could potentially have been assigned to one of a variety of candidate filamentous structures present within the cells [However, features of interest in bacterial cells are rarely several micrometers apart, and higher resolution is typically required in cryo-LM to unambiguously localize macromolecules in each diffraction-limited fluorescent spot. This was precisely the case in the study of a dynamic Type-VI secretion system (T6SS) in us cells . Ordinarhe cells . To get he cells , and typhe cells , includi2.3i.e. ordinary cryo-EM images) do not have ‘colour’ and the intensities in images are roughly correlated with the quantities and properties of scattering material in the specimen. Direct EM tags must therefore be significantly more electron dense than the surrounding biological material. The most common EM tags used thus far in the field have relied on a metal loaded protein such as ferritin [Performing cryo-LM prior to cryo-EM is nevertheless cumbersome, and even the best resolutions attainable by cryo-LM can severely limit the feasibility of some projects. To combine the spatial resolution of EM with the specificity of a tag for macromolecule localization within cellular tomographic volumes, ideally the tag should be visible directly in EM. Transmission EM images  overexpression in a olecules . In tomomembrane . Finallymembrane . Similar3B. subtilis and standard lab strains of E. coli are too thick for high-resolution cryo-ET, especially when the features of interest are located deep in the interior of the cell. In these cases, there are several thinning approaches available that convert the specimen into a viable target for cryo-ET data collection. These approaches are rapidly gaining importance as the field progresses towards tackling novel and ever more ambitious biological questions, and will be discussed in some detail. For many samples, thinning is critical for the success of in situ structural biology as it facilitates both the identification of molecules within cells, and supports high-resolution sub-tomogram averaging.Many bacteria including 3.1E. coli membranes by triggering lysis upon over-expression of a phage lysis gene [E. coli cells were obtained upon induction, which efficiently flattened during grid preparation allowing high-resolution cryo-ET imaging and sub-tomogram averaging of bacterial chemoreceptor arrays [B. subtilis cells using Lysozyme treatment [E. coli cells using penicillin treatment [Prior to vitrification, cells can be artificially thinned by a gentle lysis step. The lysis process produces thinner ‘ghost’ cells that lose some of their rigidity, allowing them to flatten during the blotting and plunge-freezing protocol in cryo-EM grid preparation. While lysis itself is relatively gentle, it nevertheless represents significant disruption to living cells prior to their cryo-fixation, and the potential for artefactual results must be considered. This approach was developed for visualizing native sis gene . A tightr arrays . Imagingreatment  and in Ereatment . Both of3.2cryo-electron microscopy of vitreous sections) [parMRC operon [E. coli cells was problematic due to thickness, particularly without the aid of direct electron detectors. CEMOVIS was therefore used to visualize the arrangement of ParMRC spindles in cells [E. coli cells containing low-copy number plasmids in the act of being segregated, showing for the first time with high-resolution cryo-EM that a ParM filament spindle mediated this process [E. coli, Pseudomonas aeruginosa and Mycobacterium smegmatis [One obvious advantage of thinning the specimen after vitrification is that no genetic or biochemical manipulation is required prior to vitrification, reducing the danger of artefacts and incorrect interpretation of cryo-ET results. To achieve this, there are two major approaches available. In one approach, vitrified specimens may be sectioned using an ultramicrotome operating at liquid nitrogen temperature, where the vitreous sections of cells are deposited on an EM grid. This technique is known as CEMOVIS (ections) , and it ections) . Each seections) . These aC operon . Direct in cells . Actin-l process . No chan process . Other Cmegmatis , 35.Despite these success stories, there are several disadvantages associated with CEMOVIS. CEMOVIS is labour intensive and sample preparation is relatively low-throughput; only a few grids may be prepared in each sectioning session. Due to the long sectioning process and additional grid transfer steps, ice contamination may build up on the grid, obscuring features of interest. Additionally, due to poor attachment of sections on the grid, cryo-ET data collection is also complicated because of section movement during tilt series data collection , 36. Fin3.3With the advent of focused ion beam (FIB) milling, sample thinning can now be achieved in a controlled manner, where regions around the feature of interest are carefully etched away to form a thin lamella or wedge suitable for cryo-ET data collection . This tePseudomonas chlororaphis bacteria were found to contain nucleus-like compartments in which nascent viral capsids are synthesised [Amoebophilus asiaticus showed bundles of T6SS in the cytoplasm arranged with hexagonal symmetry [This is evidenced by recent landmark studies using FIB-milling of bacterial cells. Phage-infected thesised . FIB milthesised . Facilitthesised  , including sub-tomogram alignment in crowded cellular environments and low signal to noise ratios.Important information regarding composition and cellular function is embedded in the high-resolution 3D structure of a macromolecular complex or assembly. Once the cellular location of the macromolecular complex is ascertained, and once the specimen is thin enough for cryo-ET imaging, then sub-tomogram averaging can be used to resolve information at the structural level. For this, copies of macromolecules visible in tomograms are computationally extracted into smaller windowed volumes known as sub-tomograms. These sub-tomograms are then averaged after 3D alignment to reveal higher resolution detail , 46. SubIn the sections below we discuss examples of sub-tomogram averaging applied to bacterial macromolecules in a cellular context, either of thin cells or cellular structures, or thicker cells where cryo-ET imaging was nevertheless successful. In all cases, the locations of the target macromolecules were either established previously from live-cell imaging, or were determined using one of the target localisation strategies discussed above.4.1E. coli mini-cells [Salmonella enterica mini-cells along with lysed E. coli, B. subtilis and H. hepaticus cells reported molecular-resolution sub-tomogram averaging maps of the chemoreceptor array into which X-ray structures of the proteins could be docked [One method adopted by researchers to produce high-quality tomograms is to use specialized bacterial strains that produce a mini-cell morphology. These mini-cells are significantly smaller and thinner than wild-type cells, and thus support high-resolution cryo-ET data collection and sub-tomogram averaging. This approach has been used to study bacterial chemoreceptor arrays, which are supramolecular transmembrane machines that localize to the cell poles, allowing cells to sense changes in their extracellular environment and instigate flagellar motility . These ani-cells . Aided bni-cells . CheW isni-cells . A parale docked . The doce docked .C. crescentus surface layer [C. crescentus cells possess such an S-layer bound to the outer membrane [C. crescentus S-layer from cell stalks led to sub-nanometre resolution reconstruction of the S-layer lattice [C. crescentus was too thick (~500 nm) for sub-nanometre resolution sub-tomogram averaging, demonstrating that targeting thin areas of cells, or obtaining thin cells can greatly facilitate in situ structural biology efforts.The advantages of imaging thin structures are illustrated by structural studies of the ce layer  on a thimembrane . Sub-tom lattice . The arr lattice . Interes4.2in situations where it disrupts the physiologically relevant state of the target complex. In such cases, tomographic data from whole cells must be used, where the best achievable resolution from sub-tomogram averaging is limited due to specimen thickness. However, molecular resolution reconstructions are still achievable, providing important architectural information about protein constitution and conformation.In many cases it is not possible to obtain a mini-cell containing the macromolecular complex of interest. Mini-cell formation is also undesirable Myxococcus xanthus showed the power of molecular resolution sub-tomogram averaging from whole cells to reveal assembly principles of macromolecular complexes in situ [An in-depth study of the Type-IV pilus (T4P) from  in situ . The T4P in situ . These m in situ  (Fig. 3B in situ  (Fig. 3BMacromolecules of interest are not always found on the cell surface, but often buried deep in the interior of bacterial cells where specimen thickness and poor tomogram quality hinder the identification of individual complexes. Here, to boost contrast at low resolutions, tilt series data are often collected with a large applied defoci. A large defocus is detrimental to sub-tomogram averaging applications however, as it results in increasing oscillations of the contrast transfer function at high resolutions. The contrast transfer function describes how, across different resolution ranges, information is transmitted or lost in the electron microscope. In practical terms, a large defocus leads to improvement in the visibility of low resolution features, but high resolution information is more difficult to recover, even with averaging. One way around this problem is to use a phase plate to obtain tomograms close to focus that preserve the low-resolution contrast while potentially supporting higher-resolution refinement in sub-tomogram averaging. Zernike phase-contrast cryo-ET was used to study phage intermediates inside cyanobacterial cells . Compare5We hope that it is apparent from the preceding text that the potential for structural cell biology studies in bacteria using cryo-ET is huge. While the developed workflows for macromolecular identification and imaging include a range of possibilities for accessing and tagging targets of interest , we expeThe potential for resolving protein structures in their cellular context represents an exciting bridge between the worlds of cellular and structural biology. We expect that through the use of cryo-FIB milling and other thinning approaches, sub-tomogram averaging will start playing a central role in cellular structural biology, although there remains a need to develop sub-tomogram averaging refinement procedures in low-signal to noise ratio environments inside cells , 61, 62.


A subgroup of patients with systemic sclerosis (SSc) develop interstitial lung disease (ILD), characterized by inflammation and progressive scarring of the lungs that can lead to respiratory failure. Although ILD remains the major cause of death in these individuals, there is no consensus statement regarding the classification and characterization of SSc-related ILD (SSc-ILD). Recent clinical trials address the treatment of SSc-ILD and the results may lead to new disease-altering therapies. In this review, we provide an update to the diagnosis, management and treatment of SSc-ILD. Scleroderma or systemic sclerosis (SSc) is a systemic multi-organ disorder characterized by autoimmunity, systemic inflammation, vascular injury, and tissue fibrosis . HippocrClassification of patients with SSc is based on the extent of skin involvement- diffuse cutaneous sclerosis (dcSSc) or limited cutaneous sclerosis (lcSSc), the latter characterized by skin sclerosis restricted to the hands, face, neck and distal extremities . AlthougThe overall incidence rate of SSc in the adult population of the United States is approximately 20 per million per year  and apprIn a review of patients with SSc-ILD, pulmonary fibrosis accounted for 19% of deaths and pulmonary hypertension (PH) in 14% . AfricanSimilar to other fibrotic lung diseases, injury to epithelial cells, activation of innate and adaptive immunity, and fibroblast recruitment and activation may lead to excessive extracellular matrix production and scarring in SSc-ILD . The facIn bronchoalveolar lavage (BAL) fluid from patients with SSc-ILD, the pro- inflammatory cytokines interleukin (IL)-8, tumor necrosis factor-a (TNF), and macrophage inflammatory protein−1a are increased . Lung biLung involvement including ILD, PH, or a combination of ILD and PH, occurs in more than 70% of patients with SSc. Pulmonary vascular disease, primarily pulmonary arterial hypertension, occurs in 10–40% of patients with SSc. Recently, coexisting PH was reported in a large SSc-ILD cohort often occurring early after diagnosis of SSc-ILD .The diagnosis of SSc-ILD is based on finding ILD on HRCT of the chest in a patient with known SSc accompanied by normal or abnormal pulmonary function tests showing restriction. Approximately one third of patients with SSc have positive anti-topoisomerase (Scl-70) antibodies; these patients have a greater likelihood of developing ILD, compared to those with lcSSc or those with positive anti-centromere antibodies . In the Figure 2). A pattern of usual interstitial pneumonia, characterized by honeycomb cysts and traction bronchiectasis may also be seen in up to a third of patients with SSc-ILD , anti- Th/To, anti-U3 ribonucleoprotein (RNP), anti- U11/U12 RNP, and rarely anti-centromere antibodies . The senPulmonary function tests may be normal at presentation, but can be helpful in the follow up of SSc-ILD . Forced Analysis of BAL from patients with SSc-ILD typically shows increased number of granulocytes, especially neutrophils and eosinophils, and sometimes an increased level of lymphocytes and mast cells . In a seThe diagnosis of SSc-ILD is based on finding ILD on the HRCT of the chest in a patient with known SSc, and with exclusion of other etiologies of pulmonary parenchymal disease such as drug induced lung toxicity, heart failure, or recurrent aspiration. A lung biopsy may be considered if there is suspicion for malignancy or granulomatous disease .p < 0.001)  was identified as a potential biomarker of SSc-ILD. It is suggested that elevated serum levels of SP-D would increase the risk of finding pulmonary fibrosis on chest images 3-fold  . CCL18 i< 0.001) .p < 0.001) (The levels of Krebs von den Lungen-6 (KL-6), a glycoprotein found predominantly on type II pneumocytes and alveolar macrophages, are elevated in the serum of patients with SSc-ILD and may correlate with the presence of pneumonitis and the radiological fibrosis score in patients with SSc . KL-6 ha< 0.001) .The importance of a decline in lung function and survival in patients with SSc was noted by Ferri . SSc-ILDThe treatment for SSc-ILD has focused on immunosuppressive therapies, particularly cyclophosphamide (CYC) and mycophenylate mofetil (MMF) based on the results of two pivotal clinical trials. Results from the Scleroderma Lung Study 1 showed a 1% change in FVC in the placebo group compared to a 2.6% change in FVC in the treated SSc subjects at 12 and 18 months (31). After 24 months, there were no differences between groups , 49. TheThe optimal treatment of SSc-ILD is not known. Developing treatments that would prevent SSc-ILD disease progression rather than disease regression is a research goal . CurrentMycophenolate mofetil (MMF)is an inhibitor of lymphocyte proliferation and is often used as first line treatment in patients with SSc-ILD who are at risk for progressive ILD . The rolIn an observational study, 13 patients received anti-thymocyte globulin plus prednisolone for 5 days, followed by MMF maintenance therapy for 12 months. Long-term MMF was well tolerated, but there was no change in mean FVC or diffusion capacity after receiving this combined therapy .Cyclophosphamide (CYC) is considered an alternative to MMF based on the results of the Scleroderma Lung Study II. The unfavorable adverse effect profile includes infertility, opportunistic infections, hemorrhagic cystitis, bladder cancer, and neutropenia . Monthlyp < 0.001) in the Azathioprine group. In the CYC group, DLCO and FVC remained unchanged , and DLCO (−11.6 ± 1.3%) were significantly worse  and a decrease in creatinine clearance . In a reBosentan, is a nonselective endothelin receptor antagonist, used in the treatment of pulmonary hypertension. It is known that the endothelin system participates in the pathogenesis of SSc, and that it could delay the progression of SSc-ILD. A prospective, double-blind, randomized, placebo-controlled, parallel group study was conducted to evaluate changes in 6 min walk test distance, FVC and DLCO changes. 163 patients were enrolled, 77 were randomized to receive Bosentan, and 86 were randomized to receive placebo for 12 months. No significant difference between treatment groups was observed for change in the 6-min walk distance. No deaths occurred in this study group. FVC and DLCO remained stable. In Conclusion, these data do not support the use of endothelin receptor antagonists as therapy for SSc-ILD .Rituximab, a monoclonal antibody that targets CD20 positive B-lymphocytes, is suggested for patients with refractory SSC-ILD . In a pia chain, is approved for treatment of rheumatoid arthritis, juvenile idiopathic arthritis, and Castleman's disease (p = 0.0368). However, no significant difference in FVC was found between the treated and control groups at 48 weeks , is an immunomodulator with antiangiogenic properties, and cytotoxic activity. Approved for the treatment of relapsed and refractory multiple myeloma . A 52 wein vitro, promotes normal repair and prevents lung fibrosis. The objective of the trial is to establish the safety and tolerability of bortezomib in SSc patients as well as exploratory effects on FVC. Participants receive MMF  and Bortezomib(1.3 mg/m2) subcutaneously once per week for the first 2 weeks vs. MMF plus placebo  for 24 weeks. The trial is planned for completion in June 2019.Bortezomib, is a FDA approved medication for the treatment of multiple myeloma. Bortezomib inhibits TGF- signaling Nintedanib and pirfenidone have anti-fibrotic effects and are approved for use in patients with idiopathic pulmonary fibrosis (IPF). In a case series of five patients with SSc-ILD, pirfenidone  was associated with a reduction in dyspnea and an increase in VC (10%) from baseline . LOTUSS,P = 0.04). Patients on a stable MMF dose did not elicit further improvement with add-on therapy with nintenadib.Nintedanib, is a tyrosine kinase inhibitor  for vascDiarrhea, the most common adverse event, was reported in 75.7% of the patients in the nintedanib group and in 31.6% of those in the placebo group . An exteLung transplantation should be considered in the early stage of respiratory failure for all patients with chronic lung disease. However, gastrointestinal comorbidities that are often seen in patients with SSc-ILD may complicate the transplant evaluation . A systeAutologous hematopoietic stem cell transplantation (AHSCT) has been proposed as a potential therapy for severe SSc disease . In a meAlthough there is no consensus statement that defines the criteria for SSc-ILD, HRCT, and PFTs serve as the primary diagnostic and staging parameters for establishing a diagnosis. Although MMF has been the initial treatment choice for SSc-ILD due to safer toxicity profiles and outcomes, more recent trials raise the option of antifibrotics or combination immunomodulatory/antifibrotic therapy as potential new treatments for patients with SSc-ILD. Lung transplant should be considered as an option, but the significant comorbidities associated with SSc including GI comorbidities should be addressed with medical and surgical evaluations prior to referring for transplant.Many questions remain unanswered. When should treatment be initiated for SSc-ILD? What treatment regimen is most efficacious? How long should the patient be treated with SSc-ILD? With the development of more sophisticated classification criteria and assessment of HRCT, availability of reliable and reproducible biomarkers and molecular profiling, answers for these questions will impact treatment strategies for patients with SSc-ILD.PB conducted literature review, conducted exploratory analysis, and helped to develop the first draft of the manuscript. MG helped in developing the first draft of the manuscript. MM conducted literature review, conducted exploratory analysis, and developed the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Surprisingly, avian taxa are an exception to this theory because correlations between life-history traits and dN/dS are apparently absent. Here we explore the role of GC-biased gene conversion on estimates of substitution rates as a potential driver of these unexpected observations.The nearly neutral theory of molecular evolution predicts that the efficacy of natural selection increases with the effective population size. This prediction has been verified by independent observations in diverse taxa, which show that life-history traits are strongly correlated with measures of the efficacy of selection, such as the dN/dS estimated from alignments of 47 avian genomes and several proxies for effective population size. To distinguish the impact of GC-biased gene conversion from selection, we use an approach that accounts for non-stationary base composition and estimate dN/dS separately for changes affected or unaffected by GC-biased gene conversion. This analysis shows that the impact of GC-biased gene conversion on substitution rates can explain the lack of correlations between life-history traits and dN/dS. Strong correlations between life-history traits and dN/dS are recovered after accounting for GC-biased gene conversion. The correlations are robust to variation in base composition and genomic location.We analyze the relationship between Our study shows that gene sequence evolution across a wide range of avian lineages meets the prediction of the nearly neutral theory, the efficacy of selection increases with effective population size. Moreover, our study illustrates that accounting for GC-biased gene conversion is important to correctly estimate the strength of selection.The online version of this article (10.1186/s13059-018-1613-z) contains supplementary material, which is available to authorized users. With his proposal of the neutral theory of molecular evolution in 1968, Kimura introduced a revolutionizing concept to evolutionary biology, which at that time was strongly influenced by the view that evolution is driven by positive Darwinian selection .s) of new mutations [Ne) [Ne and s together determine the level of genetic diversity [Ne and s determine the probability of fixation of new mutations, which ultimately affects the evolutionary rate of change [Ne is small, the effect of genetic drift is strong, and slightly deleterious mutations are more likely to reach fixation than if Ne is large [Ne will accumulate more slightly deleterious substitutions over time than species with large Ne. Here, comparison of the evolutionary rate at non-synonymous and synonymous sites allows assessment of the strength of natural selection acting on protein-coding genes, where low effectiveness of selection results in an elevated , non-synonymous to synonymous substitution rate ratio (dN/dS). Therefore, to test the validity of the nearly neutral theory, the relationship between Ne and dN/dS can be explored.After realizing the importance of nearly neutral (mainly slightly deleterious) mutations in molecular evolution, Tomoko Ohta extended Kimura’s theory to the nearly neutral theory of molecular evolution , 4. The utations . A measuons [Ne) . At the iversity , 8. Overf change , 8. Therf change . Specifiis large . ConsequNe in natural populations is a challenging task [Ne as proxies [Ne than species with a limited geographic range, such as island species [Ne than solitary species [Ne than outcrossers [Ne [Since measuring ing task –12, a co proxies –15. For  species ; social  species ; and matcrossers . It alsocrossers . Common sers [Ne .dN/dS, several studies have confirmed the prediction of the nearly neutral theory. For example, it has been observed that eusocial insects show higher dN/dS ratios than solitary species [dN/dS ratios in the chloroplasts of selfers than of outcrossing species of plants [dN/dS in mitochondrial and nuclear genes [By empirically testing for a relationship between social organization, mating system or life-history traits and  species . Anotherf plants . Moreovear genes –29. Simiar genes .dN/dS in birds [Kr/Kc) has been used as an alternative proxy for the efficacy of selection in avian lineages [Kr/Kc in birds. This observation questions whether dN/dS is an appropriate measure for the efficacy of selection in birds. However, the use of the Kr/Kc ratio as a measure of the efficacy of selection is not straightforward either given that radical and conservative mutations can be both affected by effectively neutral mutations and hence vary with Ne [dN/dS and life-history traits in birds from another angle. Specifically, they first investigated the hypotheses that life-history traits are poor proxies for Ne and that variation in life-history traits is narrow in birds. They further explored the possibility that a peculiar distribution of fitness effects of new mutations in birds could be responsible for low power to detect a correlation. However, they found evidence to reject all these hypotheses and could thus not explain the lack of a positive correlation. Botero-Castro et al. [dN/dS; after removing putative alignment errors, a positive correlation between dN/dS and longevity, but not between dN/dS and body mass, could be recovered. Moreover, they found stronger correlations when previously undetected GC-rich orthologs were included in the analysis. Taken together, observations from previous studies suggest that dN/dS might not be an appropriate measure for the efficacy of selection in birds, potentially because there is some force that biases estimates of dN/dS and thereby obscures the correlation between dN/dS and life-history traits in birds.However, not all observations are in accordance with the prediction of the nearly neutral theory. An intriguing example is the repeated failure to find a positive correlation between life-history traits and in birds , 30, 31.lineages , 31. Thio et al.  recentlyNe and recombination rate, which both vary along the genome and among species. gBGC can therefore interfere with natural selection and bias inferences of the strength and efficacy of selection based on divergence and/or diversity data [dN/dS depending on the relative impact of gBGC on synonymous and non-synonymous substitution rates.It is well documented that avian genomes are strongly impacted by GC-biased gene conversion (gBGC) –36. gBGCity data –47. The ity data , 48. If dN/dS and life-history traits in birds, and investigate if the impact of gBGC on dN/dS conceals the correlation between life-history traits and dN/dS in the avian clade. In order to distinguish the impact of gBGC on lineage-specific substitution rates from the impact of selection, we estimate dN/dS separately for different substitution categories, namely W-to-S and S-to-W changes, which are both affected by gBGC, and GC-conservative changes, i.e., S-to-S and W-to-W, which are not affected by gBGC. To do so, we adapt a recently implemented approach that explicitly allows for non-stationary base composition [dN/dS in birds. Moreover, it stresses the importance of accounting for gBGC to correctly estimate the strength of selection in comparative genomics studies, which seems particularly important when the impact of gBGC varies among lineages. Finally, we propose a new statistic, dN/dS based on GC-conservative changes, together with a program to perform estimates of the strength of selection after accounting for the impact of gBGC.Here, we revisit the relationship between position . This andN/dS based on publicly available coding sequence alignments of 7986 genes in 47 avian species and three life-history traits . To estimate non-synonymous and synonymous substitution rates, we applied methods implemented in the bio++ libraries [dN/dS [dN/dS was estimated separately for each of those categories. Briefly, for each of these categories, we used substitution mapping [ω = 1) [We explored the relationship between estimates of ibraries , 51. Firs [dN/dS . We furt mapping  to estim [ω = 1)   not only depends on the strength of gBGC but also on the relative contribution of S-to-W and W-to-S substitutions to the total number of substitutions. While the strength of gBGC is reflected in the equilibrium GC content (GC*) [dN than on dS, which will influence the net impact of gBGC on the dN/dS ratio [dN/dS based on all substitution categories together is on average decreased (compared to dN/dS based on GC-conservative changes). ΔGC synonymous and ΔGC non-synonymous showed a negative relationship with body mass , indicating that species with large Ne and thus higher strength of gBGC generally are further away from their equilibrium than species with small Ne.The overall impact of gBGC on total substitution rates (and consequently on nt (GC*) , 54, thedS ratio , 48 and dN/dS and life-history traits in birds [dN/dS and body mass among studies (including our study), which illustrates the consistent lack of a significant positive correlation if gBGC is not accounted for. Although all these studies, like us, used a substitution mapping approach to estimate branch-specific dN/dS, our study differs from previous work in three key aspects.Several previous studies have addressed the relationship between in birds , 30–32. dN/dS was separately estimated for S-to-W, W-to-S, and GC-conservative substitution categories. Comparison of the relationships between dN/dS and life-history traits among studies is therefore limited to estimates of dN/dS based on all substitutions together  and gene dataset, we re-analyzed two datasets with our revised approach, the cleaned alignments of Figuet et al.   were only marginally or non-significant (Table dN/dS based on GC-conservative changes only (which are not affected by gBGC), were strong and significant for all datasets, which suggests that gBGC has a major impact on the results regardless of the data set analyzed. In addition, as observed in the original studies, Paleognathae (ratites and tinamous) and Galloanserae (gamefowl and waterfowl) appeared to be outliers in the Figuet+HMMclean and the Botero-Castro et al. datasets  having lower overall recombination rate and lower GC content than microchromosomes . Hence, GC-rich genes are more often found on microchromosomes. Consistent with what has been described above, we observed lower dN/dS estimates for microchromosomes than for macrochromosomes  could impact estimates of ies tree . Specifiin birds , it is pnference , 34. As dN/dS and consequently the correlation between life-history traits and dN/dS in birds are alignment quality and/or missing genes [dN/dS and longevity. They also found that dN/dS was significantly correlated with body mass in the Neoaves clade, i.e., all birds except Paleognathae (ratites and tinamous) and Galloanserae (gamefowl and waterfowl). The authors suggested that the latter group of birds might be outliers, since for those birds current life-history traits might not properly reflect long-term Ne . HowevedN/dS based on GC-conservative changes were robust to variation in local GC content and chromosome location. We observed significant positive correlations for GC-rich and GC-poor genes, as well as for genes located in macrochromosomes or in microchromosomes. Estimates of dN/dS were lower for genes with higher GC content and for genes located in microchromosomes. This reduction appears to be unrelated to gBGC, as it is observed for all substitution categories including GC-conservative substitutions. We found that the reduction in dN/dS is a result of lower dN values at the same time as dS values are increased. Higher dS values for microchromosomes than for macrochromosomes have been reported before and were suggested to be a result of higher mutation rates on microchromosomes than on macrochromosomes [dN was reduced for GC-rich genes and genes located on microchromosomes. In birds, recombination rate is positively correlated with GC content and negatively with chromosome size [dN could be a result of a lower interference between selected sites (Hill-Robertson interference) due to higher recombination rates [dN/dS. Weber et al. [dS relative to dN [dN/dS ratio would be increased by sequence saturation in GC-rich regions relative to GC-poor regions. Given that we observe lower dN/dS in GC-rich regions than in GC-poor regions, we believe that sequence saturation should not be of concern for our conclusions.The correlations between life-history traits and omosomes . Still, ome size , 60. Thuon rates . Alternar et al.  investigve to dN . As a codN/dS in birds, a correlation that would be expected based on the prediction of the nearly neutral theory of molecular evolution. By estimating nucleotide substitution rates separately for different substitution categories, we observe strong positive correlations between three life-history traits  and dN/dS when the estimation of dN/dS is based on GC-conservative substitutions only. No significant correlations were observed when estimation of dN/dS was based on all substitutions together, which can be ascribed to the influence of gBGC on S-to-W or W-to-S substitutions. gBGC thus impacts the correlation between life-history traits and dN/dS, and after accounting for gBGC, we find that the efficacy of selection increases with proxies for Ne, as predicted by the nearly neutral theory. The impact of gBGC on dN/dS varies substantially among lineages, where it increases the dN/dS ratio in some lineages, but decreases it in others. Our analysis suggests that these contrasting effects are related to differences in the dynamics of base composition between non-synonymous and synonymous substitutions. Moreover, altogether our study clearly illustrates that gBGC interferes with natural selection and that accounting for gBGC is a crucial step to correctly infer measures of the efficacy of natural selection such as the dN/dS ratio in comparative genomic studies. In light of this observation, we suggest that conclusions of previous studies that ignored the impact of gBGC on molecular evolutionary rates might need a careful re-evaluation. We here provide a protocol to do this.We have explored the role of gBGC behind the apparent lack of a positive correlation between life-history traits and Haliaeetus albicilla), since the branch length between this species and another eagle species included in the data set , was too short to reliably estimate dN/dS ratios [We downloaded publicly available coding sequence alignments of 8253 orthologous genes from 48 avian genomes and their inferred phylogenetic tree from Jarvis et al. . We exclS ratios . We furtS ratios , 64, sinS ratios , 66. ThiS ratios , only coTwo additional sets of multiple sequence alignments were retrieved from Botero-Castro et al. . The firConcatenation of gene-sequence alignments into a single alignment caused problems with large computational memory for the subsequent estimation of substitution rates.dN and dS across all bins were computed to obtain genome-wide averages of dN/dS. Concatenated alignments were used if estimation of substitution rates was based on the species tree. This approach was used to increase the signal-to-noise ratio for individual estimates. We repeated the analyses for 20 bins grouped according to their GC content. To do so, we first calculated the average GC content per species for each gene. Then, we calculated the mean GC content per gene over all species and ranked the genes accordingly. In order to separate genes into two classes of GC-rich and GC-poor genes, we split the 20 bins into two groups, each containing 10 bins with the highest and lowest GC content, respectively. We further repeated the analyses by binning genes according to their genomic location; one bin of genes located in microchromosomes and one bin of genes located in macrochromosomes, respectively. To avoid analyzing genes located in intermediate size chromosomes, we included genes located in chromosomes smaller than 16 Mb  in the category of microchromosomes and genes located in chromosomes larger than 100 Mb  in the category of macrochromosomes. We excluded genes from chromosome 1 if their location in the zebra finch was chromosome 1A, representing a well-known chromosomal rearrangement, or unknown .dN and dS estimates across all bins were computed to obtain genome-wide averages of dN/dS.For the Figuet+HMM and Botero-Castro datasets, the same binning procedure as for the main analyses was performed. We randomly concatenated gene alignments into bins of roughly 400 genes each and estimated substitution rates for each bin. Subsequently, the average dN/dS for each bin by using the bio++ libraries [dN and dS, and then dN/dS [dS is the ratio of the number of synonymous substitutions performed by the model divided by the number of synonymous substitutions that would have been performed by a similar model, but set as neutral . So, the program computes the expectation of both numbers and returns the ratio as dS. In an analogous way, dN is computed for non-synonymous substitutions. Since it is possible to consider any kind of event, we also computed the ratios of the expected numbers of non-synonymous and synonymous substitutions restricted to specific substitution categories, such as S-to-S, S-to-W, W-to-S, and W-to-W. For example, dS for the W-to-S substitution category is the expected number of W-to-S synonymous substitutions, divided the number of such substitutions that would have been performed by a similar neutral model. To combine S-to-S and W-to-W substitution rates into one category, i.e., the GC-conservative category, we weighed the S-to-S and W-to-W substitution rates of each species according to the GC content at the most recent internal node of each particular tip of the tree. To reconstruct the most likely ancestral sequence at each node, we used the bppancestor program [We estimated ibraries , 51 and ibraries . As a fiibraries , T92X3 [ibraries , and L95ibraries . As a seen dN/dS , 52, 70. program .dN/dS with an additional approach, where the number of substitutions was estimated separately for each gene (instead of concatenated bins) using gene-specific phylogenetic trees (based on the first and second codon positions) from Jarvis et al. [dN, dS, and dN/dS, the sum of number of substitutions for each substitution category over all genes was computed and normalized by the sum of all the expected substitutions for the same category.To account for gene tree heterogeneity, we estimated s et al. . In ordeCurrent and ancestral GC content were estimated for 0- and 4-fold degenerate sites as proxies for non-synonymous and synonymous GC content, respectively. Current GC content was defined as the sum of G and C nucleotides in the respective lineage. As described above, ancestral GC content refers to the GC content at the most recent internal node of a particular tip of the tree. In addition, we computed equilibrium GC content for non-synonymous and synonymous changes (GC*) as W-to-S/(W-to-S + S-to-W) substitution rates separately for each lineage. Lineage-specific ΔGC was estimated as the difference between lineage-specific GC* and the lineage-specific ancestral GC content.. Briefly, the authors retrieved body mass estimates from the CRC Handbook of Avian Body Masses [Estimates of body mass (grams), maximum longevity (years), and age of sexual maturity (days) were retrieved from Figuet et al. . Brieflyy Masses , while ly Masses ). All esAll statistical analyses were performed in R v. 3.2.2 (R core team 2015). Pearson’s correlation coefficient was used to test for correlations among variables. To account for phylogenetic relationships of species in correlation analysis, we used the ape package in R in order to compute phylogenetically independent contrasts following the method described by Felsenstein .Additional file 1:Table S1. Species specific dN/dS estimates for different substitution categories. (XLSB 56 kb)Additional file 2:Supplementary Tables S2-S6, and Supplementary Figures S1-S8. (DOCX 608 kb)


Dietary protein is effective for body-weight management, in that it promotes satiety, energy expenditure, and changes body-composition in favor of fat-free body mass. With respect to body-weight management, the effects of diets varying in protein differ according to energy balance. During energy restriction, sustaining protein intake at the level of requirement appears to be sufficient to aid body weight loss and fat loss. An additional increase of protein intake does not induce a larger loss of body weight, but can be effective to maintain a larger amount of fat-free mass. Protein induced satiety is likely a combined expression with direct and indirect effects of elevated plasma amino acid and anorexigenic hormone concentrations, increased diet-induced thermogenesis, and ketogenic state, all feed-back on the central nervous system. The decline in energy expenditure and sleeping metabolic rate as a result of body weight loss is less on a high-protein than on a medium-protein diet. In addition, higher rates of energy expenditure have been observed as acute responses to energy-balanced high-protein diets. In energy balance, high protein diets may be beneficial to prevent the development of a positive energy balance, whereas low-protein diets may facilitate this. High protein-low carbohydrate diets may be favorable for the control of intrahepatic triglyceride IHTG in healthy humans, likely as a result of combined effects involving changes in protein and carbohydrate intake. Body weight loss and subsequent weight maintenance usually shows favorable effects in relation to insulin sensitivity, although some risks may be present. Promotion of insulin sensitivity beyond its effect on body-weight loss and subsequent body-weight maintenance seems unlikely. In conclusion, higher-protein diets may reduce overweight and obesity, yet whether high-protein diets, beyond their effect on body-weight management, contribute to prevention of increases in non-alcoholic fatty liver disease NAFLD, type 2 diabetes and cardiovascular diseases is inconclusive. The prevalence of obesity and its associated co-morbidities, such as non-alcoholic fatty liver disease (NAFLD), type 2 diabetes and cardiovascular diseases, has increased in a growing number of countries , 2. Enerad libitum high-protein diet relative to baseline  , 60. MecDe novo synthesis of glucose in the liver from gluconeogenic precursors including amino acids is stimulated by a high protein diet in the fed state . To counteract poor compliance, dietary restraint is necessary . In seve as well . In gene as well , 131.Taken together, Clifton et al.  concludeOlder studies, in the perspective of composing meal replacers to be used as energy restricted diets showed strong energy restriction effects on body composition, in relation to the percentage from dietary protein. The protein content of a formula diet was varied from 0 to 50 g/d resulting in a protein loss of between 1202 and 91 grams, respectively, over 28 days . Loss ofIf the protein-induced effects on appetite and energy expenditure observed during energy restriction also hold under non-restricted conditions, then, increasing protein intake with a usual diet may prevent overweight and obesity. A 12-week intervention study was performed comparing high-protein (30% of energy from protein) and low-protein (5% of energy from protein) diets, in weight stable individuals . In thisHumans with overweight or obesity may show co-morbidities, such as a nonalcoholic fatty liver disease, type 2 diabetes, or cardiovascular diseases. Whether a high-protein diet may be protective against these co-morbidities, independent of, or in addition to effects of weight loss is still under debate.de novo lipogenesis and stimulates hepatic ketogenesis  seems to be a marker of type 2 diabetes , 157. NeTaken together, when protein diets are applied during energy restriction aiming at weight loss and subsequent weight maintenance, the latter usually shows favorable effects in relation to insulin sensitivity, although some risks may be present. That a higher protein diet would promote insulin sensitivity beyond its effect on body-weight loss and subsequent body-weight maintenance seems unlikely.Parameters that indicate cardiovascular risks usually change in a favorable direction during body weight loss. The question remains whether the type of diet, especially a protein diet, would affect favorable changes in cardiovascular parameters. Atherosclerosis lies at the root of cardiovascular complications, and the main indicators are the HDL- and LDL cholesterol. Certain proteins may exert a greater effect on blood cholesterol levels than other . PossiblThere is a long-held view that high-protein intake might interfere with calcium homeostasis by increasing the acid load. It is hypothesized that this could be partially buffered by bone, subsequently resulting in bone resorption and hypercalciuria . In geneWith respect to body-weight management, the effects of diets varying in protein differ according to energy balance. During energy restriction, sustaining protein intake at the level of requirement appears to be sufficient to aid body weight loss and fat loss Figure . An addiHigh protein-low carbohydrate diets may be favorable for the control of IHTG in healthy humans, likely as a result of combined effects involving changes in protein and carbohydrate intake. When protein diets are applied during energy restriction aiming at weight loss and subsequent weight maintenance, the latter usually shows favorable effects in relation to insulin sensitivity, although some risks may be present. That a higher protein diet would promote insulin sensitivity beyond its effect on body-weight loss and subsequent body-weight maintenance seems unlikely.At least high-protein diets do not seem to have adverse effects on these co-morbidities. In conclusion, higher-protein diets may reduce overweight and obesity, yet whether high-protein diets, beyond their effect on body-weight management, contribute to prevention of increases in NAFLD, type 2 diabetes and cardiovascular diseases is inconclusive Figure .The sections of the manuscript were written by MD, LT, BG-C, TA, and MW-P. The review is partly an update of Westerterp-Plantenga et al.  and WestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diagnosis of cancer as an emergency is associated with poor outcomes but has a complex aetiology. Examining determinants and time trends in diagnostic routes can help to appreciate the critical role of general practice over time in diagnostic pathways for patients with cancer.To examine sociodemographic, cancer site, and temporal associations with type of presentation among patients with cancer diagnosed as emergencies.Analysis of Routes to Diagnosis data, 2006–2015, for patients with cancer in England.The authors estimated adjusted proportions of emergency presentation after emergency GP referral (GP-EP) or presentation to accident and emergency (AE-EP), by patient sex, age, deprivation group, and year of diagnosis using multivariable regression.n = 130 372) presented as GP-EP, 62% as AE-EP (n = 346 192), and 14% (n = 78 057) through Other-EP sub-routes. Patients presenting as emergencies were more likely to have been GP-referred if they lived in less deprived areas or were subsequently diagnosed with pancreatic, gallbladder, or ovarian cancer, or acute leukaemia. During the study period the proportion and number of GP-EPs nearly halved , while that of AE-EP increased .Among 554 621 patients presenting as emergencies, 24%  are over-represented among cases whose emergency presentation involved GP referral. Reductions in diagnoses of cancer through an emergency presentation likely reflect both the continually increasing use of 2-week-wait GP referrals during the study period and reductions in emergency GP referrals. First, about two-thirds of all patients with cancer who are diagnosed as emergencies would have had prior GP consultations with relevant symptoms, often leading to investigations or referrals.9,12,9,10,8In England, the frequency of emergency presentations  is routinely monitored through the ‘Routes to Diagnosis’ programme of the National Cancer Registration and Analysis Service (NCRAS) of Public Health England.2This study aims to characterise sociodemographic, cancer site, and temporal associations with emergency presentation sub-routes among patients with cancer diagnosed as emergencies. The objective was to examine patient groups that are either over- or under-represented in emergency presentations directly involving a GP emergency referral to hospital, and establish related temporal trends and their likely contribution to overall changes in how patients with cancer are being diagnosed.The authors studied Routes to Diagnosis data for 2006–2015 on patients aged ≥25 years diagnosed with any of 35 common and rarer cancers, responsible for 95% of incident cases. The ‘diagnostic route’ of each registered tumour is assigned by NCRAS using a rules-based  approach, which incorporates information from linked Hospital Episode Statistics, Cancer Waiting Times, and NHS Cancer Screening Programme data.Exposure variables included sex, age , social deprivation , cancer (35 different sites as defined by ICD-10 codes), and year of diagnosis.n = 554 621) diagnosed via AE-EP, GP-EP, and Other-EP by sex, age group, deprivation status, cancer site, and year of diagnosis. Adjusted proportions were predicted from a multivariable multinomial logistic regression model where the outcome was AE-EP and Other-EP (baseline category: GP-EP) and all of the exposure variables were included as independent variables; reference categories: male, aged 70–79 years, least deprived, colon cancer, 2006.The analysis had two objectives. The first is to describe associations between exposure variables and each emergency presentation sub-route. The researchers reported the number and proportions (both crude and adjusted) of emergency presenters  referral, non-emergency GP referral, screening, and ‘Other’ (baseline category: GP-EP), and including the same independent variables as in the first multinomial logistic regression model.The second objective was to describe temporal trends in each emergency presentation sub-route, also taking into account time trends across all other diagnostic routes.n = 130 372) had presented through GP-EP, 62% (n = 346 192) had presented through AE-EP, and 14% (n = 78 057) through the Other-EP sub-route. Absolute numbers and crude and adjusted proportions of emergency presentations by GP-EP, AE-EP, and Other EP sub-routes, by sociodemographic variables, cancer site, and year of diagnosis are available from the authors on request.Among the 554 621 patients presenting as emergencies during 2006–2015 , 24% (n n = 17 157) versus 21% (n = 7509) for ages 25–49 years (adjusted proportions for age groups for Other-EP are available from the authors on request). The likelihood of GP-EP decreased with increasing levels of deprivation, for example, adjusted proportions for least versus most deprived were 25% (n = 22 997) versus 19% (n = 22 775), while, in contrast, that of AE-EP increased  . There was little variation in the likelihood of Other-EP by levels of deprivation . Given the large sample size, there was evidence (P<0.001) for variation in emergency presentation sub-route by variable category for all above variables .There was limited variation in emergency presentation sub-route by sex. Patients who were older and presented as emergencies were more likely to be diagnosed both via GP-EP and via AE-EP compared with younger patients, reflecting that patients who were younger and presented as emergencies were more likely to be diagnosed via the Other-EP sub-route; adjusted proportion for those aged ≥80 years was 11% (P<0.0001) for variation in emergency presentation sub-route by cancer site.Patients presenting as emergencies with pancreatic, acute myeloid and acute lymphoblastic leukaemia (ALL), ovarian, and gallbladder cancers were more likely to be diagnosed via the GP-EP sub-route (adjusted proportions of GP-EP ≥27%) . In contP<0.0001) for variation in emergency presentation sub-route by year of diagnosis.The number of incident cases for the studied cancers, adjusted for changes over time in sociodemographic characteristics and cancer site distributions, increased each year, for example, from 237 799 cases in 2006 to 284 660 cases in 2015, an increase of 20%, while the number of emergency presentations each year decreased slightly , while the opposite was true for AE-EP . Relatedly, against a slight overall decrease in the absolute numbers of emergency presentations of any type between 2006 and 2015 (n = 17 364 to n = 9155) and that of AE-EPs slightly increased (n = 31 049 to n = 36 868). Absolute numbers and crude adjusted proportions of emergency presentations by GP-EP, AE-EP, and Other EP sub-routes for all years of diagnosis are available from the authors on request.The changing proportion of emergency presentations overall was accompanied by a changing composition of EP sub-routes . GP-EP wand 2015 , the absOver a recent decade, and against an overall continuous decrease in the percentage of patients diagnosed with cancer through an emergency presentation (from 24% in 2006 to 20% in 2015), the likelihood of emergency presentation via GP referral decreased, coupled with an increased likelihood of emergency presentation via A&E, even after accounting for sociodemographic and cancer site case mix over time. Among patients presenting as emergencies, patients living in areas of greater deprivation were less likely to be diagnosed via the GP-EP sub-route, and more likely to be diagnosed via the AE-EP sub-route. There was large heterogeneity in sub-route profile by cancer site.The authors used a large population-based dataset covering a 10-year period and presented adjusted proportions of emergency presentation sub-routes by each patient characteristic, cancer site, and year of diagnosis, respectively. All variables included in this study come from high-quality cancer registration data. Diagnostic route, used to capture emergency presentation status and sub-route type, is derived via an algorithmic approach using linked routine population-based datasets.2–17As common in observational studies, other variables  could at least partially account for some of the reported sociodemographic, cancer site, and temporal variation in emergency presentation sub-routes. It could be revealing to examine the potential interplay between stage at diagnosis and emergency presentation, as advanced stage is associated with greater likelihood of emergency presentation,15,10A limitation inherent to all research using routine data is that it does not allow for exact circumstances to be taken into account. For example, a patient presenting for emergency may have attended A&E following verbal GP advice without a formal referral. This hypothetical patient would have likely been assigned an AE-EP sub-route, while GP-EP sub-route may have been more apt, which would introduce misclassification error. The magnitude of such a misclassification is difficult to quantify but is likely to be small.9,There are no other population-based studies of emergency presentation sub-routes for cancer cases in England covering both common and rarer cancers with which to compare the present results.12,21The findings overall indicate that certain cancers that can be associated with painless symptoms that may be tolerable to the patient but where GPs may appropriately request an urgent specialist assessment — for example, pancreatic or gallbladder cancer presenting with painless jaundice; acute leukaemia presenting with pallor; ovarian cancer presenting with abdominal distension — were associated with the highest likelihoods of GP-EP in this study. These findings underline the importance of tumour factors as a contributor to emergency presentations in some patients. As these symptoms have relatively high predictive values for cancer,20As more deprived patients presenting as emergencies were less likely to have been referred by their GP and more likely to have presented to A&E, help-seeking patterns among otherwise similar patients subsequently diagnosed with the same cancer seem to vary by socioeconomic status.–26,29,Against an overall decreasing proportion of patients with cancer who are diagnosed through emergency presentations, the number of patients diagnosed through the GP-EP sub-route is decreasing, while that of patients diagnosed through AE-EP and non-emergency routes is increasing. This decreasing trend in emergency presentations overall has occurred in spite of other evidence for opposite, (increasing) trends in both general A&E attendance and emergency hospital admissions.22