For Europe's urban agglomerations to be economically competitive, it is vital that international destinations be easily accessible. Although much has been invested in the construction of European rail infrastructure over the past century, passenger transport by rail has not grown as fast as transport by road and air. So why do people not use international trains more, even though they have an extensive international rail network at their disposal? Based on a series of in-depth interviews with relevant public and private stakeholders and two expert meetings, we identify the main bottlenecks and constraints. In order to understand the complexity of international rail transport, we have divided the existing bottlenecks into four groups corresponding to four layers of the rail transport system: mobility services, transport services, traffic services, and the physical and digital infrastructure. We formulate concrete policy recommendations for improvements to be made in the various components of the rail transport system. In recent decades, the volume of long-distance travel between Europe's metropolitan agglomerations has increased dramatically, especially by road and air , 37. TheInternational rail services so far account for only a modest share of all trips made , which iReplacing road and air transport with rail transport still proves difficult in practice because the "intermodal competitiveness" of rail is weak. Choosing to travel by air  rather In this context we formulated two research questions aimed at what is needed to increase the market share of international passenger rail transport. Our first question focuses on what is currently known about international traveler mode choice. This was based on literature study. To improve the position of the international railsector in serving that market, the second question focuses on the obstacles to grow their market share, given the answer to the first question, and what can be done to overcome them. This was based on structured expert consultation. These are the main research questions we aim to answer in this paper.We concentrate on passenger rail transport, excluding freight. Freight transport, although also relevant, is only discussed in sofar as it affects passenger transport. When studying the bottlenecks, we also start from the perspective of the international rail passenger, and less from the perspective of the railway industry . We limit our geographical scope  to the main connections within the international railway networks of Belgium, the Netherlands, France, the United Kingdom, Germany and Austria. We have focused on the main cross-border connections, high-speed rail connections and international night trains. International rail services that are largely of regional interest have not been included.The paper is structured as follows. In Sect. A thorough search of Scopus and Web of Science on international travel mode choice shows very quickly that there has been far less research into the mode choice behaviour of travellers who decide to make long-distance journeys by rail compared to studies that focus on identical journeys by car, plane or coach/bus. The reasons are quite simple: long-distance rail travel—defined here as trips of 100 km or more —is less For long-distance travel—based on work by Limtanakool et al.  for the In addition to travel time, travel costs are an important factor in deciding which mode of transport to use. These costs consist of several components (which differ according to the mode), such as fuel, parking and maintenance costs (for car travel), the ticket price and the costs for travelling to and from the station or airport (for rail or air travel). The higher the transport costs, the less likely this mode of transport will be chosen. Cheaper wins from more expensive. As with travel time, the different cost components are perceived differently by the traveller , but little is known about these cost perceptions for long-distance travel. An important consideration is whether or not the traveller takes all costs  into account when deciding on the transport mode to use. Not infrequently the cost of petrol is compared one-to-one with the cost of a public transport ticket, not taking into account parking and maintenance costs of the car.The comfort level of a means of transport is a third component. It refers to the subjective  assessment of the journey. Comfort has to do with convenience, safety and even luxury while "on the road", but also with everything that happens before and after the journey. To illustrate: if it is easier to book online a flight from Amsterdam to South Bolivia—consisting of three to four flights operated by two or three airlines—than a train ticket from Amsterdam to Stockholm, this does not contribute to a high level of comfort. Studies that do take the comfort factor into account show that it is an important factor in the choice of transport mode and that, for example, the higher comfort of an overnight train weighs heavily in its favour compared to car, bus and air travel (e.g. ). HigherIn short, the modal choice behaviour of travellers is not black and white; all the factors discussed above play a role. If a certain mode of transport has a shorter journey time than another, this does not automatically mean that all travellers will base their decision solely on this factor. Empirical data , 18 showIn order to promote international rail passenger transport, it is important to emphasize competitive journey times and ticket prices, together with the higher level of comfort in comparison with driving or flying as a distinguishing factor. Where international rail transport is not competitive with other modes, measures need to be taken to improve journey times, fares or comfort. To understand how and where these changes can be made, we present a layered model of the rail transport system in the next section.The world of international rail transport is extremely complex. Not only are there many countries involved, but within each country the operation of international rail services depends on close cooperation between public and private parties. But what exactly is the international rail transport system? To help us understand the rail mobility system, we use the four-layer model . Each laMobility services are the first layer. These consist of a whole range of services with the traveller at the centre to plan, purchase and make their journeys. They include the more or less traditional services such as traffic information and route planners, but also new, often digital services such as real-time route information and Mobility as a Service (MaaS) concepts that are built around the journey from A to B rather than a specific mode of transport. It is about travel. The second layer of the rail transport system is formed by the transport services with the operator at the centre. These concern the operators of the rail transport system (such as railway companies) and the rolling stock and transport services they use to transport their passengers. It is about running trains. The third layer consists of traffic services with the network. This is the set of measures and instruments for optimal and safe use of capacity on the railway network. They include the allocation of train paths to the various operators and traffic management on the available infrastructure, as well as the various systems to promote traffic flows and safety, such as the European Rail Traffic Management System (ERTMS). It is about use of rail capacity. The fourth layer is the physical and digital infrastructure. This is the basis of the rail transport system: the system of tracks, stations, shunting yards and digital hardware on which and along which journeys are made. It is about providing capacity.The layer model reveals the dependencies between the layers. The lower layers (here layer 4) facilitate what happens in the layers above them. In other words, the layer above makes demands on the delivery of the specific functions and services in the layer below. Without tracks no transport services; without digital infrastructure no train protection system. So without traffic services no transport services, and without transport services no mobility services.It can be said that each of the four layers of the rail transport system (and its subsystems) is organised in its own way, either between organisations  or within one organisation  . A majorTo get a full picture of international rail passenger transport, document analyses and a series of consultations and interviews with relevant stakeholders were organized between October 2019 and June 2020. The information collection process is outlined in Table The information from these consultations was used as input for two expert meetings, attended by 22 experts  (both held on 22 January 2020). The expert meeting served to validate the findings from the consultations. The following stakeholders were present and represent the main international rail transport corridors from Belgium, the Netherlands, France, the United Kingdom, Germany and Austria  that emerged from the consultations and expert meetings of representatives of traveler organisations and international travel sectors. We give an overview of the bottlenecks from the passenger's point of view (5.1), and of the bottlenecks related to the four-layer model (5.2).The main goal of the European policy to improve international rail services is to optimise the use of the existing railway infrastructure. This is reflected in the two pillars of the European policy: open access and technical harmonisation. The aim of the railway packages is to contribute to the improvement of the quality, competitiveness and efficiency of the European railway sector, the idea being that this will serve the interests of the international rail passenger. However, the assumption that this policy would lead to a large increase in international rail passenger transport has not materialised. In fact, the relative share of rail in the European transport market has actually declined in recent years. This is understandable from the perspective of the international rail traveller, as they still face numerous bottlenecks. It is therefore only logical that travellers do not currently regard the train as a realistic alternative to the car or plane. International travellers are independent and make their own decisions about how to travel; governments can influence that choice by responding to travellers' needs—but have not yet done enough in that respect.The problem is clearly not limited to the lack of infrastructure alone. International travellers have an extensive European rail network at their disposal. For example, passengers from the Netherlands have direct access to the railway network in Belgium, France and the United Kingdom via the high-speed HSL trains, and to the German railway network via the high-speed ICE trains to Frankfurt and the Intercity to Berlin. These connections give the traveller access to the whole of Europe, albeit with one or more changes. Yet the potential of the train for travellers in Europe is not fully exploited due to at least four major bottlenecks:Travel time is an important factor when choosing a mode of transport. The train is more attractive if the travel time is competitive with alternative options. The greatest time savings are achieved when trains run on dedicated tracks, allowing for limited stops and high cruising speed. Many European countries already have such high-speed lines, but on large parts of the infrastructure trains cannot yet run at their maximum cruising speed, either through line speed limitations or additional stops. Moreover, the rail networks have been planned and built to serve national interests, making choices that make perfect sense from a national point of view , but resulting in longer travel times for international travellers. A good illustration can be found in the slower operating speeds for high speed trains in the intensively used rail networks of Ile de France, London, Flanders, and the Ruhr area. This means that the travel time savings to international travellers that the European high-speed network provide are curtailed, despite the billions that were invested (including from EU funds).The second barrier experienced by the traveller is the price of the train ticket. Many people consider the price of a train ticket to be high compared to the price of air travel. However, it is by no means certain that this is the case (leaving aside the dumping practices in the low-cost airline sector as a result of COVID-19). It is difficult to accurately compare the cost of train and plane tickets because the prices of the tickets on offer vary so much, but a Consumer Association sample of ticket prices  shows thHaving to change trains during a journey is one of the biggest sources of stress or discomfort for travellers . Changinhttp://www.railteam.eu) do provide fairly robust passenger rights for many international journeys with regard to missed connections, but switching from one operator to another can be risky: unlike airlines, operators of rail services have rather limited agreements on taking over each other's passengers in case of delays or cancellations and if it is there, the process to the traveller is far less supported than when missing a flight.Another reason why international travellers are reluctant to choose train journeys with one or more changes is that they feel that passenger rights are inadequate when changes are involved. For example, if your train is delayed and you miss a connection, can you take the next train without having booked a seat? And if there is a delay on part of the journey, can you claim compensation for the whole journey? Notably, services like RailTeam journey planner  leave much to be desired. There seems to be a clear lack of passenger-friendly access to the system, information and tickets. Travel information (compared to other modes) is poor, booking and rebooking procedures are complicated or impossible, and timetables are unreliable.The main bottleneck seems to be the booking system. Booking international train journeys is difficult because there is no easy and transparent system for finding and booking international train tickets. Obtaining tickets for main destinations and direct services is reasonably easy, but buying tickets for indirect services or more distant destinations is considerably more complicated. The ticket information landscape is fragmented and technically complex, and the cost–benefit ratio of developing a passenger-friendly platform has so far proved unattractive to potential providers. There are hardly any independent travel information and ticketing providers in Europe. People who want to plan and book a journey get caught up in a confusing maze of rules and information. Railway undertakings generally only offer tickets for their own trains; tickets for other companies' trains, even for connecting trains, are only offered in limited cases (e.g. for services of partner companies). Moreover, each railway undertaking has its own sales channels and digital systems, which means that some tickets can only be purchased online, by phone or at a ticket office.Finding and buying airline tickets is now much easier, as booking one or more flights is made easy by platforms such as skyscanner, cheaptickets, gotogate, and google flights. When choosing a trip, people are influenced by how easy it is to find and book a trip. If finding a ticket proves difficult, they are likely to choose another travel option for which they can easily obtain a ticket. Airlines work together internationally in broad alliances; railway companies take a different approach. For example, NS  is not allowed to sell the cheap tickets of Ouigo, a subsidiary of SNCF . NS International sells tickets to London and Kent, but not to other destinations in the UK. The national carriers generally do not sell tickets for services of new entrants to the railway market and vice versa. As a result, international train journeys often have to be split up into a series of shorter journeys. This is not only inconvenient to book but also affects passengers' rights in the event of delays or missed connections.There is no uniform European reservation system for train journeys. In Europe, there are two main systems for reserving trains: In one system, the traveller buys a ticket for a certain route on a certain day, whereby different trains can be used (this flexible system is often referred to as the "German system"). This is advantageous for travellers who miss a connection. In the other system, the traveller buys a ticket for a specific train on a specific day . In this system, the journey must be made at a specific time and seat reservation is mandatory. In practice, it is difficult to combine these two reservation systems. When passengers change from a French to a German reservation, they do not know for sure whether there will be a seat available for them on the connecting train, and if so, in which coach they can find it. And when changing from a German reservation to a French reservation, passengers who miss their connection are not allowed to board the next train (because they did not reserve a seat). None of the providers of tickets for journeys within their own operating area are confronted with this structural defect in the system. The passengers are left to sort it out themselves. There is no direct incentive for the providers of tickets to come up with an overarching system. However, a uniform, public-friendly booking system for international trains would encourage more people to travel by train. As such, this situation represents an indirect, but so far unacknowledged, incentive for greater coordination and cooperation. Greater uniformity of data and data systems across Europe would make access to ticket information and ticketing much easier. It could lead to a system similar to that of the airlines, working on the basis of alliances and code-sharing. However, at the moment it is not in the interest of individual ticket providers to take the initiative in creating such a unified system: the necessary investments are too large and the prospects for recovering the benefits and for third-party entry into the system are too uncertain. Therefore, an umbrella system can only be created by all parties working together.With regard to transport services (layer 2), but also traffic services (layer 3) and infrastructure (layer 4), there seems to be a strong dominance of national interests through the dominance of national operators and network managers focussing largely on the domestic market. Although some of the first railway lines in Europe were built for cross-border transport, over the past century the rail passenger transport sector has provided value mostly in dense areas and concentrated on transport within national borders. As a result, the interests of international passengers have been under-represented in decision-making.Over the last decades the international rail services like dwindled. And despite recent examples of a shift, like the ÖBB starting the Nightjet, most players influencing the rail transport system are still nationally oriented. In carrying out their responsibilities, they are nationally oriented: they strive to optimise the national rail transport system without paying much attention to the effects at the international level. COVID-19 also illustrated this. Strong local support for the national railway, less for international connections. They largely neglect the international dimension of the rail transport system because they are 'rewarded' primarily for their domestic performance. For these players, the benefits of international transport performance are mostly intangible and largely irrelevant to performance reviews. The national governments of the Member States underestimate the economic and other interests of metropolitan agglomerations in their decisions on international transport services. In addition, there is a worrying lack of decisive international coordination in striking a balance between the needs of national and international rail transport. This affects the quality and thus the competitiveness of international rail passenger transport.In addition, the capacity constraints in traffic services (layer 3) are due to a) the different technical, train protection and control systems and the lack of a common language, and b) the problematic distribution of capacity on certain sections between national and international services and between passenger and freight transport. The technical differences between rail systems in Europe have been a major bottleneck for a long time. They concern track gauge, platform height, the electrical power supply and the train control and safety systems. These differences force travellers to transfer or limit operational speed. Technically, the differences between the systems can all be overcome. However, it makes things much more complex for the rolling stock. The consequences are higher costs for the whole system, longer lead times for rolling stock and greater complexity (with the risks involved in obtaining certification).In relation to infrastructure (layer 4), speed limits are a major bottleneck due to the limited capacity and quality of existing infrastructure and stations. Because the development of the railway infrastructure in the European countries has been concentrated for years on the domestic networks, a fully interconnected international high-speed network has not been established at that spatial scale. This has led to insufficient attention being paid to the development of international high-speed corridors. In the past twenty years, the European Commission has produced four 'railway packages' of legislation for more competition and uniform technical solutions to promote the harmonisation of the railway transport system. However, these European policies have not always led to the desired improvements, either in the market pillar (aimed at open access to rail corridors) or in the technical pillar (aimed at interoperability across the European rail network). Solutions that work well for services to Germany do not work, or work differently, for services to Belgium and France. This can largely be explained by the differences in technical and other systems between the countries, but the considerable differences in culture and procedures also play a role. The railways have a long history, infrastructure managers are considered rather inflexible and operators are not particularly willing to cooperate with each other.From travel demand research it seems that travellers are positive about international train travel. However, barriers mentioned here hamper the introduction of better services on the main links—the corridors—between Europe's major conurbations, to make good on that promise. Passengers will therefore benefit from a policy focus on making improvements to the core network . At present, however, policy attention is not focused on these corridors but is spread over the entire network. This situation is at odds with the priorities of the European Commission, which recently set itself the target of completing the core (trans-European transport network) network by 2030 and then the comprehensive network by 2050 .With short-haul flights gradually losing public acceptance and increasing road congestion, there is an excellent opportunity to fundamentally rethink the whole issue of international rail travel. Whether COVID-19 will be a game changer here remains to be seen. Van Wee and Witlox , in theiA corridor coordinator is needed to ensure that all parties work together to create better connections between international transport links and train paths . Corridor coordination will eventually lead to the demand for a corridor authority. This transport authority within a corridor will ensure that all stakeholders know what level of service is required on each specific section of the corridor . In the long run, the need will arise to merge the corridor authorities into a single European rail traffic management and capacity management system for cross-border services.The market for international trains has proved difficult to develop. If the policy of open access does not lead to the introduction of the desired international transport services, the corridor coordinator should be able to tender new supranational public service obligations for major routes. Ultimately, this could be carried out by the new corridor authority. An even better service could be achieved if the corridor coordinator were later given the task of increasing the operating speed of the services by solving technical problems and removing infrastructure bottlenecks. European funds can be called upon to provide all or part of the necessary financing. The experience of the Rhine Commission  could beThe European rail transport policy must ensure that the rail market is opened up to different transport services and that trains run faster. The train passenger should benefit from this. But if the desired train connection cannot be found or booked, much of the benefit is lost. Much remains to be done in this regard. Specifically, travel and passenger information must become more accessible and international journeys much easier to book. International train travellers want a user-friendly booking procedure that brings together the services and prices of all train operators on a route (former public operators and new market entrants) in one convenient format. As travellers do not travel from station to station but from door to door, they want integrated travel advice and ticketing (cf. MaaS) . There aAll operators should allow third parties (not just former public operators) to sell tickets. The aim should be to standardise the format of ticket information and conditions, along the lines of the airline situation. This will make it easier, more attractive and economically viable for all train companies, online ticketing platforms and travel agencies to sell third-party tickets. None of the individual national companies has sufficient financial or other interest in dismantling the status quo, so a cooperative approach is needed. International train tickets are usually only available from three months before the date of travel. This excludes the train for travellers who want to book their journey much further in advance. Operators should be encouraged to make international train tickets available earlier than the current three months before the date of travel. A period of at least nine months should be feasible. When tendering for and awarding concessions and public service obligations, the authorities should raise this with the operators and hold them accountable to the extent possible.Like airlines, rail operators must make agreements among themselves to take over each other's passengers in the event of delays or cancellation of flights (through ticketing). The European Commission can make binding agreements on this in the Rail Passenger Rights Regulation, which is currently being recast. Unfortunately, our interviews showed that some Member States (under the influence of their railway companies) are watering down the agreements, limiting a stronger traveller focus.Part of the railway network capacity that could be utilised by international services is currently unfilled, mostly situated outside of Europe’s densely populated areas. The EU's open access policy is intended to stimulate market players to use this space. However, this has led to the emergence of only a few new operators, most of whom also operate in regional markets and are in fact subsidiaries of the national operators . Successful new entrants to the rail market operate in Italy, Austria and the Czech Republic: NTV/Italo, Westbahn and Regiojet. These and other new operators, united in ALLRAIL (Alliance of Rail New Entrants), point out that the national operators, as former state-owned companies, enjoy much greater market protection. National governments—in defiance of EU policy—tend to protect their national operators from competition. Stimulating new services on a European level, beyond open access is required.Many international travellers still do not see the train as an attractive form of transport. The opening of the market has apparently not given train operators enough incentive to meet the needs of international travellers. A question that arises is what can be done to make train travel more attractive to passengers. Of course, the operators have an important role to play in marketing their products. As the greening of transport is an important government objective, it is conceivable that the national government could  finance an information campaign to bring international train travel to the attention of the public. In addition, the government could use flanking policy to bring about a shift in passenger numbers to more sustainable modes of transport. An example is the French government that makes its support of Air France dependent on a ban on domestic short-haul flights. Companies and governments can boost the rail market by banning such short flights within Europe for their staff. They could also consider ending the tax deduction for business trips by short-haul flights.Travellers base their choice of transport partly on the perceived price. As discussed above, train journeys are by no means always more expensive than flying. Nevertheless, attractive pricing might encourage travellers to take the train. A reduction of the VAT rate on train tickets on a European level, which would also contribute to the objectives of the European Green Deal. This also applies to the oft-repeated suggestion of reducing rail access charges levied by infrastructure managers for international long-distance trains. For long-distance connections in Europe, a degressive tariff could be applied: the longer the distance, the lower the charge per distance unit.The management of railway capacity is based on a decades-old system that has been continuously adapted and expanded in a series of incremental improvements. This system does not make it easy to free up more space for international trains. More intelligent use can be made of existing capacity. Such a scheme seems reasonable to plan and fill capacity, but it hinders the flexibility needed to increase the frequency of international services on all international routes. By way of illustration, Eurostar argues that it should be possible in the short term to gain at least 16 min on the journey from Amsterdam to London, and that the five infrastructure managers on the Amsterdam-London route are all seeking to optimise the use of capacity within their own areas. This time saving can be achieved if there is better coordination between the available international timetables. The travel time would then be reduced from 3 h 58 min to 3 h 42 min, which would make the train much more competitive with the plane. If there are questions about the distribution of capacity between freight and passenger transport and/or between national and international connections, these options are not operational but political considerations. If there is a scarcity of capacity, the decision on how to allocate priorities is a political one. On many international services, time could be saved if the timetables allocated by different infrastructure managers were better coordinated. According to Eurostar, just a small variation in the timetable can cut 16 min off the travel time and will make the difference for travellers in choosing between flying or taking the train. In such cases, the coordination body could better balance national and international interests when allocating capacity.The capacity of the railway network can be significantly increased through better use of information technology, which can make rail transport safer and more reliable. Speeds can also be increased, which would facilitate cross-border rail traffic. The introduction of a uniform electricity supply could also contribute to a more intensive use of the railway network. The EU has chosen to implement the ERTMS system for train control and safety management throughout Europe. However, the problem is complex and difficult to grasp, and many stakeholders are concerned about the slow progress of implementation and rising costs. Here we note that the priority application and harmonisation of information technology on the main railway lines will give a significant boost to international rail transport.Wherever possible, railway capacity should be increased primarily through the use of intelligent safety systems such as ERTMS, which make better use of capacity. However, on some sections physical extension may be necessary. The construction of new infrastructure should be limited to cases where capacity is insufficient or speed is insufficient to meet the required quality standards. Improving the infrastructure is possible and feasible. The unbundling of regional, national and international rail transport on some routes will facilitate traffic flows and allow higher operating speeds.Research in international travel, on routes where trains and planes compete, is mainly concerned with modal share and travellers' preferences for both options. These studies show a promising position for rail in the market. But this promise is not being fulfilled. Some research also looks at the development of services and their quality. The research looks at what the reasons are for the low demand. There are clear obstacles in the way the management of the railway sector is structured at national and international level. These barriers manifest themselves in the rail services offered, not only in the transport itself, but also in many other aspects of the travel experience. There is a discrepancy between the promises made in research and the reality on the tracks.This study aims to broaden the research perspective to understand not only what is promised, but also what opportunities exist to strengthen international rail travel as an alternative for routes up to 800 km. It is based on a broad perspective as presented by relevant stakeholders in the relevant sectors. Moreover, it has a clear starting point in the Netherlands, a country from which long-distance rail journeys are always international journeys. Countries such as France, Germany, Italy, Spain and the UK are indeed developing their high-speed networks on a national scale, without any subset of the barriers described here. However, in other countries such as Austria, Belgium, Denmark and the Netherlands, and thus in the European Union as a whole, these barriers limit the potential of rail transport, despite the promising research on modal preferences.The barriers presented here are of course based on consultation with a limited number of key stakeholders in this Dutch context. A better understanding of these barriers will help to promote the future of European railways. Therefore, we would like to present this here as a first research agenda to address additional research topics in the field of international travel beyond the current main topics of air travel and mode choice. We propose to extend the analysis to other countries and to explore, beyond stakeholder consultation , the potential of new ways to drive the future of international rail travel in Europe.


Establishing fecal microbiota transplantation (FMT) to prevent multifactorial diarrhea in calves is challenging because of the differences in farm management practices, the lack of optimal donors, and recipient selection. In this study, the underlying factors of successful and unsuccessful FMT treatment cases are elucidated, and the potential markers for predicting successful FMT are identified using fecal metagenomics via 16S rRNA gene sequencing, fecal metabolomics via capillary electrophoresis time-of-flight mass spectrometry, and machine learning approaches.Selenomonas was identified as a microorganism genus that showed significant donor–recipient compatibility in successful FMT treatments. A strong positive correlation between the microbiome and metabolome data, which is a prerequisite factor for FMT success, was confirmed by Procrustes analysis in successful FMT . Additionally, weighted gene correlation network analysis confirmed the positively or negatively correlated pairs of bacterial taxa (family Veillonellaceae) and metabolomic features  responsible for FMT success. Further analysis aimed at establishing criteria for donor selection identified the genus Sporobacter as a potential biomarker in successful donor selection. Low levels of metabolites, such as glycerol 3-phosphate, dihydroxyacetone phosphate, and isoamylamine, in the donor or recipients prior to FMT, are predicted to facilitate FMT.Specifically, 20 FMT treatment cases, in which feces from healthy donors were intrarectally transferred into recipient diarrheal calves, were conducted with a success rate of 70%. Overall, we provide the first substantial evidence of the factors related to FMT success or failure; these findings could improve the design of future microbial therapeutics for treating diarrhea in calves.Video abstractThe online version contains supplementary material available at 10.1186/s40168-021-01217-4. Clostridioides difficile can be treated using FMT with a high success rate Metabolomics analysis using fecal samples was conducted at the Human Metabolome Technologies, Inc. . SpecifiThe levels of IgA, IgM, and IgG in feces were measured using ELISA. Specifically, feces were suspended in PBS (10 μl per mg), and the supernatants were collected using centrifugation. Next, 96-well ELISA plates were coated with 500 ng/ml of sheep anti-bovine IgA (Bethyl), IgM (Bethyl), or IgG (Bethyl) overnight at 4 °C. After blocking with 0.05 % (v/v) of Tween-20 for 1 h at RT, diluted fecal suspensions were incubated in the plates for 2 h at RT. After washing, the plates were treated with 100 ng/ml of HRP-conjugated sheep anti-bovine IgA (Bethyl), IgG (Bethyl), or IgM (Bethyl) for 1 h at RT. Bovine reference serum containing IgA, IgM, and IgG with known concentrations was used as a standard.To describe the correlations of microbiomes and metabolites associated with the success or failure of FMT, coexpression networks were constructed using the weighted gene correlation network analysis (WGCNA) (v1.70-3) package in R . WGCNA iP-values were corrected for multiple testing using a false-discovery rate  method [Multivariate statistical tools, including unsupervised principal component analysis (PCA) and supervised partial least squares discriminate analysis (PLS-DA), were employed to reduce and visualize the complex metabolomics datasets . In PLS-) method . ProcrusAlpha and beta diversity statistics were obtained using the QIIME 2 scripts diversity plugin . In the C. perfringens, C. parvum, rotavirus, and/or coccidia in multiple calves with diarrhea  at day 0, whereas the family Veillonellaceae and genera Selenomonas, Acidaminococcus, and Collinsella were significantly more abundant 7 days after FMT (R-7-success)  , suggesting that it may act as a signature microbe and could have the potential to ensure donor–recipient compatibility   was condnts Fig. a. Discriss) Fig. b, c. In re) Fig. a, b. It ity Fig. a, c. Mority Fig. d. Pathwaity Fig. e.Fig. 3ITo investigate the effects of FMT-induced changes in the gut microbiome on intestinal metabolism, most fecal samples collected from 12 FMT treatments  were analyzed using CE-TOFMS. In total, 366 peaks composed of cations 214 peaks) and anions (152 peaks), and 264 peaks, including 159 cations and 105 anions, were attributable to known standard metabolites that could be quantified  between the microbiota and metabolites was observed in successful cases  was observed in unsuccessful cases  and successful recipients 7 days after FMT   in successful cases, whereas Succinivibrio was negatively correlated with the presence of pimelic acid and P-aminobenzoic acid in unsuccessful cases , whereas Clostridium, but not Roseburia, was positively correlated with the presence of 2-aminoethylphosphonic acid in unsuccessful cases (R-0-failure)   or failure (R-1-failure) of FMT. In recipients, 7 days after FMT, Ruminococcus was negatively correlated with the presence of adenine, thymine, and 1,3-diaminoporopane in successful cases (R-7-success), but it was positively correlated with the presence of adenine in unsuccessful cases (R-7-failure)  Fig. SA-H. Furtses Fig. c. In recre) Fig. c. In recss) Fig. c. ClostrCollinsella, Prevotella, Gemmiger, Acidaminococcus, and Selenomonas . The trait for taurocholic acid was associated with MEblue and MEturquoise    Fig. SB. At dayoup Fig. c, showedoup Fig. . In conture Fig. , were siure Fig. b. Thus, Campylobacter, Actinobacillus, and Sporobacter were identified based on the mean decrease in accuracy; Campylobactor, Sporobacter, and Streptococcus were identified as the most discriminating predictors based on the mean decrease in the gini criteria  FMT treatments. Another important discovery was that of the genus Lactobacillus, which was abundant in recipients prior to FMT in successful (but not unsuccessful) cases. Lactobacillus promotes the growth of commensal microbiota and may participate in a mutualistic relationship between the host and microbes in the gastrointestinal tract of successful recipients after FMT [Lactobacillus rhamnosus CRL1505 has been known to modulate antiviral immune responses in intestinal epithelial cells and antigen-presenting cells (APCs), such as macrophages and dendritic cells, to increase resistance against intestinal viral infections [Lactobacillus rhamnosus CRL1505 also augments the Th1 response triggered by TLR2 signaling and increases the expression of MHC class II molecules as well as IL-1β, IL-6, IL-10, and IFN-γ in APCs for modulating intestinal immunity and thereby improving immune-health status [Spirochaetes, which is responsible for mucohaemorrhagic colitis in swine [Spirochaetes in the occurrence or progression of CD; nevertheless, these results suggest that this phylum may negatively affect the efficacy of FMT. Overall, Selenomonas, Lactobacillus, and Spirochaetes seem to be microorganisms associated with donors and/or recipients that lead to the success or failure of FMT; thus, a convenient strategy for detecting the presence of such bacteria in the field should be established to increase the rate of FMT success.In this study, an important advance was the discovery of beneficial and nonbeneficial bacteria identified from successful and unsuccessful FMT treatments. The genus tic acid , could b pathway . WGCNA after FMT . In analfections . Lactobah status . In contin swine , was abuCollinsella is capable of producing butyric acid [Collinsella and butyric acid were both discriminately abundant 7 days after successful FMT treatments. Therefore, the intestinal microbiota appears to coevolve with the host and play a crucial role in host nutrient absorption and metabolism [Bacillaceae and genera Methanobrevibacter, Eggerthella, and Clostridium) were positively correlated with the levels of branched-chain amino acids  as well as with levels of arginine, histidine, phenylalanine, and tryptophan. Given that the amino acids present in feces have been positively correlated with the severity of Crohn’s disease [Entamoeba, Giardia, Acanthamoeba, and Balamuthia, and to exhibit a distinct kinetic feature of their survival [Giardia duodenalisis, a worldwide parasite that causes giardiasis  [This study also confirmed dynamic changes in metabolites belonging to successful and unsuccessful FMT treatments. In successful cases, short-chain fatty acids, especially butyric acid, and medium-chain fatty acids, such as octanoic acid, were enriched after FMT treatments. The genus ric acid ; consisttabolism . Furthertabolism , it is ptabolism , 6, 13; tabolism , 46; con disease , the higsurvival . Dihydrodisease) . To ensuSporobacter as a potential biomarker for donor selection using RF classification and LEfSe analysis; Sporobacter was also positively correlated with the production of succinic acid in R-1-success, suggesting that it may play an important role in the interactions of microbiota and metabolites in potential donors. Establishing discriminative characteristics using the novel prediction model applied in this study to identify calves with diarrheal incidence that were at high risk of failure even after FMT therapy may also be useful for veterinary physicians in relation to their management plans. Thus, our study emphasizes the use of a machine learning approach to help protect livestock. Further studies are needed to determine whether the donor FMT itself induces the complete resolution of CD or whether it helps promote a spontaneous recovery. Indeed, it will be important to develop a robust understanding of the gut microbiome and metabolome across individuals in general.Notably, the establishment of FMT as a veterinary practice for the effective treatment of CD remains difficult due to safety concerns regarding the transmission of virulence factors . It is aThis study was the first to examine multidonor FMT as a strategy for CD treatment through the remediation of gut microbiota with an emphasis on the personalization of interventions; additionally, the inherent individualized variability in microbiota, metabolites, and other physiological features was considered. Our findings provide new insights into developing successful FMT strategies with a multiomics approach, and they may help advance research into microbial therapeutics for CD treatment.Sporobacter and Selenomonas in donor calves and Lactobacillus in recipient diarrheal calves in successful FMT cases; thus, the presence of these genera may predict FMT success. These findings have enormous significance for the livestock industry because FMT could eventually address the challenge of CD prevention as well as the use of excessive antibiotics.In this study, we demonstrated that FMT is an effective treatment option for the prevention of diarrhea in calves, specifically by identifying a beneficial microbial cluster as well as functional metabolites. We showed that FMT success or failure depends on the microbial composition of both donors and recipients. We also consistently identified the microbial genera Additional file 1: Table S1. Demographic information of the calves selected for FMT. Table S2. Unweighted distance matrix among groups. Table S3. P values of the Tukey’s multiple comparison test from the one-way ANOVA between PC1 scores of the PCA. Table S4. Demographic information of the selected healthy and diarrheal calves. Table S5. Unweighted unifrac distance matrix analysis.Additional file 2. Datasets 1-10.Additional file 3. Module; trait.Additional file 4: Fig. S1. Overview of farm specific cow and disease condition. The effect of FMT on enteritis and weak calf syndrome disease prevention in successful and unsuccessful treatments. (A) Farm specific cows’ number. (B) Success rate evaluated by presence and absence of enteritis and weak calf syndrome before and after FMT for recipient (n = 14) in successful treatments and recipient (n = 6) in unsuccessful treatments. ‘+, presence; ‘-, absence. Fig. S2. Clinical parameter. Clinical efficacy of FMT on successful and unsuccessful treatments based on day-0 (before FMT) and day-7 (after FMT). Data are expressed as mean±SD, shown for R-0-success (n = 14), R-0-failure (n = 6), R-7-success (n = 14), and R-7-failure (n = 4). γ-GT, Gamma-Glutamyl Transferase; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. *P <0.05 (Student’s t-test). Fig. S3. Microbial composition based on phylum level. Relative abundances are shown as mean ± SEM for donor (n = 13), recipient 0 (n = 13), recipient 1 (n = 13), and recipient 7 (n = 13) in successful treatments, and donor (n = 4), recipient 0 (n = 4), recipient 1 (n = 4), and for recipient 7 (n = 4) in unsuccessful treatments. P-values  indicate statistical significance either successful or unsuccessful treatments by one-way ANOVA followed by tukey multiple comparison test. P-values  indicate statistical significance by Student’s t-test. Fig. S4. Microbial composition based on family level. Relative abundances are shown as mean ± SEM for donor (n = 13), recipient 0 (n = 13), recipient 1 (n = 13), and recipient 7 (n = 13) in successful treatments, and donor (n = 4), recipient 0 (n = 4), recipient 1 (n = 4), and for recipient 7 (n = 4) in unsuccessful treatments. P-value (*P <0.05) indicates statistical significance either successful or unsuccessful treatments by one-way ANOVA followed by tukey multiple comparison test. P-values  indicate statistical significance by Student’s t-test. Fig. S5. Microbial composition based on genus level. Relative abundances are shown as mean ± SEM for donor (n = 13), recipient 0 (n = 13), recipient 1 (n = 13), and recipient 7 (n = 13) in successful treatments, and donor (n = 4), recipient 0 (n = 4), recipient 1 (n = 4), and for recipient 7 (n = 4) in unsuccessful treatments. P-values  indicate statistical significance either successful or unsuccessful treatments by one-way ANOVA followed by tukey multiple comparison test. P-value (†P <0.05) indicates statistical significance by Student’s t-test. Fig. S6. Heatmap analysis based on metabolites in concentration level identified in all the groups. Each column represents a sample and each row represents a metabolite. Colors indicate fold increases or decreases in concentrations, as indicated in the key. Data are shown for donor (n = 9), recipient 0 (n = 9), recipient 1 (n = 9), and recipient 7 (n = 9) in successful treatments, and donor (n = 3), recipient 0 (n = 3), recipient 1 (n = 3), and for recipient 7 (n = 3) in unsuccessful treatments. The parameters that were used for the analysis were Euclidean distance measure and Ward cluster algorithm, using MetaboAnalyst 4.0 software. Fig. S7. Partial Least-Squares Discriminant Analysis (PLS-DA) parameter for the comparison of successful and unsuccessful treatments. (A) D-success vs D-failure ; (B) R-0-success vs R-0-failure ; (C) R-1-success vs R-1-failure , and (D) R-7-success vs R-7-failure , shown for donor (n = 9), recipient 0 (n = 9), recipient 1 (n = 9), and recipient 7 (n = 9) in successful treatments, and donor (n = 3), recipient 0 (n = 3), recipient 1 (n = 3), and for recipient 7 (n = 3) in unsuccessful treatments. Fig. S8. Effect of FMT on amino acid level in successful and unsuccessful treatments. Amino acid concentration obtained from fecal sample in each group on before and after FMT. Data are shown as mean ± SEM for donor (n = 9), recipient 0 (n = 9), recipient 1 (n = 9), and recipient 7 (n = 9) in successful treatments, and donor (n = 3), recipient 0 (n = 3), recipient 1 (n = 3), and for recipient 7 (n = 3) in unsuccessful treatments and were analyzed using the unpaired t test with Welch’s correction. *P <0.05; **P <0.01. Fig. S9. Effect of FMT on selected metabolites in successful and unsuccessful treatments obtained from fecal sample in each group on before and after FMT. Data are shown as mean ± SEM for donor (n = 9), recipient 0 (n = 9), recipient 1 (n = 9), and recipient 7 (n = 9) in successful treatments, and donor (n = 3), recipient 0 (n = 3), recipient 1 (n = 3), and for recipient 7 (n = 3) in unsuccessful and were analyzed using the unpaired t test with Welch’s correction. *P < 0.05. GABA, gamma-Aminobutyric acid. Fig. S10. Abundant metabolites in lipid and fatty acid metabolism based on their relative area obtained from fecal sample in each group on before and after FMT. Data are shown as for donor (n = 9), recipient 0 (n = 9), recipient 1 (n = 9), and recipient 7 (n = 9) in successful treatments, and donor (n = 3), recipient 0 (n = 3), recipient 1 (n = 3), and for recipient 7 (n = 3) in unsuccessful treatments. Data are shown as mean ± SEM and were analyzed using the unpaired t test with Welch’s correction. *P < 0.05. Fig. S11. Effect of FMT on polyamines. Polyamines (spermidine and putresine) and diamine (cadaverine) in successful and unsuccessful treatments based on their concentration are obtained from fecal sample in each group on before and after FMT, for donor (n = 9), recipient 0 (n = 9), recipient 1 (n = 9), and recipient 7 (n = 9) in succeed treatments, and donor (n = 3), recipient 0 (n = 3), recipient 1 (n = 3), and for recipient 7 (n = 3) in unsuccessful treatments. Data are shown as mean ± SD. were analyzed using the unpaired t test with Welch’s correction. *P < 0.05. Fig.S12. Microbiota-metabolome correlation based on group specific procrustes analysis. Procrustes analysis in A) D-success group , B) R-0-success , C) R-1-success , D) R-7-success , E) D-failure , F) R-0-failure , G) R-1-failure , H) R-7-failure . Fig. S13. The weighted gene co-expression network analysis (WGCNA) to identify microbiota-metabolites correlation. (A) Module trait relationship among microbiota, clinical data, metabolites and fecal immunoglobulins . Each row corresponds to a module. Each column corresponds to a trait labeled below. Each cell at the row-column intersection contains the correlation coefficient and P-value (in brackets) between the module and that trait. A highly positive correlation between a specific module and a trait is indicated by dark red, and green indicates negative correlation. Data are shown for donor (n = 9), recipient 0 (n = 9), recipient 1 (n = 9), and recipient 7 (n = 9) in successful treatments, and for donor (n = 3), recipient 0 (n = 3), recipient 1 (n = 3), and recipient 7 (n = 3) in unsuccessful treatments. (B) Heatmap analysis from WGCNA analysis based on modules-trait relationships. Each column represents a sample and each row represents a microbial taxon from the selected modules. Colors indicate increases or decreases in abundances, as indicated in the color key (donor and recipient n numbers in successful and unsuccessful treatments as shown above). Fig. S14. Heatmap based Identification of the potential microbial taxa by module trait relationship in donor cases in WGCNA analysis. Each column represents a sample and each row represents a microbial taxon from the selected modules. Colors indicate increases or decreases in abundances, as indicated in the color key, for D-success (n = 9) in successful treatments and D-failure (n = 3) in unsuccessful treatments, respectively. Fig. S15. The weighted gene co-expression network analysis (WGCNA) to identify microbiota-metabolites correlation before FMT. (A) Module trait relationship among microbiota, and selected metabolites. Each row corresponds to a module. Each column corresponds to a trait labeled below. Each cell at the row-column intersection contains the correlation coefficient and P-values (in brackets) between the module and that trait. A highly positive correlation between a specific module and a trait is indicated by dark red, and green indicates negative correlation. (B) Heatmap analysis from WGCNA analysis based on modules-trait relationships. Each column represents a sample and each row represents a microbial taxon from the selected modules. Colors indicate increases or decreases in abundances, as indicated in the color key, shown for recipient 0 (n = 9) in successful treatments, and recipient 0 (n = 3) in unsuccessful treatments, respectively. Fig. S16. The weighted gene co-expression network analysis (WGCNA) to identify microbiota-metabolites correlation after FMT at day 1. (A) Module trait relationship among microbiota, and selected metabolites. Each row corresponds to a module. Each column corresponds to a trait labeled below. Each cell at the row-column intersection contains the correlation coefficient and P-value (in brackets) between the module and that trait. A highly positive correlation between a specific module and a trait is indicated by dark red, and green indicates negative correlation. (B) Heatmap analysis from WGCNA analysis based on modules-trait relationships. Each column represents a sample and each row represents a microbial taxon from the selected modules. Colors indicate increases or decreases in abundances, as indicated in the color key, shown for recipient 1 (n = 9) in successful treatments, and recipient 1 (n = 3) in unsuccessful treatments, respectively. Fig. S17. Heatmap based Identification of the potential microbial taxa by module trait relationship after FMT in recipient cases in WGCNA analysis. Each column represents a sample and each row represents a microbial taxon from the selected modules. Colors indicate increases or decreases in abundances, as indicated in the color key, for recipient 7 (n = 9) in successful treatments, and donor (n = 3), recipient 7 (n = 3) in unsuccessful treatments, respectively. Fig. S18. Metagenomics analysis for potential biomarker identification for a successful FMT. (A) Diarrheal score comparison between diarrheal group for diarrhea (n = 49), R-0-success (n = 14), and R-0-failure (n = 6). Data are expressed as mean±SEM and were analyzed using the 1-way ANOVA with Tukey’s post hoc multiple comparisons (*P <0.05). (B) Microbial composition of overall healthy and diarrheal group for healthy (n = 105), diarrhea (n = 46), D-success (n = 13), D-failure (n = 4), R-0-success (n = 13), and R-0-failure (n = 4). Relative abundance (%) of (C) Sporobacter, (D) Camphylobactor. Data are expressed as mean ± SEM for healthy (n = 105), diarrhea (n = 46), D-success (n = 13), D-failure (n = 4), R-0-success (n = 13), and R-0-failure (n = 4).


The effects of mixing orders of tannic acid (TA), starch, and α-amylase on the enzyme inhibition of TA were studied, including mixing TA with α-amylase before starch addition (order 1), mixing TA with pre-gelatinized starch before α-amylase addition (order 2) and co-gelatinizing TA with starch before α-amylase addition (order 3). It was found that the enzyme inhibition was always highest for order 1 because TA could bind with the enzyme active site thoroughly before digestion occurred. Both order 2 and 3 reduced α-amylase inhibition through decreasing binding of TA with the enzyme, which resulted from the non-covalent physical adsorption of TA with gelatinized starch. Interestingly, at low TA concentration, α-amylase inhibition for order 2 was higher than order 3, while at high TA concentration, the inhibition was shown with the opposite trend, which arose from the difference in the adsorption property between the pre-gelatinized and co-gelatinized starch at the corresponding TA concentrations. Moreover, both the crystalline structures and apparent morphology of starch were not significantly altered by TA addition for order 2 and 3. Conclusively, although a polyphenol has an acceptable inhibitory activity in vitro, the actual effect may not reach the expected one when taking processing procedures into account. Postprandial hyperglycemia is an important factor that causes disturbance of glucose metabolisms, like type II diabetes. Starchy foods are the main source of carbohydrates for human beings, the digestion of which decides the changes of postprandial blood sugar level to a large extent. α-Amylase is a key carbohydrate-hydrolyzing enzyme that initially catalyzes starchy components, producing reducing sugars, such as maltose, maltotriose, maltooligosaccharides, etc., and these reducing sugars are further hydrolyzed by α-glucosidase to glucose that is finally absorbed by enterocytes in the small intestine . TherefoNatural polyphenols or phenolic extracts from plant foods have been reported to develop the inhibitory activity against α-amylase, retarding starch digestion both in vitro and in vivo . NotablyUsually, to characterize the inhibiting effect of a polyphenol against α-amylase, the polyphenol is mixed with the enzyme firstly, followed by addition of substrates  ,10. ThisTM Ultra Amylase Assay Kit was purchased from Life Technologies Co. . Maize starch and p-hydroxybenzoic acid hydrazide  were obtained from Yuanye Biotech Co. . Other reagents were of analytical grade.Tannic acid (TA), porcine pancreatic α-amylase , and phosphate- buffered saline (PBS) tablets were purchased from Sigma-Aldrich Co. . EnzCheckThe three mixing procedures applied in this study  were as v and v0 are the initial reaction velocity of starch digestion in the presence and absence of TA, respectively, which were obtained from the slopes of the linear correlations between the reducing sugar contents and digestion time.The inhibition effects of TA for the three mixing procedures were initially characterized by determination of the initial reaction velocity of starch digestion in the absence and presence of TA . The cont is the digestion time; tC is the fraction of digested starch at digestion time t; C∞ is the fraction of digested starch at the end point of the reaction; and k is the digestion rate constant. To obtain the value of k, Equation (2) can be transformed into a logarithm of slope (LOS) plot in which there is a linear correlation between ln(tdC/dt) and k as follows  is the TA concentrations; I is α-amylase inhibition at each TA concentration; maxI is the maximum inhibition.IC follows :(5)I=ImaicK and the uncompetitive inhibition constant iuK were obtained from the Dixon (6) and Cornish-Bowden (7) equations respectively as follows  is the quencher (TA) concentration.The fluorescence spectra of α-amylase in the absence and presence of TA with various concentrations were determined by using a spectrofluorometer . The fluorescence quenching constant,  follows :(8)F0F=1B, the adsorption amount of TA per mass of starch) of starch was calculated according to Equation (9) as follows:C0 and tC are the respective TA concentrations outside the dialysis bag of control and TA-starch samples at a dialysis time t; V is the solution volume in the beaker; m is the mass of gelatinized starch in the dialysis bag.It seems inconvenient to measure the adsorption property of TA onto gelatinized starch directly. Thus, a dialysis method was introduced in this study for two preparation procedures including (1) mixing TA with pre-gelatinized starch and (2) co-gelatinizing starch with TA. Both the gelatinization processes were conducted at 90 °C for 20 min. The final concentration of starch in the gelatinized system was 10 mg/mL, and the TA concentrations were in a series of 2, 4, 6, and 8 mg/mL. The respective gelatinized TA-starch solutions for two procedures were placed into a dialysis bag with the membrane cut-off molecular weight of 7 kD . To start the dialysis process, the dialysis bag was put into a beaker containing PBS buffer, and the TA concentrations outside the dialysis bag at individual time points were determined by the Folin–Ciocalteu method with a standard curve of absorbance value against TA concentration. The dialysis of TA in the absence of starch was used as the control. Then, the binding capacity  can be transformed into a logarithm of slope plot (11) in which there is a linear correlation between ln(tdC/dt) and tk as follows:To describe the free diffusion property of TA, the first-order pharmacokinetics Equation (10) was applied to analyze the correlation between the TA concentration outside the bag and dialysis time as follows ,25:(10)CTo characterize the binding interactions between TA and starch, the gelatinized TA-starch complexes were prepared based on two procedures including (1) mixing TA with pre-gelatinized starch and (2) co-gelatinizing starch with TA. For procedure (1), 40 mL of 10 mg/mL pre-gelatinized starch was mixed with 1 mL of TA with a series of concentrations (10–60 mg/mL), while for procedure (2), 40 mL of 10 mg/mL raw starch suspension was mixed with 1 mL of TA with a series of concentrations (10–60 mg/mL), followed by co-gelatinization at 90 ℃ for 20 min. Both the TA-starch gelatinized complexes were then lyophilized, ground, and sieved (200-mesh). The obtained powder was analyzed by FTIR, XRD, and SEM to characterize the changes in molecular structures and apparent morphology of starch caused by TA addition, from which the binding interactions between them may be also reflected.−1, and the sample was scanned 32 times with the resolution of 4 cm−1.Before FTIR analysis, all the samples were dehydrated at 45 °C for 3 h to minimize the impact of the residual water component. Then, the samples (~5 mg) were mixed with KBr (1:100) and pressed into a semitransparent pellet. The FTIR spectra were recorded by use of an IR spectrometer  within the frequency range of 4000 to 400 cmX-ray diffraction (XRD) was conducted to explore the crystalline structure of the TA-starch complex samples. After drying the samples at 45 °C for 3 h, they were further ground finely and scanned from 10° to 60° at a step size of 0.02 and a time interval of 0.5 s per step by using a Powder X-ray Diffractometer  equipped with a CuKα lamp and a nickel filter.For SEM observation, the lyophilized starch samples were fractured and sprayed with gold powders at the surfaces, followed by being imaged by use of an Environmental Scanning Electron Microscope  with a magnification of 4000×. The operation was performed in a high vacuum mode and 20 kV.p < 0.05, the data is considered as statistically significant and thus marked with different superscripts.One-way analysis of variance (ANOVA) by Graphpad Prism 6 software was applied to analyze the significant difference between the data obtained. When k1) that mainly represents the hydrolysis of rapidly digestible starch [The activity of α-amylase in the presence and absence of TA for the different mixing orders of TA, starch, and α-amylase were indicated by the initial reaction velocity A. Based e starch  followed50 value, inhibition kinetics, and fluorescence quenching were applied. It was found that the enzyme inhibition of TA was dependent on the phenolic concentration  at the respective TA concentrations  that describes the dissociation of TA-amylase binary complex [icK value for order 2 (25.912 mg/mL) was significantly higher than order 1 (FQK (9.209 × 103 M−1), and the bimolecular quenching constant, qk (3.1 × 1012 M−1s−1), were obtained according to the Stern-Volmer equation  in the anhydroglucose ring mainly caused the peak at 1080 cm−1 [−1 that represents the stretching vibration of C=O (in O-C=O of TA) [−1   became m TA) [−1 A,B. In agranules  was not k) were obtained to reflect the catalytic ability of α-amylase, especially for the first phase as the substrate (rapidly digestible starch) is able to bind with the enzyme efficiently and thus can be catalyzed efficiently in this stage [k1 (digestion rate constant in the first phase) suggests a higher enzymic ability and thus a lower inhibitory activity [k1 value of order 1   . Notablyorder 1) B,C, indiorder 1) B. This iR2) of the fitting model applied for diffusion of TA inside the dialysis bag to outside in the absence and presence of TA were all above 0.96  than that in the absence of starch   od starch . Therefod starch . On the with TA) , indicatwith TA) . This iswith TA) B. Therefwith TA) , causingwith TA) B.−1  were observed for all the TA-starch samples (−1 to 1022 cm−1) [Furthermore, to illustrate the effect of TA binding on the microcrystalline structures and apparent morphology of starch, FTIR, XRD, and SEM were applied in characterization of lyophilized gelatinized starch in the presence and absence of TA. As there were no new peaks observed in TA-starch complexes (both for pre-gelatinization before TA addition and co-gelatinization with TA), compared to the respective FTIR spectra of TA and starch A,B, the  samples A,B, sugg22 cm−1)  was not 22 cm−1) . Therefo22 cm−1) . MoreoveThe three orders in this study correspond to three supplementary modes of polyphenols in diets, e.g., (1) intaking polyphenols, phenolic extracts, or foods rich in polyphenols before meals (order 1). In this case, polyphenols have more opportunity for binding with α-amylase, developing the inhibitory activity and delaying digestion of starch in meal; (2) intaking polyphenols (extracts or foods) together with meal. In this case, the phenolic compounds could be adsorbed onto the gelatinized starch in meal, which affects the binding of polyphenols with α-amylase and thus decreases the inhibition effects; (3) starchy foods are processed together with polyphenols for healthy food production. In this case, the different polyphenol/starch mass ratios may cause different polyphenol binding capacity of starch, and this further affects the amounts of unbound polyphenols that have the inhibitory activity against α-amylase. Notably, the addition of polyphenols discussed here did not significantly change the microcrystalline and integral morphology of lyophilized starch due to the relatively weak interactions between them. However, for the polyphenols that are able to interact with starch molecules under some certain external filed forces through relatively strong non-covalent forces, like hydrogen bonding and hydrophobic force, the changes in starch digestibility and digestion velocity may be attributed to the increase in relative crystallinity of starch ,40. FurtBy taking three common processing procedures of polyphenols, starch, and α-amylase in actual case into account, the effects of mixing orders on the inhibitory activity of tannic acid against α-amylase were explored in this study. Mixing TA with α-amylase before starch addition caused the highest inhibiting effects at each TA concentrations because TA had more opportunity for contacting and binding with α-amylase before the enzyme specifically bound with and catalyzed starch. As for two procedures where mixing TA with starch before α-amylase addition , gelatinized starch could adsorb TA through weak non-covalent interactions like Van der Waal’s force, which decreased the binding of TA with the active site of α-amylase. However, the adsorption capacity of the pre-gelatinized starch was lower than the co-gelatinized starch at a low TA concentration, and higher than the co-gelatinized starch at a high TA concentration, which may result from the different TA-starch adsorption mechanisms for the two gelatinization procedures. This resulted in the difference in the inhibitory effect of unbound TA. To evaluate the inhibitory activity of a phenolic compound in diets, in addition to molecular structure of the polyphenol and other existing components, supplementary modes of the polyphenol may be also considered because different mixing orders of the inhibitor, enzyme, and substrate are supposed to cause different inhibition effects as suggested in this study.


Philip Alexander, MD, is a native Texan, retired physician, and accomplished musician and artist. After 41 years as an internal medicine physician, Dr. Phil retired from his practice in College Station in 2016. A lifelong musician and former music professor, he often performs as an oboe soloist for the Brazos Valley Symphony Orchestra. He began exploring visual art in 1980, evolving from pencil sketches—including an official White House portrait of President Ronald Reagan—to the computer-generated drawings featured in this journal. His images, which first appeared in this journal in the spring of 2012, are his own original creations.Methodist DeBakey Cardiovascular Journal, submit your creation online at journal.houstonmethodist.org as a “Humanities” entry.If you would like to see your art published in the


Hestina persimilis and Hestinalis nama were obtained via sanger sequencing. Compared with other mitogenomes of Apaturinae butterflies, conclusions can be made that the mitogenomes of Hestina persimilis and Hestinalis nama are highly conservative. The phylogenetic trees build upon mitogenomic data showing that the relationships among Nymphalidae are similar to previous studies. Hestinalisnama is apart from Hestina, and closely related to Apatura, forming a monophyletic clade.In this study, the mitogenomes of Hestina persimilis and Hestinalis nama  were acquired. The mitogenomes of H. persimilis and H. nama are 15,252 bp and 15,208 bp in length, respectively. These two mitogenomes have the typical composition, including 37 genes and a control region. The start codons of the protein-coding genes (PCGs) in the two mitogenomes are the typical codon pattern ATN, except CGA in the cox1 gene. Twenty-one tRNA genes show a typical clover leaf structure, however, trnS1(AGN) lacks the dihydrouridine (DHU) stem. The secondary structures of rrnL and rrnS of two species were predicted, and there are several new stem loops near the 5′ of rrnL secondary structure. Based on comparative genomic analysis, four similar conservative structures can be found in the control regions of these two mitogenomes. The phylogenetic analyses were performed on mitogenomes of Nymphalidae. The phylogenetic trees show that the relationships among Nymphalidae are generally identical to previous studies, as follows: Libytheinae\Danainae +  + Danainae\Libytheinae + ((Heliconiinae + Limenitidinae) + ))). Hestinalisnama is apart from Hestina, and closely related to Apatura, forming monophyly.In this study, the complete mitochondrial genomes (mitogenomes) of  Hestina persimilis and Hestinalis nama belong to the lepidopteran family Nymphalidae, Apaturinae and mainly distribute in the Palaearctic and Oriental region. Their adults inhabit mountainous broad-leaved forests and present the habit of sipping tree juice and water in wetlands. The larvae were reported as a kind of agriculture pest of the host plants, Ulmaceae. At present, there are only one species (H. nama) of the genus Hestinalis and three species  of the genus Hestina distributed in China. Hestinalis nama was originally described as Diadema nama by Doubleday in 1844 . Ad. AdtrnS1ogenomes . Previou TΨC arm . Missingof tRNAs  or not. rrnL gene is found between trnL (CUN) and trnV, while the rrnS gene is located between trnV and the control region. The lengths of rrnL genes of H. persimilis and H. nama mitogenomes are 1334 bp (AT content 84.29%) and 1327 bp (AT content 83.44%). The sizes of rrnS genes of H. persimilis and H. nama are 776 bp (AT content 85.03%) and 774 bp (AT content 84.39%). The secondary structure of rrnL genes include six structural domains except for that domain III is absent in arthropods . There are several new stem loops near the 5′ of rrnL secondary structure, and these loops were not found in other insects.The thropods . The rrn domains . Both thellifera , Graphol molesta , Manducaca sexta , etc. ThDashes, black dots and circles indicate the Watson-Crick base pairings, G-U bonds and U-U, A-A, A-C and A-G bonds, respectively.H. persimilis, and 11 intergenic spacers  are found in H. nama. It has been reported that Lepidopteran mitogenomes usually have two typical and relatively conservative intergenic spacers. The longer one is located between trnQ and nad2 genes, with the length of 91 and 69 bp in H. persimilis and H. nama. Previous studies found that the nucleotide sequence of the trnQ-nad2 spacer and the nad2 gene have a highly similarity. It has been inferred that the trnQ-nad2 spacer may come from the nad2 gene [trnS2(UCN) and nad1 genes, with the length of 22 and 13 bp in H. persimilis and H. nama, respectively, sharing a conserved sequence of ATACTAA.It has been proposed that mitogenomes tend to be highly economized in size by eliminating or reducing intergenic spacers . Howeverad2 gene . The othH. persimilis, and fourteen overlapping spacers  are found in H. nama. ATP8 and ATP6 overlap with the ATGATAA motif in the two mitogenomes, which had also been reported in many other lepidopterans [Comparing with the intergenic spacers, the overlapping regions are more conservative . Fourteeopterans .H. persimilis and 390 bp in H. nama) in the two mitogenomes are located between rrnS and trnM. The AT content is also the highest in mitogenomes (91.23% in H. persimilis and 88.72% in H. nama). Previous studies indicated that the control region is the segment with fastest evolutionary rate and can be used as an important molecular marker for animal population genetics. The control region, also known as the A + T-region or D-loop, is always the largest intergenic spacer in animal mitogenomes and considered as the initial region for replication . The conrrnS followed by 19 bp Poly-T stretch, a poly-A stretches (9 bp in H. persimilis and 6 bp in H. nama) at the upstream of trnM (10 in H. persimilis and (AT)6 in H. nama), and the repeated sequences (23 bp in H. persimilis and 25 bp in H. nama). All these characteristics are generally considered to be related to the transcription or replication of mitogenomes [There are generally four conserved structures in the control region, including a motif of ATAGA located at downstream of  of trnM , the micogenomes . Althougogenomes . Accordiogenomes .Hestina and Euripus, Sasakia come together to form a branch; Apatura and Hestinalis form a branch; Chitoria, Timelaea and Herona form a branch. Early on, Apatura included Chitoria [Chitoria is distant from Apatura. Ohshima et al. (2010) has published an updated work on Apaturinae butterflies, considering that Chitoria should been removed from Apatura [H. persimilis, H. assimilis and the genus Euripus (represented by Euripus nyctelius) form a branch, of which the result is the same as the morphological study. Hestinalis is a clearly distinct lineage within the genus Hestina, not together with H. persimilis and H. assimilis, and closely related to Apatura, that is considered to be a result of mimicry [The phylogenetic analyses are performed on concatenated nucleotide sequences of 13 PCGs  derived from 54 available Nymphalidae mitogenomes, with two Papilionidae species serving as outgroups. These 54 sequences represent 9 subfamilies: Apaturinae; Biblidinae; Calinaginae; Danainae; Heliconiinae; Libytheinae; Limenitidinae; Nymphalinae and Satyrinae. The BI  and ML tChitoria , while i Apatura , their g mimicry , which iH. persimilis and H. nama were obtained using sanger sequencing. Comparing them with other mitogenomes of Apaturinae butterflies, the conclusion can be drawn that the mitogenomes are highly conserved, sharing the same gene order, gene location, codon usage, nucleotide composition and AT-biased pattern. The secondary structures of rrnL and rrnS of two species are roughly similar to other lepidopterans. Although the control regions vary greatly in length, their structure has not changed much, which includes four basic conservative regions. The topology of phylogenetic analyses are generally identical to those of other studies. Hestinalisnama is not grouped with Hestina, and is closely related to Apatura, which is consistent with early studies.The mitogenomes of


To determine the predictive performance of the integrated model based on clinical factors and radiomic features for the accurate identification of clinically significant prostate cancer (csPCa) among Prostate Imaging Reporting and Data System (PI-RADS) 3 lesions.A retrospective study of 103 patients with PI-RADS 3 lesions who underwent pre-operative 3.0-T MRI was performed. Patients were randomly divided into the training set and the testing set at a ratio of 7:3. Radiomic features were extracted from axial T2WI, diffusion-weighted imaging (DWI), and apparent diffusion coefficient (ADC) images of each patient. The minimum redundancy maximum relevance (mRMR) and least absolute shrinkage and selection operator (LASSO) feature selection methods were used to identify the radiomic features and construct a radiomic model for csPCa identification. Moreover, multivariable logistic regression analysis was used to integrate the clinical factors with radiomic feature model to further improve the accuracy of csPCa identification, and the two are presented in the form of normogram. The performance of the integrated model was compared with radiomic model and clinical model on testing set.p < 0.001; testing set: p = 0.035). Multivariable logistic regression analysis showed that age and PSA could be used as independent predictors for csPCa identification. The clinical–radiomic model produced the receiver operating characteristic (ROC) curve (AUC) in the testing set was 0.88 , which was similar to clinical model  (p = 0.048) and higher than the radiomic model  (p < 0.001). The decision curve analysis implies that the clinical–radiomic model could be beneficial in identifying csPCa among PI-RADS 3 lesions.A total of four radiomic features were selected and used for radiomic model construction producing a radiomic score (Radscore). Radscore was significantly different between the csPCa and the non-csPCa patients (training set: The clinical–radiomic model could effectively identify csPCa among biparametric PI-RADS 3 lesions and thus could help avoid unnecessary biopsy and improve the life quality of patients. Prostate cancer (PCa) is one of the most common cancer and the second leading cause of cancer deaths among men . The Provia avoiding unnecessary biopsies and overtreatment.Currently, the classification of PI-RADS category 3 lesions has not been clearly defined, which represents a “gray zone” that contains benign, indolent, and invasive lesions. Since considered as positive MRI finding, PI-RADS 3 lesion should always be biopsied according to European Association of Urology guidelines, which results in a diagnosis of csPCa in 3%–50% of the patients , 5. HoweRecent studies have shown that patient age, high prostate-specific antigen density (PSAD), PSA velocity, low apparent diffusion coefficient (ADC) signal, and even genetic risk are associated with the existence of csPCa , 7, 9. FTherefore, the objective of this study was to construct a nomogram that integrate radiomics based on bpMRI and clinical information to identify csPCa in PI-RADS 3 lesions.The Institutional Review Board approved this retrospective study performed at a single medical institution and waived the requirement of informed consent . A retrospective collection of 1,675 patients who underwent prostate MRI examination due to PSA elevation in our hospital from January 2016 to January 2019 was conducted. All MRI images were assigned PI-RADS V2.1 score by two genitourinary radiologists experienced in urological diagnosis (3- and 6-year experience). Screened out lesions identified as PI-RADS 3 and sent to another experienced genitourinary radiologist (more than 10-year experience) for review. All the radiologists were blind to the histopathological results at the time of reading. If there is a disagreement on the diagnosis, the three radiologists will discuss it until a consensus is reached. PI-RADS V2.1 defined the score 3 lesion as the focal low signal of ADC and/or high signal of DWI with high b-value in the peripheral zone (PZ) or the uneven signal of T2WI sequence in the transitional zone (TZ) with blurred edges. In the course of the discussion, 25 cases were downgraded to PI-RADS 1–2, and 39 cases were upgraded to PI-RADS 4–5. The final scoring results showed that 859 patients (51.3%) had PI-RADS scores of 1 and 2, and 153 patients (9.1%) had PI-RADS scores of 3; the other 663 cases (39.6%) had PI-RADS scores of 4 and 5.Exclusion criteria were as follows: (1) PI-RADS 1–2, PI-RADS 4–5, or PI-RADS 3 coexisted with other types of lesions ; (2) poor image quality (N = 8); (3) MRI findings were not confirmed by histopathological results(N = 11); (4) intervention prior to MRI examination, such as biopsy, surgery, or hormone therapy (N = 24) Figure 12) and/or dynamic-contrast-enhanced T1WI. Based on the DWI images of 1,500 b-values, the ADC icons were calculated by extended single exponential fitting model. The specific scanning parameters were recommended by PI-RADS V2.1  with 32-channel body phased line coil was adopted. The scanning sequence included T1WI, axial T2WI (no fat-saturated), sagittal T2WI, coronal T2WI, DWI  fusion-guided prostate biopsy with a navigation system (HIVISION Noblus/TopicPath). At least two biopsy samples were obtained from each targeted lesion. The 12-core systematic biopsies were routinely performed following the targeted biopsy procedure. If patients underwent radical prostatectomy, their pathological ISUP grades, instead of biopsy ISUP grades, were used to define csPCa. The pathological results were evaluated by expert urologists, and the location of the lesions were recorded to ensure the correspondence to the suspicious lesions on MRI. csPCa was defined as ISUP Class 2 or higher (Gleason = 3 + 4 or higher). Lesions with GS = 3 + 3 were defined as clinically insignificant prostate cancer (ciPCa), which were categorized in the same group as benign lesions .2) DWI, and ADC were co-registered using Elastix software package (v.4.10) to ensure that DWI and ADC images have the same resolution, field of view (FOV), and orientation as T2WI. The two radiologists involved in the image evaluation used Insight Segmentation and Registration Toolkit  to manually draw the region of interest (ROI) on the T2WI image layer by layer, then copy it to DWI and ADC images to ensure the consistency of volume of interest (VOI) sketches in different sequences. To ensure the stability and repeatability of the annotations, the same radiologists repeated the annotation procedure after a week, and all the annotations were re-examined by another senior radiologist with 15 years of experience in prostate MRI diagnosis.First, histogram-based intensity standardization method was used to standardize the bpMRI, and the voxel size of image was resampled to 1 mm × 1 mm × 1 mm. Then, the axial T2WI, high b-value  was used to extract and select the features of each mode with reference to VOI . A totalAll samples were randomly divided into the training set and the testing set at a ratio of 7:3. First of all, most of the features were excluded by using the mRMR method, and only 30 features with the least redundancy and the greatest correlation with the target label were retained. The radiomic signature was constructed based on the features selected from the training set. Due to the large number of features extracted, there was redundancy between features, and some features had little or no correlation with the modeling object. Then, least absolute shrinkage and selection operator (LASSO) was used to select the most useful features. Finally, a logistic regression was trained using the remaining features, and a radiomic score was calculated as the linear combination of the selected radiomic features and corresponding coefficients.In addition, we have employed the multivariate logistic regression analysis to combine clinical characteristics, including age, PSA value, zone of the lesion, prostate volume, with radiomic features, and identify the independent predictors of csPCa among PI-RADS V2.1 category 3 lesions in the training set. Furthermore, a clinical–radiomic nomogram was built to provide clinicians with a quantitative tool for csPCa identification. The area under receiver operating characteristic (ROC) curve (AUC) was adopted to evaluate the accuracy of the nomogram in identifying csPCa.AUCs of clinical, radiomic, and clinical–radiomic model were basically evaluated with histopathological manifestations in both training and testing set. Moreover, DeLong’s test was used to compare the ROC curves of the nomogram, radiomic, and clinical model, and Hosmer–Lemeshow test was performed to evaluate the goodness-of-fit of the calibration curve between nomogram and the pathological results. Furthermore, decision curve analysis (DCA) was used to quantify the net benefit of each predictive model.www.Rproject.org) was used for quantitative feature analysis. The Shapiro–Wilk test was performed to evaluate the normality of data. According to the results of normality test, independent sample t-test or Mann–Whitney U-test was used to detect the difference of clinical characteristics between non-csPCa group and csPCa group. Multivariable logistic regression was performed using “rms” package to construct the clinical–radiomic nomogram. ROC curves and AUCs were established using “PROC” package to evaluate the diagnostic accuracy of each predictive model. Calibration curve and Hosmer–Lemeshow test were performed with “ModelGood” and “DescTools” packages, respectively. Finally, the “rmda” software package was used for decision curve analysis. p < 0.05 indicated that the difference was statistically significant.To identify csPCa among PI-RADS 3 lesions on bpMRI, we trained the binary classification models by labeling csPCa as 1 and both ciPCa and benign lesions as 0. R language software , 70 cases of benign hyperplasia (67.9%), and 5 cases of ciPCa (4.9%); 44.7% (46/103) lesions were located in TZ, and 55.3% (57/103) lesions were located in PZ. The prevalence of csPCa located in TZ and PZ was 11.7% (12/103) and 15.5% (16/103), respectively. Among the 28 patients with csPCa, 13 cases were ISUP grade 2 (GS = 3 + 4), 6 cases were ISUP grade 3 (GS = 4 + 3), 7 cases were ISUP grade 4 , and 2 cases were ISUP grade 5 (GS = 5 + 4). The diameter of lesions ranged from 5.2 to 18.6 mm, with an average diameter of 9.5 mm ± 3.4 mm Table 2.p = 0.041). Therefore, these two clinical factors can be used as independent predictors , which indicated that the probability of csPCa was positively correlated with Radscore. The radiomics feature model has an above-average predictive efficiency for csPCa in PI-RADS 3 lesions, with an AUC of 0.71  and inert PCa (GS < 4 + 3). Zhang et al. developed and verified a non-invasive radiomic model based on MRI to distinguish between inert and invasive PCa before treatment. They finally selected nine radiomic features to construct imaging tags with 0.944 for sensitivity, 0.786 for specificity, and 0.901 for AUC in the validation set . RadiomiGiambeluca et al. introduced the concept of texture analysis into the study of PI-RADS 3 lesions for the first time . They fop < 0.05). In our opinion, the reason may be that the sample size of csPCa in the testing set was too small (only six cases) to accept comprehensive verification, thus caused a certain randomness in the result. In addition, malignant epithelial cells of sectional csPCa in PI-RADS 3 lesions were sparsely arranged and distributed along the acinar, which overlapped greatly with some benign diseases such as inflammation, hyperplasia, and fibrosis, resulting in insignificant changes in MRI signal. Age was associated with Gleason score, and the older the age, the higher the risk of poor histology. Studies have shown that the ORs and 95%CI of poor histological prognosis of prostate were 2.21 (1.30–3.76) and 1.58 (0.90–2.76) in men over 80 years old compared with those under 70 years old, respectively  Special Program for Diagnosis and Treatment Technology of Clinical Key Diseases in Suzhou (LCZX202001), (2) Gusu Health Talent Project of Suzhou (GSWS2020003), (3) Suzhou Key Laboratory of Health Information Technology (SZS201818).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the VEST IV trial, the author concluded that external stenting of saphenous vein graft mitigates its remodeling and also significantly reduces the diffuse intimal hyperplasia and development of lumen irregularities at 4.5 years after coronary artery bypass grafting surgery. We also have valuable a suggestion in addition to external stenting that might nullify the pathology caused by the stent and might enhances graft patency. We have read the VEST IV trial  by DavidWe would like to bring a major pathology incurred by stent for the author’s notice. In VEST IV trial, they have shown that early graft failures in both groups  were comparable. Restrictive annuloplasty for mitral regurgitation would result in functional mitral stenosis/ patient–prosthesis mismatch [We recommend few modifications in the VEST trial, which will help them, achieve better venous graft patency.The arterial system is closed, pressured, and with a potentially bidirectional flow. The presence of a valve in the arterialized venous conduit may hinder this functionality. Whitney et al. did a detailed study using time-lapse angiography throughout 11-year and demonstrated that venous valves cause turbulence and dilatation of the vein segment nearer to the valve .With a flow rate of 10–30 ml/min, valves begin to close and the vein valve’s closure creates a pressure trap in the segment distal to the valve. Segmental hypertension results in an accelerated atherosclerotic process in vein grafts . Mills  found thA simple valvulotomy may be used to split these valves allowing a more even flow and avoiding the air trapping and thrombus formation in the valve recesses. A potential cause for perioperative infarction or early graft failure may be avoided. The potential risk factor for valvulotomy was intimal injury and residual valve inside the vein segment. Using modern valvotomes, studies show that the intimal injury was only minimal and the remnant valves also get resolved during the due course .A study by Monsefi et al. revealed that patients with valvulotomized SVG (v-SVG) had good clinical outcomes with a patency rate of all v-SVG was 97.1 versus 95.8% of arterial grafts at 18 ± 6 months postoperative period [In 2018, Anli et al. revealed that the mid-term patency of v-SVG was 96.1% in comparison with 96.7% for the arterial grafts at 3.1 ± 2 years [Lajos et al.  found thSVG on average will have 10–12 valves with more valves located in a below-knee position . While pIt’s evident from the VEST IV trial that intimal hyperplasia was hindered by applying external stents thereby delayed graft failure. By deploying external stents over the vein in addition to valveless vein/v-SVG, we can avoid the potential risk factors for both early and late venous graft failure.