GWP) and cumulative energy demand (CED) of developing a coastal ecosystem enhancement programme in the estuary region of Galicia, north-western Spain. The focus is on the processes involved in creating green artificial reefs (GARs): manufacture, transport and installation. The starting point is the supply chain for the green artificial reef (GAR) units; greenhouse gas emissions (GHG) and energy needs for each phase are analysed. Various scenarios are considered to determine which options are indeed available when it comes to establishing the supply chain. Different types of energy supplies, different options for the location of production centres, as well as different means of transport were studied. Results reveal the critical phases for selecting how the GAR units must be produced, transported by road and sea and then installed in their permanent location.The principle of sustainability should condition a project in which artificial reefs are being installed to protect biodiversity as well as enhance costal ecosystems. In particular, this principle should be taken into account in the logistical processes related to manufacture and transport. This study assesses the global warming potential ( The coastal regions of Galicia (NW Iberian Peninsula) stand out for their geographical features and for the economic resources they provide. Indeed, geography experts use the term “ría” in English because of the unique way in which the finger-like Galician estuaries are formed with the contrasting heights of the land and sea. In this singular environment, the ecosystem boasts a high level of natural resources: marine species make up the first link of the food chain and are crucial to a thriving local economy .Nonetheless, Galician estuaries also need protection. On the one hand, the fishing and seafood gathering industries, which have elevated levels of production and excellent economic prospects, can have a negative effect on the environment. The impact of other human activity can also be felt, including building and water sports, among others . With thOne way to protect biodiversity and marine species is to install artificial reefs (AR). They behave in a similar way as their natural counterparts by offering a refuge for different species to reproduce. These are called production modules. At the same time, ARs in the form of protection modules provide a shield; fishing methods severely harmful to biodiversity are thus impeded . For theA GAR  programme is needed to enhance coastal ecosystems in Galicia. For such a purpose, groups of GAR units (GAR groups) are necessary for the entire estuary region. The reader should bear in mind that, at the time of producing a GAR group, a careful planning is a key factor for reducing the potential impacts on the environment . In factCarral et al.  developeGWP) and cumulative energy demand (CED) indicators associated with a programme to enhance the coastal ecosystems of the Galician estuaries. This programme is based on the use of green artificial reef (GAR) groups. Manufacturing, transport and installation of the units are within the scope of this study (GHG), as well as the solution that requires the least amount of energy. To the best of the authors’ knowledge, this is the first time that the production and distribution life cycle stages of GARs are analysed from an environmental point of view. The results of this work together with the ones presented in previous studies [The main objective of this study is to assess both the global warming potential  and cumulative energy demand (CED) are the two environmental indicators used in this study. The reader should bear in mind that the European Union (EU) prioritises the reduction of both energy consumption and emissions [GWP indicator is an index for estimating the extent to which an activity has an impact on global warming through the atmospheric emission of greenhouse gases (GHG) [2-eq. As indicated in missions . On the es (GHG)  and the CED is useful to measure the primary energy used throughout the entire supply chain for the reef units. In other words, this indicator calculates the energy that is used during a specific life cycle stage of a product [CED is measured in megajoules (MJ).On the other hand,  product . It firs product ,15. CED GWP and CED factors used in this study are a proposal from the authors based on the existing literature. All the results of this work are referred to a GAR unit (or to a certain number of GAR units). In other words, the GAR unit  can be considered as the functional unit (FU) used here. The mass of each GAR unit is 7.5 tonnes (average mass). Consequently, it can also be said that the functional unit is a quantity of mass. According to Boschiero et al. [Consequently, this study’s scope covers the moment in which the components for the GAR units arrive at the manufacturing plant to the final phase in which GAR units are transported by sea to their permanent locations fixed to the seabed . The proo et al. , in a coGHG emissions are assigned to the total quantities of manufactured product, which is the only outcome of the system. The manufacturing and distribution system examined in this study is divided into the phases outlined in It is assumed that all components will enter and exit the manufacturing plant. Thus, the energy consumption and The manufacturing of GAR units entails several phases within the supply chain . This caMany of the areas in which the ecosystem enhancement will take place have estuaries with small fishing ports whose pier surface area is too small for manufacturing and storing the GAR units. Such piers can only be used for specific, one-off tasks.If production is centralised , road transport is required to carry the units to the port. To minimise the distance travelled, Gayo Romero  recommenIn terms of maritime transport, the points of departure will be determined with the goal of minimising the time needed . The lifSmall vessels are generally provided with lifting equipment suitable for loading and unloading at ports and piers unequipped with cranes. In the case of GAR units, a 10 t safe working load (SWL) is needed for the crane to carry out port activities and take part in fastening the reef at sea. While the work boat has on-deck storage, the mini bulk carrier uses a cargo hold with a sliding hatch. The different cargo systems affect the number of GAR units that can be stowed. However, the vessel cranes have similar capacities for hoisting and lowering. The reader can find in For the installation process (positioning and anchoring), the vessel has to change its position, anchoring at different points. The mini bulk carrier is larger than the special service work boat, consequently, its manoeuvrability is expected to be hindered during the task of fixing the unit to the seabed. The system for lowering the GAR units is by means of a cable. Great precision is required during this stage. Consequently, it must be supervised by divers in constant communication with the vessel to guide the entire process.López-Davalillo  classifiIt is also possible to make a more detailed classification by zones: (i) Northern zone including Ribadeo, Foz, Vivero, Barquero, Ortigueira, and Cedeira estuaries; (ii) Costa Ártabra formed by Ferrol, Ares, Betanzos, and La Coruña estuaries; (iii) Costa da Morte (or Costa de la Muerte in Spanish) with Corme y Lage, Camariñas, Corcubión, Muros, and Noya estuaries; (iv) Rías Bajas including Arousa, Pontevedra, Aldán, and Vigo estuaries. The Northern zone belongs to the Northern Estuaries, while Costa Ártabra and Costa da Morte are part of the Central Estuaries. Finally, Rías Bajas is the only zone of the Southern Estuaries.i is used to distinguish among the four different zones. Subindex j is used for a similar purpose but, this time, for distinguishing among the estuaries of a specific zone. Each estuary needs a specific number of GAR groups. Each GAR group consists of 60 GAR units. Consequently, subindex k is used for differentiating among all GAR groups of a specific estuary. For instance, Ribadeo will need 180 GAR units, that is, three GAR groups. Therefore, for Ribadeo, k will vary between 1 and 3. Letter A is used for identifying a GAR group. For practical purposes, A always presents three subscripts  with the objective of indicating the zone, the estuary as well as the number of the group, respectively. For example, Ribadeo is the first estuary (j = 1) of the Northern zone (i = 1) and it has three GAR groups: 1,1,1A, 1,1,2A and 1,1,3A. As each GAR group has 60 GAR units, the following notation is used for identifying a specific GAR unit: i,j,k,lA. Subindex l always varies from 1 to 60. By way of example, 1,1,1,1A is one of the 60 GAR units belonging to the first GAR group (k = 1) to be installed in Ribadeo (j = 1), first estuary of the Northern zone (i = 1).Subindex H, that is, the point of departure. Consequently, all harbours present two subscripts i and j for identifying the zone and the estuary, respectively. In other words, 1,1H is the harbour of the first zone  for the first estuary . It is important to note that, on a practical level, two different estuaries can have the same harbour, as is the case for Ferrol, Ares, Betanzos, and La Coruña estuaries in 2,1H = 2,2H = 2,3H = 2,4H = Lorbé harbour), among others. All the GAR groups  and their corresponding GAR units  of a specific estuary have the same point of departure . Nevertheless, each GAR group  (and their corresponding GAR units) belonging to a specific estuary has its own point of arrival. Consequently, the points of arrive will be designated as i,j,kPA. It will be assumed that the point of arrival will be the same for all the GAR units belonging to a specific GAR group k.Each estuary has an associated harbour The reader can find in i,j,k,lA) are transported to the corresponding port  from the manufacturing plant. Two scenarios are considered for the manufacturing process. In the first case, each zone i has its own manufacturing plant. Consequently, the manufacturing plant will be designated as iMP. In the second one, the same manufacturing centre is used for all zones. It will be noted as CMP .The enhancement of each region entails installing several GAR groups according to surface area and the peculiarities of each case. A total number of 4080 GAR units are needed for the Galician estuaries . All GAR units  belonging to a specific GAR group  must be transported from the manufacturing plant (iMP or CMP) to the harbour  and, after that, to the point of arrival in the sea . The first is a road transport in which the distance only depends on the estuary j and the zone i. In the case of the second scenario, subindex i is only relevant for the harbour, since the location of the manufacturing plant is always the same for the four zones. The distance between the manufacturing plant and the harbour will be noted as i,jLd. The second is a maritime transport. In this case, the point of arrival  also changes according to subindex k, as explained before. In other words, the distance between a specific harbour  and the point of arrival of a specific GAR group will depend on subscripts i, j and k. Consequently, it will be noted as i,j,kMd. For estimating i,jLd it is first necessary to determine the exact location of the manufacturing plants for both scenarios. This will be explained later in Taking into account all the above, each GAR unit (iMP and CMP), a harbour  and the point of arrival  for a specific GAR group .The reader can find in iMP and CMP). For both cases, a static location method is employed. This method is based on the use of the centre of gravity to determine the coordinates [As previously indicated in rdinates . It pinprdinates .In most cases, the location determined by this method is not ideal, but it is a good starting point. Thus, once the theoretical location has been obtained by using the corresponding equations , only ontEI) across the different phases (t). They are included in In the particular case of the manufacturing process, it is necessary to consider the energy inputs (GWP (electricityGWP) as well as the consumption or use factor for CED (electricityCED) associated with the production of 1 kWh of electricity. The Spanish electricity grid mix is used for such a purpose [It is now necessary to determine the emission factor for  purpose , and the purpose ,26,27,28GWP for manufacturing 1 GAR unit is estimated from Equation (1) and it is measured in kg CO2-eq./FU.Therefore, CED for manufacturing 1 GAR unit is calculated from Equation (2). The units of measurement are MJ/FU.Similarly, 2-eq. and MJ, respectively) derived from the complete enhancement programme in the estuary region of Galicia (4080 GAR units):It is also possible to estimate the total value of both indicators  associated with the production of 1 kWh of electricity are included in On the other hand, a second scenario is considered in which only wind, hydroelectric, solar photovoltaic, and solar thermal alternatives participate in the Spanish electric grid mix. This is an ideal scenario that will be used to determine the extent to which results could be improved by using the most common renewable sources. It is important to note that this scenario may not be realistic. The corresponding emission factors  and (2), replacing electricityGWP and electricityCED by electricity−idealGWP and electricity−idealCED, respectively. After that, Equations (3) and (4) can also be applied.GWP and CED. In this sense, this study only takes into account direct energy outputs and emissions. In other words, other related activities such as building or recycling the vessels, among others, are out of the scope of this work. Road and maritime transport will be analysed separately. It is a question of converting the distance covered and the time spent on loading (including stowage of cargo), unloading and installation operations (positioning and anchoring the GAR units) into an amount of consumed fuel. In the case of transport and installation phases, it is preferable to directly estimate the total value of both indicators derived from the complete programme in the estuary region of Galicia (4080 GAR units):On the other hand, transport and installation phases can also present a considerable impact in terms of both Transport−InstallationTotal − GWP is the total number of kg of CO2-eq. emitted during the transport and installation phases of all GAR units. RoadTotal − GWP measures the number of kg of CO2-eq. emitted during the ground operations derived from the complete enhancement programme in the estuary region of Galicia (4080 GAR units). MaritimeTotal − GWP is equivalent to RoadTotal − GWP but, this time, for the maritime operations. Similarly, Transport-InstallationTotal − CED is measured in MJ and it is associated with the transport and installation phases of all GAR units (Equation (6)). Consequently, it is the sum of the energy consumed during ground  and maritime operations .In Equation (5), RoadTotal − GWP:iN is the number of estuaries j belonging to zone i. It can be deducted from i,jLd is the distance between the manufacturing plant and the harbour and it is measured in km. Parameter i,jn is the number of trips needed to transport all GAR units  is used for estimating MaritimeTotal − GWP:Equation (7) is used for estimating AR units  linked tjN is the number of GAR groups k belonging to estuary j and it can be deducted from i,j,kMd is the distance between the harbour  and the point of arrival  for a specific GAR group . It is measured in nautical miles (Nm). Parameter i,j,kn is the number of voyages needed to transport all the GAR units  belonging to a specific GAR group . NmGWP is the emission factor for 1 Nm of maritime transport and it is measured in kg CO2-eq./Nm. i,j,kT is the time in hours necessary for loading, unloading and installing the GAR units transported in each voyage and, for calculation purposes, it can be split between the outward and return trips. Finally, hGWP is the emission factor per hour for the loading, unloading and installation operations and it is measured in kg CO2-eq./h. RoadTotal − CED can be estimated by using Equation (9):kmCED is the amount of energy consumed per km in road transport, measured in MJ/km. Similarly, MaritimeTotal − CED is calculated through Equation (10):In Equation (8) NmCED is the energy use or consumption factor for 1 Nm of maritime transport and it is measured in MJ/Nm. hCED is the energy factor per hour for the loading, unloading and installation operations and it is measured in MJ/h.In Equation (10), kmGWP, in fact, depends on the type of vehicle used for the road transport. As previously indicated in i,jn. Consequently, it is not necessary to distinguish between trips with and without cargo, since kmGWP can be the average value for the two possible types of trip, as is the case in this study.With the objective of applying Equations (7)–(10), it is necessary to know the values that the different emission and energy use factors adopt. NmGWP also depends on the type of vessel used for the maritime transport  4 GAR units per hour in their final locations , while the special service work boat allows the installation of 6 GAR units per hour. Furthermore, the loading process in the port, in terms of time consumed, is the same for both types of vessel. In other words, it is possible to load 15 GAR units per hour. This process includes stowage of cargo. Consequently, both types of vessels will need 272 h for such a purpose. Therefore, the values that i,j,kT adopt for each type of vessel can also be determined. Moreover, in this study it is assumed that the distance between a specific harbour  and the point of arrival  is always the same: 3 Nm. Therefore, i,j,kMd is always equal to 3 Nm.In this study, it is assumed that an articulated truck can transport 3 GAR units in each trip while a rigid truck can only transport 2 GAR units. As previously indicated in With the information provided so far, it is possible to perform certain intermediate calculations that will be used to obtain the results presented in the following sections. By way of example, it is possible to estimate the total number of nautical miles that each type of vessel has to travel for transporting all the GAR units. In the particular case of the mini bulk carrier, 68 outbound trips are needed for transporting all the GAR units (4080/60 = 68). This implies that there will also be 68 return trips, that is, 136 voyages of 3 Nm. Consequently, mini bulk carrier travels 408 Nm (136 × 3 = 408); GWP (kg CO2-eq.) and CED (MJ) derived from the complete restoration of Galician estuaries can be estimated by using the following equations:Finally, the total value for iMP and (ii) CMP. The coordinates provided for iMP refer to the following industrial estates: Xove (near Celeiro), Espíritu Santo (south of Lorbé), Cee (outside Corcubión), and Nantes (on the outskirts of Portonovo). The coordinates for CMP are the ones of Santa Comba industrial estate. The distances  between the manufacturing plant and the corresponding harbours are also shown in iMP, the articulated truck travels 83,600 km while the rigid one travels 125,400 km. On the other hand, for the centralised manufacturing plant (CMP), With the information included in The results for the manufacturing process are included in It is also possible to show the results separately for each one of the subprocesses contained in iMP and CMP).The reader can find in GWP and CED indicators. This is also true for maritime transport (iMP or CMP), the articulated truck always outperforms the rigid one. It is important to remember that the articulated truck can transport 1 GAR unit more than the rigid option per trip. Consequently, the distance to be covered by the articulated truck is considerably lower than that for the rigid truck. In other words, the difference in the kilometres between the two types of trucks is so large that the rigid option never performs better for a specific scenario, although it presents the lowest emission and energy consumption factors (CMP) is associated with a longer transport distance in comparison with the other manufacturing option (iMP). Consequently, GWP and CED results are always better for the iMP scenario.From the results presented in ransport . These r factors . In the Similar results are included in GWP and CED indicators [2 emitted by the mini bulk carrier (diesel) is the sum of emissions from transport  and the emissions from the other activities . In all cases, the time used for port operations and the installation of GAR units presents a larger contribution to GWP and CED indicators than the maritime transport itself. This is the reason why, for a specific type of fuel, the service work boat always presents better results than mini bulk carrier. This is very relevant since the service work boat has to travel four times the distance covered by the mini bulk carrier, while it only presents a time reduction of about 25% compared to mini bulk carrier. In other words, the time spent on installation and port operations resulted to be more relevant than the maritime distance. While in road travel the higher load capacity stands out, the superior manoeuvrability of the vessel is the deciding factor for maritime transport.From the results included in dicators ,33,34,35GWP and CED. At the same time, it is also interesting to know the value that these two environmental indicators adopt for the worst option. Multiple scenarios can be defined by combining the different options for the electricity grid mix, the type of truck and its fuel, the type of vessel and its fuel, and the type of manufacturing plant model . Nevertheless, from all the potential scenarios, what is important is to select the combination that produces the least impact in terms of both GWP and CED results for the scenarios defined in The GWP and CED indicators. Although there is some difference between the worst and best scenarios, the improvement cannot be said to be as significant (in relative terms) as in other phases. Manufacture is a relevant stage under the Spanish electricity grid mix scenario. Nevertheless, under the alternative electricity grid mix, manufacturing 4080 GAR units hardly contribute to both impacts compared to the rest of phases analysed in this study. In other words, the use of renewables in the manufacturing process is an obvious way to reduce both GWP and CED. Nevertheless, the electricity grid mix of a specific country depends on economic, social and geopolitical issues that are not necessarily in line with environmental targets. It is important to note that the results for road transport change considerably from one scenario to another. For the two worst case scenarios, its contribution to GWP and CED is about 1.8 and 2.8 times  lower than the ones for maritime operations. Despite this, it is by no means negligible. However, for the two best scenarios, road transport contributes around 4.5 and 6 times  less than maritime phases. This highlights the importance of selecting the best scenario for road transport. Based on the results shown in GWP) and the cumulative energy demand (CED) indicators were analysed for the complete coastal ecosystem enhancement in Galicia (North-western Spain) by using green artificial reefs (GAR units). In particular, manufacture, transport and installation phases were studied. Different alternatives were considered in terms of energy sources, location of the manufacturing plants and transport. Consequently, it was possible to determine the best scenario. The most important conclusions drawn from this study are:GWP and CED is the maritime one. Road transport presents a considerable variability between the worst and best possible scenarios.The results show that the phase that has the greatest potential impact in contributing to GWP and CED indicators.The use of renewables in the electricity grid mix has a positive impact on the manufacturing of GAR units in terms of both GWP and CED), since they imply the shortest distance transport.In terms of road transport, the key factor is to reduce the distance. Consequently, production by zones and the articulated trailer resulted to be the best options (GWP and CED indicators.Manoeuvrability resulted to be more important than distance in terms of maritime transport and operations. Consequently, the special work boat obtained better results than mini bulk carrier for GHG emissions and energy consumption.The use of LNG as fuel for vessel and truck engines lead to reductions in In this study, the global warming potential (As for future work, it would be extremely worthwhile to continue exploring ways to optimise maritime transport and to improve the design of auxiliary machines used during the installation of GAR units. Other lines of work can be focused on studying a greater number environmental indicators.


Heroin can be detected and quantified by certain analytical methods, however, forensic professionals and criminal laboratories study for cheaper and faster detection tools. Surface-enhanced Raman spectroscopy (SERS) rises as a possible alternative tool with its widening application spectra. There are few studies regarding Raman and SERS spectra of heroin and its metabolites, which are unfortunately controversial. In this study, we compared five different surfaces in order to find out more efficient Raman-active substrate for opiate detection and rapid quantification of heroin and its metabolites in saliva.Morphine standard material was used to identify proper surface for SERS analysis of opiates. Heroin and its metabolites  were calibrated between 50 ppb and 500 ppm and quantified on AuNRs with signal enhancement of silver colloids in saliva. Raman microscope with a 785-nm laser source was used.Obtained results showed that heroin and its metabolites can be detected and quantified in saliva samples using a SERS-based system. Additionally, the present study revealed that synergetic effect of a specific gold nano-surface with ability controlling liquid motion and silver nanoparticles increase band numbers and intensities. Therefore, we suggest a fast, accurate and cost-effective method to detect and quantify heroin in biological fluids. Papaver somniferum [1]. In a survey study from the United States [2], it has been stated that heroin use is extending in the population from city centers to urban areas and to different socio-cultural classes. According to World Drug Report 20171United Nations Office on Drugs and Crime (2018). World Drug Report 2017 - 2 [online]. Website https://www.unodc.org/wdr2017/field/Booklet_2_HEALTH.pdf [accessed 27 03 2018]. and European Drug Report 20172EMCDDA (2017). European Drug Report 2017: Trends and Developments [online]. Website u2987 [accessed 16 12 2019]. heroin is still the leading substance for addiction among high-risk opioids. Heroin addiction is still a widespread problem as a significant cause of morbidity and mortality due to increased risk of cardiopulmonary diseases, chronic infections including Hepatitis C and hepatic failure2. Abuse of illicit drugs, including high-risk opioids, is still a major problem globally. It is responsible from increasing mortality and morbidity, particularly in young population. Heroin is one of the most commonly abused illicit drugs, which is diacetyl product of morphine that most commonly extracted fromThe use of surface enhanced Raman spectroscopy (SERS) increases in forensic sciences applications. SERS is a potentially fast and cost-effective analytical method requiring very little amount of sample. There is a large number of SERS based studies dealing with identification of chemical trace evidences and certain body fluids [3–10]. On the other hand, there are few SERS studies regarding identification of toxicological substances including morphine, cocaine and heroin directly dealing with toxicological analysis of heroin and its metabolites with SERS in different biological mediums [11–15]. However, these studies dealt with identification of chemical structure of drugs without measuring their concentrations in biological fluids.Detection and quantitation of heroin and its metabolites in biological fluids is essential for both healthcare facilities and legal authorities. In healthcare facilities including psychiatric institutions devoted to orehabilitation and treatment of alcohol and drug addiction, enzymatic methods and paper-based tests are used for drug detection, since they are cheaper and easier. However, it is impossible to use these methods for legal purposes because of their false results. In toxicology laboratories, liquid chromatography (LC) coupled with tandem mass spectrometry and gas chromatography mass spectrometry systems are the leading analytical methods to analyze illicit drugs for legal purposes. These quantitative methods are more reliable compared to enzymatic methods, while systems and analyses are quite expensive and time consuming compared to other methods . However, SERS is a promising method that allows an accurate identification and quantification of illicit drugs, which can potentially be used, in both healthcare facilities and in toxicology laboratories performing analyses for legal purposes. Therefore, this study based on the question of “Is it possible to measure the concentration of a drug or its metabolites in a biological fluid?”The aims of this paper are to find out most proper Raman active surface for opiates and accurate SERS spectra of heroin and its metabolites, and develop a potentially more selective and faster method for detection and quantification the concentration of heroin and its metabolites based on SERS measurements in medium of saliva. MM (morphine monohydrate) was used to find out the proper nano-surface for SERS analysis of opiates since morphine has similar chemical structure with many other opiates including heroin and other heroin metabolites. MM as standard material in methanol was diluted with methanol to obtain 250, 500 and 1000 ppm concentrations. These concentrations were used as controls for each other. Diluted MM solutions were mixed with AgNP colloids with 1:1 volume and vortexed. 2 µL of final solutions containing MM concentrations of 125, 250 and 500 ppm were dropped on AgNRs, AuNP/nanocellulose surface, AuNP/polyanilinesurface, TLC silica gel surface and AuNRs. Following an incubation period of 10 minutes, SERS spectra were obtained from at least 5 different area of each sample. Instrument parameters were as follows: 10x objective, 30 μm laser spot size, 150 mW laser power, and 40 s acquisition time. Baseline correction was performed for all measurements. Methanol for lab analysis, and acetonitrile gradient grade for LC were purchased from Merck KGaA . Heroin standard, in powder form, in 99.8% purity. Morphine monohydrate (MM) 1.0 mg/mL calibrated standard solution in methanol, monoacetyl morphine (6MAM) 1.0 mg/mL calibrated standard solution in methanol and morphine-3-β-glucuronide (M3B) 1.0 mg/mL calibrated standard solution in methanol was obtained from Lipomed AG . Aniline (C6H5NH2) was obtained from Sigma-Aldrich Chemie GmbH  and it was distilled under vacuum before using. Lithium perchlorate (LiClO4) was purchased from Sigma-Aldrich Produktions GmbH . Nanocellulose, hydroxylamine hydrochloride (NH2OH.HCl), and hydrogen tetrachloroaurate (HAuCl4) were obtained from Sigma-Aldrich Chemie GmbH . Polyethylene glycol (PEG) 400, methanol, silver nitrate (AgNO3), and sodium hydroxide (NaOH) were purchased from Merck KGaA . AgNS was developed using technique that previously described in the study by Yilmaz et al. [18]. The nanocellulose covered AuNPs was prepared by reducing HAuCI4 in the presence of the PEG 400 in an aqueous solution according to the previously reported procedure in the literature [19]. 100 µL of 1 × 10-2 M concentrated aqueous solution of HAuCI4, 0.6 g nanocellulose, 2 mg PEG and 7 mL deionized water were mixed and heated at 90 °C for 20 min to obtain a wine-red solution. The resulting solution was centrifuged, and AuNPs were separated from the unreacted nanocellulose and PEG solution.Reactions were generally carried out in 50 mL vials. 1 mmol of aniline was dissolved in 10 mL of carbon tetrachloride and stirred for a while to obtain a homogenous solution (A) then a certain amount 0.86 g nanocellulose and 1 ml of 1 × 10-2 M concentrated aqueous solution of HAuCI4 were dissolved in 10 mL of 1 M HAuCl4 aqueous solution and after several minutes of stirring, a homogenous solution (B) was obtained. Then solution (A) was transferred into solution (B), to acquire an interfacial reaction between these two phases. After 10 min, green PANI (polyaniline) formed at the interface and then gradually diffused into the aqueous phase. The reaction at the interface was allowed for 2 h. After 2 h, the entire aqueous phase was filled with dark green PANI, while the organic layer was an orange color. Then, the prepared PANI was collected by filtration and washed several times with methanol, acetone, and distilled water. Finally, it was dried in a vacuum oven at 40 °C for 12 h [20]. The aluminum side of commercial TLC Silica gel aluminum sheets  was used to obtain SERS signals of MM in this study.Anisotropic gold nanorod arrays were produced on the BK7 glass slides via the oblique angle deposition (OAD) technique as previously described in the study by Yilmaz et al.[18]. Due to oblique placement of nanorods the surface had ability to control liquid motion and prevent uncontrolled aggregation.The silver nanoparticles were synthesized by utilizing chemical reduction method suggested by Leopold and Lendl [21]. All chemicals were prepared in double distilled water. In typical experiment, the concentration of silver nitrate was set to 10-3 M in the prepared reaction mixtures. 1.5 × 10-3 M hydroxylamine hydrochloride was used in the final reaction mixture for reduction of silver nitrate. In our experiments, NaOH added to the hydroxylamine solution can adjust the final pH of solution. Solution was mixed vigorously during this process and the color change was distinguishable . Finally, this solution was kept for incubation overnight in the dark place.After the formation of five different SERS-active substrates  array, silver nano-surface (AgNS), AuNPs/nanocellulose and AuNPs/polyaniline) for MM detection, MM mixed with silver colloids were dropped on to the substrates, SERS spectra were obtained and given in Figure 1. DeltaNu Examiner Raman microscope  with a 785-nm laser source, a motorized microscope stage sample holder, and a CCD detector was used to detect heroin. Instrument parameters were as follows: 10x objective, 30 μm laser spot size, 150 mW laser power, and 40 s acquisition time. Baseline correction was performed for all measurements.After the decision of most proper SERS surface, varying concentrations of MM, M3B and 6MAM samples were detected using AuNRs as SERS substrate. The SERS spectra corresponding to each concentration were recorded. The SERS intensity versus concentration calibration curves for MM, M3B and 6MAM (50 ppb–500 ppm) were obtained by calculating the average reading of the response of 5 different areas. The relation between the MM, M3B and 6MAM concentrations and the peak intensity at 627 cm−1 was used for prediction of exponentially. The coefficient of determination (R2) and the detection limit of heroin and its metabolites were calculated. Heroin in powder form was dissolved in methanol in 5000 ppm concentration. The solution was diluted with methanol to achieve concentrations of 10 ppb, 50 ppb, 1 ppm, 100 ppm, 500 ppm, and 1000 ppm heroin solutions. Equal amount of AgNP solution was added to heroin samples, and final solutions of heroin were in concentrations of 5 ppb, 25 ppb, 500 ppb, 50 ppm, 250 ppm, 500 ppm and 2500 ppm. The relation between the heroin concentrations and the peak intensity at 627 cm−1 was used for prediction of exponentially. The coefficient of determination (R2) and the detection limit of heroin and its metabolites were calculated. Being one of the most common biological fluids analyzed in terms of heroin and its metabolites detection, saliva was used to simulate biological specimens. Saliva samples were obtained from a healthy adult volunteer of free from drug or alcohol use. Stock solution of heroin in methanol in concentration of 1000 ppm was diluted with methanol. 200 µL sample was spiked with heroin, MM, 6MAM and M3B solutions in different concentrations, and vortexed for 5 min. 400 µL of acetonitrile was added to spiked samples to aggregate proteins and to achieve chemical homogenization, and vortexed for 5 min. Solution was centrifuged for 20 minutes in 12,000 rpm. 200 µL of supernatant was taken and mixed with 200 µL AgNP solution and vortexed for 5 min. Solution with AgNP was dropped on to gold surface in amount of 2 µL to gather SERS spectra. Final solution was in concentration of 125 ppm, the similar procedure was performed for the other concentrations . After an incubation period of 10 min under normal room conditions, SERS spectra were gathered from 5 different points of the spiked sample stains. Mean values of bands’ intensities of 5 spectra were used for each heroin concentration.Spiked heroin, MM, M3B and 6MAM samples in blank saliva matrix were prepared in concentrations of 650 ppb, 30 ppm and 100 ppm to evaluate calibrated formulae and achieve measurements. The author that analyze samples kept blind and did not informed about the concentrations of samples. Spectra of samples were obtained from five different areas and concentrations were evaluated according to intensity-concentration match point in calibration curve for saliva matrix. Local Ethics Board of Hacettepe University approved this study with decision number 2016/06-02.The SERS spectra of each developed SERS-substrates were examined according to blank and MM samples . After statistically determining extreme values as strong bands and excluding bands associated with surface itself, TLC surface showed six strong bands at 532, 627, 726, 967, 1049 and 1332 cm-1 . There were also seven strong bands gathered MM spectra from AgNS surface at 443, 532, 627, 934, 1049, 1109 and 1332 cm-1, respectively . AuNRs nano-surface produced the most intense bands at eight different SERS bands at 321, 443, 534, 627, 823, 935, 1033 and 1332 cm-1. AuNPs/polyanniline surface revealed the most intensive noise amongst all five surfaces. There was no identifiable band exceeding the noise level . AuNPs/nanocellulose surface showed the least back noise . However, it revealed only two strong bands at 627 and 797 cm-1after exclusion of bands assigned to surface.After determining the most proper surface for opiates by evaluating SERS signals of MM, AuNRs were used in detection and quantification of heroin and its metabolites. The spectra of heroin, M3B and 6MAM obtained using SERS is shown in Figure 3. Heroin and its metabolites spectra have one prominent and consistent band at 627 cm-1 and this could be identified in extremely low concentrations. Heroin spectra also showed strong bands at 443, 531, 1107, 1245 and 1338 cm-1. Being the most stable band, intensities of the band at 627 cm-1 were utilized for calibration curves for heroin and its metabolites . The analyses of parameters for MM, M3A, 6MAM and heroin samples using AuNRs as SERS substrate were evaluated in Table 1. Since the saliva samples had the most consistent and stable bands with higher intensities, saliva matrix used in quantification studies. Gathered spectra of five different concentrations in saliva were used to create calibration curves for heroin, MM, M3B, and 6MAM-spiked saliva. To measure heroin and its metabolites concentration in saliva, blind samples of spiked saliva were prepared by one of the authors, and a blinded author quantified heroin concentration in three samples. Spectra of blind samples were gathered from five different area of the staining and mean values of the intensities of the bands at 627 cm-1 used in calculation. Concentrations were calculated using intensity-concentration match point in calibration curve. For this reason, calculations were made according to equation of “CH = (I/327.12)1/0,45” for heroin, “CMM = (I/130.42)1/0,494” for MM, “CM3B = (I/119.46)1/0,834” for M3B and “C6MAM = (I/108.13)1/0,478” for 6MAM .Spiked concentrations of heroin, MM, M3B and 6MAM in saliva matrix and calculated concentrations are presented in Table 2. Total duration for analysis of heroin concentration in a heroin-spiked saliva sample using SERS was approximately 20 min. This is much lower compared to conventional instrumental analyses procedure in daily routine of toxicology labs. Abuse of illicit drugs or narcotic medications has been a challenging issue for both healthcare and legal systems for several centuries [2]. Therefore, identification and/or quantification of illicit drugs or certain medications is crucial for healthcare and forensic professionals. For this purpose, there are several methods investigated in the literature, however there is still need for cheaper and faster methods for toxicological analyses . SERS spectra of MM obtained in current study were compatible with Raman and SERS spectra revealed in the previously conducted studies [24–26]. Li et al. [26] used Ag film as a Raman-active material and gathered SERS spectra of MM using 633-nm laser source. They showed the most intense bands at 566, 633, 850, 1123, 1205 and 1284 Raman wavenumbers. Rana et al. [25] studied SERS spectra of MM using Ag colloids with both 785- and 633-nm laser sources. They stated that characteristic MM’s Raman bands detected by 633-nm laser source were more remarkable, which were seen at 462, 542, 609, 625, 1350, 1469 and 1698 cm-1. The results in the present study were compatible with the previous studies and showed the most intense and characteristic bands at 321, 443, 534, 627, 823, 935, 1033 and 1332 cm-1.Ag nanoparticles are known to intensify Raman signals of MM as well as AuNRs . Inscore et al. [12] studied 80 illicit drugs and their metabolites using SERS and stated that best results can be achieved with AuNRs to reveal SERS spectra of MM. In addition to the literature, we achieved to increase reliability of analysis by avoiding uncontrolled aggregation of studied sample due to a surface characterized with angled AuNRs that potentially control liquid motion. A comparison of Raman-active quality of studied surfaces in terms of SERS findings and related comments is shown in Table 3.In the literature, there are few previous studies dealing with Raman spectra of heroin and its metabolites as an illicit drug. Hodges et al. [27] studied Raman spectra of certain drugs of abuse including cocaine and heroin, and they have shown the most prominent band between 620–630 cm-1for heroin, in their study. A similar band formation has been shown in Ryder et al.’s study [28], which had also showed Raman spectra of heroin with more identifiable bands gathered from solid mixtures. However, these studies had not revealed an exact Raman spectrum for heroin. Since it is more sensitive and specific compared to conventional Raman spectroscopy, SERS analyses would reveal more convenient and useful data.To the best of our knowledge, this is the first ever study dealing with SERS spectra of heroin and its metabolites measurement in saliva medium. The surface produced according to the method by Yilmaz et al. [18] and AgNP were utilized to reveal better intensities in extremely low heroin concentration in order to use it for identification and quantification of heroin in complex media. In our study, band at 627 cm-1 was the most prominent and convenient band of which intensity was also identifiable in low concentrations for all four analytes.SERS is a fast and accurate way to identify and quantitate illicit drugs. Handheld spectroscopy also has potential to provide opportunity to analyze any samples in the field in a fast and accurate way Ocean Optics (2017). Identifying Illicit Drugs using Surface Enhanced Raman Spectroscopy [online]. Website http://blog.oceanoptics.com/illicit-drugs-identification-sers [accessed 04 09 2018]. . Although there are many commercial handheld Raman systems being sold for detection of illicit drugs in the market, most of these systems cannot overcome fluorescence effectively and have relatively lower signal/noise ratios . SERS aids in overcoming fluorescence and increasing signal/noise ratios . Therefore, SERS needs to be used to identify and measure heroin concentrations in complex media such as body fluids namely saliva, urine, and blood. Inscore et al. studied identification of certain illicit drugs in saliva with SERS including heroin without measuring their concentrations. They have stated that heroin is Raman active on gold nano-surface . Andreou et al. [29] also studied detection of illicit drugs in saliva with a micro-fluidic device using SERS and AgNPs. In this study, we have proposed a SERS method to detect and quantify heroin with a specific gold nano-surface produced with gold nano-arrays in a specific position to control liquid motion [18] and AgNP as Raman signal intensifiers. SERS spectra obtained with our method showed significantly higher intensities and low signal/noise ratios in bands specific to heroin. A study from China [22] revealed that drugs could be identified in urine samples via SERS-based systems. A recent study [13] showed that morphine identification is possible in human urine with SERS. Similarly, our results supported the information that identification of heroin and morphine is possible in saliva samples. Currently utilized quantitative analytical toxicology procedures are more reliable compared to enzymatic methods, while systems and analyses are quite expensive and time consuming. A routine analysis of heroin concentration in a biological fluid with LC–MS or GS–MS took 60 to 90 minutes, while the presented SERS procedure took approximately 20 min [32]. Therefore, the present study suggests a potentially cheaper and faster method than routine heroin analysis systems for heroin analysis in biological fluids. As a major limitation of the study, the utilized method might not be able to provide similar results in patients with certain diseases affecting the quality and contents in biological fluids, namely saliva. In conclusion, obtained results showed that heroin, MM, M3B and 6MAM can be detected and quantified in saliva samples using a SERS-based system. Additionally, the present study revealed that synergetic effect of a specific gold nano-surface with ability controlling liquid motion and silver nanoparticles increase band numbers and intensities. Therefore, we suggest a fast, accurate and cost-effective method to detect and quantify heroin in biological fluids, saliva in particular.This work was supported financially by Hacettepe University (Grant Number: THD-2018-17241). This article is based on studies of PhD thesis of Ramazan AKÇAN in Bioengineering Department of Graduate School of Science and Engineering. This study was presented in Taiwan-Turkey Science Summit that was held in Ankara, Turkey, on 1-4 April 2018.  Authors would to acknowledge Assoc. Prof. Gökhan DEMİREL for his technical support and material supply support.There is no need informed consent about this work.This study was presented at the Taiwan-Turkey Science Summit entitled “Translation of Cells, Nanomaterials and Signaling Molecules into Regenerative Medicine” between April 1 to 3, 2018.


It is not yet known how antibiotics may affect Serious Bacterial Infections (SBI). Our aim is to describe the presentation, management, and serious bacterial infections (SBI) of febrile children on or off antibiotics.Retrospective, cohort study of febrile Emergency Department patients, 0–36 months of age, at a single institution, between 2009and 2012.Seven hundred fifty-three patients were included: 584 in the No-Antibiotics group and 169 (22%) in the Antibiotics group. Age and abnormal lung sounds were predictors for being on antibiotics (OR 2.00 [95% CI 1.23–3.25] and OR 1.04 [95% CI 1.02–1.06] respectively) while female gender, and lower temperatures were negative predictors (OR 0.68 [95%0.47–0.98] and OR 0.47 [95% CI 0.32–0.67] respectively). Antibiotics were prescribed by a physician 89% of the time; the most common one being Amoxicillin/Clavulanic Acid (39%). The antibiotic group got more blood tests (57% vs 45%) and Chest X-Rays (37% vs 25%). Overall, the percent of SBIs (and pneumonias) was statistically the same in both groups (6.5% in the No-antibiotic group VS 3.6%).Children presenting on antibiotics and off antibiotics were significantly different in their presentation and management, although the overall percentages of SBI were similar in each group. Further investigations into this subgroup of febrile children are needed. Children with fever constitute a substantial proportion of ambulatory emergency department (ED) visits . SeriousThe objective of this study was to describe previously healthy children, presenting to the ED with fever, stratified by previous antibiotic use or not; and to describe the distribution and types of SBI in those two groups.We carried out a retrospective, cohort study of patients 0–36 months of age presenting with fever to the ED of the American University of Beirut Medical Center in Beirut, Lebanon, between July 1, 2009 and June 30, 2012. Institutional Review Board approval was obtained. This is an ED of a tertiary care center, in a middle-income country where pediatric patients during the time of our data collection where seen primarily by pediatricians with or without intensive care training.We included all patients 0 to 36 months of age, with fever  measured in the ED, at home or at the pediatrician’s office. We retrieved the records of patients with one or more of the following chief complaints, ED discharge diagnoses or hospital admission/discharge diagnoses: fever, cough, sore throat, runny nose, fussy, lethargy, decreased activity, seizure activity, vomiting, diarrhea, pneumonia, urinary tract infection (UTI), viral illness, tonsillitis, pharyngitis, cellulitis, abscess, meningitis, encephalitis, sepsis, septic shock, bacteremia.We excluded all patients with an underlying immunosuppressive disease or immunosuppressive medication; with an underlying chronic disease (that may impact fever management); with a previous UTI; and admitted to the ED or hospital within the last 2 weeks.We included information on: patient demographics, clinical presentation, and management. Data was collected by 4 physicians who had a training by the principle investigator in order to use the same terminology and categorize signs and symptoms in the same way.3) by sterile catheterization or SPA and > 100,000 cfu/ml for clean catches [Urinary Tract Infection: a positive urine culture > 5000 cfu/ml for suprapubic aspiration (SPA), > 10,000 cfu/ml for a sterile catheterization in children < 2 months old; > 50,000 cfu/ml AND pyuria by urinalysis , which were considered contaminants unless treated as true infections per documentation , 16, 17.Meningitis: a positive cerebrospinal fluid culture other than coagulase negative Staphylococcus which was considered a contaminant, unless treated as true infections per documentation , 18.We defined Serious Bacterial Infection (SBI) as one of the following:We defined Pneumonia as a Chest X-Ray reported by a radiologist as definite or probable for a pneumonia  irrespective of microbiological results as these are low yield . This deWe defined tachypnea and tachycardia as values above the upper limit of normal for age, as per Additional file We defined abnormal perfusion as any documentation of mottled skin, or capillary refill greater than 3 s, or a flash capillary refill consistent with possible warm shock.The “Antibiotic” group included all children who were on antibiotics prior to the ED visit as per care giver’s report or who had received antibiotics within the past 2 weeks. The “No-Antibiotic” group included the children who had not received any antibiotics prior to the ED visit.P-value of 0.05 was set for the entry of potential predictors into the model, whereas a p-value of 0.1 was set for removal from the model. The results were presented by the odds ratio (OR) and 95% confidence interval (CI). P-value of < 0.05 was considered statistically significant. Missing data was left empty.The Statistical Package for Social Sciences (SPSS), version 24.0 was used for data cleaning, management and analyses. Descriptive statistics were summarized by presenting the number and percentage for categorical variables, whereas continuous ones were presented by mean and standard deviation (±SD). In the bivariate analysis, the association between antibiotic use and other categorical variables was assessed using Chi-Square test, whereas Student’s t-test was used for the association with continuous variables. Multivariate regression analysis was used to adjust for potentially confounding variables. Variables which were statistically significant in the analysis or clinically important were included in the multivariate analysis. The stepwise logistic regression analysis assessed the association between antibiotic use and the different predictors. The datasets analyzed during the current study are available from the corresponding author on reasonable request.We retrieved 1427 patients from the medical records; 753 met our inclusion criteria and were analyzed: 584 in the No-Antibiotics group and 169 (22.4%) in the Antibiotics group , in fact none of the children < 90 days of age had received antibiotics prior to presentation in this sample. In addition, the Antibiotic group was mostly of male gender , and had a longer duration of fever prior to presentation . Interestingly, associated symptoms presented as frequently in both groups except for a sore throat: 10.1% in the Antibiotic group compared to 4.8% in the No-Antibiotic group (p = 0.01)  were still takings antibiotics at presentation; and 10.8% were taking multiple. The mean days of antibiotic use was 3.5 ± 3.0 days. The antibiotic was prescribed by a Medical Doctor in 89.3% (101/113) of the cases. Finally, the most common antibiotic used was an oral 3rd generation cephalosporin at 33.2% followed by a combination of penicillin/beta-lactamase inhibitor at 31.9%.. Interestingly, up to 10.2% had received intravenous (IV) or intramuscular (IM) 3rd generation cephalosporin,, prior to the ED visit.p < 0.0001); to have abnormal lung sounds , an abnormal tympanic membrane ; and abnormal tonsils . While the No-Antibiotic group were more likely to have an abnormal mental status  and to be looking more sickly .When comparing the two sub-groups Table , we notep = 0.01) and chest radiography  (see Table p = 0.02); a more frequently positive urine analysis  and to have influenza (p = 0.03). Interestingly, the frequency of fluid boluses and admissions was the same in both groups.The Antibiotic group was more frequently tested by blood work . However, UTIs were statistically more common in the No-Antibiotic group   and was perhaps started on antibiotics for that reason; this may be explained by the fact that upper respiratory infections (URTI) are the most common reason for being on outpatient antibiotics . The No-The most common source of antibiotic prescription in our country remains the physician but only at 89.3%. It is worth noting that our Lebanese pharmacies can still issue an antibiotic without a prescription. This fits with results from a recent Lebanese and Middle Eastern report that antibiotics were one of the most common medications self-prescribed by patients , 23.Finally, in our sample, the most common antibiotics used were broad spectrum antibiotics, such as a 3rd generation cephalosporin. As we noted that most patients on antibiotics had abnormal lung sounds, tympanic membranes or tonsils, perhaps these were to treat a pneumonia, otitis or Streptococcus tonsillitis. This is an interesting choice given the American Academy of Pediatrics (AAP) guidelines to treat these primarily with Amoxicillin –26. HoweIn this retrospective study our data is limited by the accuracy and completeness of the medical records, therefore no inferences were made on immunization and vital signs because of this. We don’t have the exact timing of the laboratory draws, but all reported laboratory results were done during the sentinel ED visit. We also do not have information on the duration of antibiotic pretreatment, nor why it was given and therefore cannot determine its exact impact on cultures and laboratory results. In addition, the SBI rates of the pretreated group may be underreported as the antibiotics could have influenced the culture results. However, this reflects the reality of our clinical practice and decisions we have to make.In conclusion, this is the first study of its kind to describe febrile children already on antibiotics presenting to the ED compared to those not on antibiotics. It generated interesting preliminary data that opens doors to further investigations on predictors for testing febrile patients on antibiotics, on understanding how to interpret the test results and more importantly to understand predictors of SBI and SBI outcomes in this group.Additional file 1.


GAA) with methylation by S-adenosylmethionine. Because methyl groups are used for the conversion of GAA to creatine, methyl group deficiency may occur as a result of GAA supplementation. With this study, the metabolic responses of cattle to post-ruminal supplementation of GAA were evaluated with and without methionine (Met) supplementation as a source of methyl groups. Six ruminally cannulated Holstein heifers (520 kg) were used in a split-plot design with treatments arranged as a 2 × 5 factorial. The main plot treatments were 0 or 12 g/d of l-Met arranged in a completely randomized design; three heifers received each main plot treatment throughout the entire experiment. Subplot treatments were 0, 10, 20, 30, and 40 g/d of GAA, with GAA treatments provided in sequence from lowest to highest over five 6-d periods. Treatments were infused continuously to the abomasum. Heifers were limit-fed twice daily a diet consisting of (dry matter basis) 5.3 kg/d rolled corn, 3.6 kg/d alfalfa hay, and 50 g/d trace-mineralized salt. Plasma Met increased (P < 0.01) when Met was supplemented, but it was not affected by supplemental GAA. Supplementing GAA linearly increased plasma arginine  and plasma concentrations of GAA and creatinine (P < 0.001). Plasma creatine was increased at all levels of GAA except when 40 g/d of GAA was supplemented with no Met . Plasma homocysteine was not affected by GAA supplementation when heifers received 12 g/d Met, but it was increased when 30 or 40 g/d of GAA was supplemented without Met ; increases were modest and did not suggest a dangerous hyperhomocysteinemia. Urinary concentrations of GAA and creatine were increased by all levels of GAA when 12 g/d Met was supplemented; increasing GAA supplementation up to 30 g/d without Met increased urinary GAA and creatine concentrations, but 40 g/d GAA did not affect urine concentrations of GAA and creatine when no Met was supplemented. Overall, post-ruminal GAA supplementation increased creatine supply to cattle. A methyl group deficiency, demonstrated by modest increases in plasma homocysteine, became apparent when 30 or 40 g/d of GAA was supplemented, but it was ameliorated by 12 g/d Met.Creatine stores high-energy phosphate bonds in muscle, which is critical for muscle activity. In animals, creatine is synthesized in the liver from guanidinoacetic acid ( GAA) is a precursor for creatine, which is a compound that allows the storage of high-energy phosphate bonds in muscle  is an essential amino acid (AA) that is often limiting for cattle (S-adenosyl-l-methionine which is the body’s primary methyl group donor. Homocysteinemia can result from conditions where methyl groups are limiting (Brosnan and Methionine (r cattle . ActivatOur hypotheses were that 1) GAA supplementation could create a methyl group deficiency in cattle and 2) Met supplementation could prevent the methyl group deficiency.All experimental procedures involving cattle were approved by the Institutional Animal Care and Use Committee at Kansas State University.l-Met arranged in a completely randomized design; three heifers received each main plot treatment throughout the entire experiment. Subplot treatments were 0, 10, 20, 30, and 40 g/d of GAA, with treatments provided in sequence from the lowest to the highest level. Treatments were increased over time due to the possibility that the GAA might cause negative health effects on the heifers, and the increasing doses would allow the detection of any problems at the lowest amount that was problematic. In addition, the sequential increase of GAA amounts partially reduced the need for an extended adaptation period because the heifers were adapted to the next greatest amount of GAA prior to initiating each treatment. Treatments were all provided as continuous infusions into the abomasum to preclude the potential for ruminal degradation. Treatments were infused into the abomasum through Tygon tubing  passed through the ruminal cannula, the reticulo-omasal orifice, and the omasum and held in the abomasum with a circular rubber flange (10 cm diameter) at one end. A peristaltic pump  was used to make the infusions.Six ruminally cannulated Holstein heifers  were used in a 44-d experiment, composed of 14 d for adaptation to facilities and diet and five 6-d periods, with samples collected on days 3 and 6 of each period. The experiment used a split-plot design. The main plot treatments were 0 or 12 g/d of l-Met, it was added to the solution for each heifer and shaken until dissolved.The treatment solutions of GAA were prepared as 1% solutions in water; the GAA was initially solubilized with 0.22 g of 6 M HCl/g GAA (pH approximately 2.5), and then 0.11 g of 50% (wt/wt) NaOH/g GAA was added, which raised the pH to approximately 3.1. The GAA was then diluted, as necessary, so that the final weight of the solution was 4 kg/d. For those heifers receiving DM)] basis) 5.3 kg/d of rolled corn, 3.6 kg/d of alfalfa, and 50 g/d of trace-mineralized salt. Using the TDN], 65% ruminally undegraded CP, 2.1% Met in CP; alfalfa = 60% TDN, 20% ruminally undegraded CP, 1.35% Met in CP). Because the diet contained no animal products, it was assumed devoid of GAA and creatine. Animals were observed daily for any potential symptoms of toxicity such as inappetence or depressed attitude, but no health problems were observed during the study.Heifers were housed in tie-stalls with free access to water and were limit-fed twice daily (0600 and 1800 hours) a diet  providinNDF), N, and ash. The NDF content was measured according to the technique of Samples of the feed ingredients  were collected and frozen (−20 °C) on days 3 and 6 of each period for subsequent analysis. Samples were mixed within the period to obtain composite samples, dried in a 55 °C forced-air oven for 72 h to determine partial DM, ground to pass through a 1-mm screen , and stored for subsequent analysis. Feed samples were analyzed for DM, neutral detergent fiber  to harvest plasma. Plasma samples were frozen at −20 °C for later analyses of AA (day-6 samples only), homocysteine (day-6 samples only), GAA, creatine, and creatinine. On day 6 of each period, urine samples were collected, following stimulation of the vulva to induce urination, three times at 0700, 1000, and 0100 hours, composited, and frozen at −20 °C for later analyses of GAA, creatine, and creatinine.On days 3 and 6 of each period, blood samples were collected from the coccygeal vein into 10-mL heparinized blood collection tubes  at 4 h after the morning feeding (1000 hours). Samples were stored on crushed ice immediately after collection and then centrifuged  after deproteinization by mixing plasma with equal volumes of 10% (wt/vol) sulfosalicylic acid containing norleucine as an internal standard  were placed in polyethylene tubes, and disulfide bonds were reduced with the addition of 200 µL 2-mercaptoethanol, which was mixed and allowed to set at 20 °C for 5 min. Samples were then deproteinized with 2 mL of methanol, which was mixed for 30 s. Samples were then centrifuged at 4,000 × g for 15 min at 20 °C. Supernatants (2.25 mL) were then dried in 2-mL microcentrifuge tubes under N2, then reconstituted with 50 µL water and 50 µL 2-mercaptoethanol. Carboxymethyl derivatives were produced by the addition of 200 µL of 2 M iodoacetic acid (mixed in 0.1 M sodium borate buffer adjusted to pH 11.5); sample blanks, which were subtracted from concentrations of derivatized samples, were treated with 200 µL of 0.1 M sodium borate buffer adjusted to pH 11.5. Following mixing and a 5-min reaction period, 400 µL of lithium diluent  was mixed with each sample, which was subsequently centrifuged at 17,000 × g for 5 min at 20 °C and filtered through a 0.2-µm syringe filter. The HPLC analysis used a cation-exchange column (Pickering Laboratories) maintained at 33 °C, a 20-µL sample loop, and a flow rate of 0.6 mL/min. The eluant was a lithium eluant at pH 2.75  for 20 min, followed by a lithium column regenerant  for 5 min, and re-equilibration of the column with LI275 for 20 min prior to the next sample injection. Post-column derivatization of eluted compounds with o-phthalaldehyde and 2-(dimethylamino)ethanethiol (both from Pickering Laboratories) was followed by fluorescence detection .Plasma homocysteine was analyzed as the carboxymethyl derivative formed as described by g, 10 min, 4 °C), and then filtering through a 0.2-µm syringe filter into HPLC vials for injection. For urine sample preparation, 100 µL of sample was diluted with 900 µL diluent, which consisted of 0.9 g of ammonium phosphate and 1.01 g of sodium 1-heptane sulfonic acid in 1 liter of deionized H2O with pH adjusted to 2.2 with H3PO4. Diluted samples were filtered through a 0.2-µm syringe filter into an HPLC vial. The components of the sample were separated on a 25 cm × 4.6 mm Discovery BIO Wide Pore C18 column . The mobile phase consisted of 1.01 g sodium 1-heptane sulfonic acid, 0.9 g ammonium phosphate, 35 mL methanol, and 70 µL triethylamine made to 1 L with deionized H2O and adjusted to pH 2.8 with 7.5 M H3PO4. Compounds were detected by absorbance at 200 nm. To achieve chromatographic separation, 5 µL samples were injected to the column at 20 °C with a flow rate of 0.5 mL/min for 14 min, and then with a flow rate of 1.2 mL/min. Sample separation was completed at 25 min, and the column was flushed with 100% methanol for 10 min at 1.2 mL/min and re-equilibrated with the mobile phase for 19 min. The flow rate was then returned to 0.5 mL/min for 1 min prior to the next injection. Total run time was 55 min.Urine and blood GAA, creatine, and creatinine were determined using HPLC according to Glomerular filtration was calculated under the assumption that creatinine is entirely filtered from the blood and not reabsorbed from glomerulus. Renal reabsorption of GAA was calculated as: 1 − [(urinary GAA concentration/urinary creatinine concentration)/(plasma GAA concentration/plasma creatinine concentration)], and renal reabsorption of creatine was calculated similarly with concentrations of creatine replacing those of GAA.Treatments were arranged as a 5 × 2 factorial and included five amounts of GAA  and two amounts of Met (0 and 12 g/d). Initial analyses were completed with plasma GAA and creatine concentrations to determine if cattle had adapted to treatments by day 3. Data from samples collected on days 3 and 6 of each period were analyzed with a repeated-measures analysis with a model including fixed effects of Met, GAA, day, and all interactions. Heifer within Met level was included as a random effect that served as the error term for testing effects of Met. Day was treated as a repeated measure with a covariance structure of compound symmetry.Data from day 6 were analyzed with a model including fixed effects of Met, GAA, and their interaction. The GAA supplementation was considered as a repeated measure with the covariance structure of autoregressive. Heifer within Met level was included as a random effect that served as the error term for testing effects of Met. Means were separated using polynomial contrasts to test the linear and quadratic effects of GAA as well as the interactions of Met with those effects.S-adenosylmethionine, which provides the methyl group for synthesis of creatine from GAA. Because regulation of creatine synthesis occurs at the level of GAA synthesis, the use of methyl groups in the conversion of GAA to creatine is an unregulated process , GAA . Because the plasma data demonstrated that cattle had not completely adapted to treatments by day 3 of each period, only data from day 6 were used for analyses discussed in this paper.Data  demonstrP = 0.003) for plasma homocysteine  with GAA supplementation, with all amounts of supplemental GAA elevating plasma creatine except when 40 g/d of GAA was provided with no supplemental Met. It is possible that methyl group deficiency may have limited the synthesis of creatine from GAA when no Met was provided, preventing cattle from maintaining the higher rates of creatine synthesis when large amounts of GAA were provided.Plasma creatine concentrations increased quadratically (P = 0.001) plasma GAA concentrations from 1.08 to 2.26 mg/L, but increases in plasma GAA concentrations tended (P = 0.06) to be less when Met was provided, perhaps reflecting that Met produced a more effective uptake of GAA to support creatine synthesis. Some authors have reported increased concentrations of plasma GAA in humans (P = 0.001) with GAA administration. Because of the conversion of creatine to creatinine, it was not surprising that plasma creatinine was elevated by GAA supplementation.In our study, supplemental GAA linearly increased  urinary concentrations of GAA. The shapes of the curves tended to be affected by Met supplementation  because heifers receiving no supplemental Met demonstrated a decrease in urinary GAA concentrations when 40 g/d GAA was supplemented, but heifers receiving 12 g/d Met did not. Considering an estimated urine output of 10 L/d, urinary GAA excretion was increased by not more than 0.4 g/d, even when the highest amount of GAA was supplemented, demonstrating that the preponderance of supplemental GAA was methylated to creatine.In our study, supplemental GAA  quadratiP = 0.02) in response to GAA supplementation in parallel with increases in plasma creatine. Like plasma creatine, when 40 g/d of GAA was provided without supplemental Met, there was a decrease in urinary creatine concentrations, but this decrease was not observed when 12 g/d Met was supplemented . The increases in urinary creatine concentrations in response to GAA supplementation were associated with elevated plasma creatine concentrations, which likely resulted from increased creatine synthesis. When the body is faced with increased GAA supply, much of the GAA is methylated to creatine, but the excess creatine will be eliminated through renal excretion which is the major clearance mechanism for the body  with GAA supplementation. The reason for the reduction of urinary creatinine in response to increasing amounts of GAA is unclear, but it is possible that increases in urine volume could be responsible. Increases in urine volume, however, probably could not account for the decrease in urinary creatine concentration in heifers receiving 40 g/d GAA without Met because the magnitude of drop was not as great for creatinine as for creatine.Urinary creatinine concentration linearly decreased (P < 0.01) and quadratically (P < 0.01) decreased creatine reabsorption. We also observed a significant interaction between Met and GAA for creatine reabsorption  because heifers receiving 40 g/d GAA without Met demonstrated much greater reabsorption than those that received 12 g/d Met along with 40 g/d GAA. Renal reabsorption of creatine might be expected to decrease as the availability of creatine increases and the body retains less of the available creatine. Although a portion of the changes in urinary creatine concentrations in response to GAA provision can be attributed to changes in plasma concentrations, differences in renal reabsorption were more quantitatively important in affecting urinary creatine concentrations.There was a Met × GAA-linear interaction for renal reabsorption of GAA  because Heifers excreted significant amounts of creatine in the urine, even when no GAA was supplemented. Assuming urine outputs of 10 L/d, control heifers excreted about 5 g/d of creatine, which would seem to be an insensible loss of both N and methyl groups. Previous work has shown that cattle , as wellAs a whole, there were notable increases in plasma and urinary concentrations of both GAA and creatine as increasing amounts of GAA were supplemented. However, two of the three heifers receiving 40 g/d of GAA without supplemental Met demonstrated marked decreases in urinary concentrations of GAA and creatine; urinary creatinine was also lower for those two heifers, but the magnitude of drop was not large. Only one of those two heifers demonstrating low urinary GAA and creatine concentrations had a low plasma creatine concentration and neither had strikingly low plasma GAA. Although we did not observe any evidence of health problems in any of the heifers, it is possible that the unusual plasma and urinary concentrations of GAA and creatine could have been a precursor to problems that could have developed if the treatment had been extended for more than 6 d. As noted above, the treatment providing 40 g/d GAA without Met was characterized as having greater renal absorption than would have been predicted from the responses to GAA supplementation up to 30 g/d, demonstrating that the urinary concentrations of GAA and creatine could not be fully explained by changes in plasma concentrations, particularly for GAA. This suggests that some metabolic effects downstream of creatine synthesis may have been affected by the methyl group deficiency noted for that treatment. A precise explanation for the metabolic changes in those two heifers remains unclear.P = 0.01; The effect of GAA and Met supplementation on plasma AA  is shown in P < 0.001), but GAA supplementation had no effect on plasma concentrations of Met, which suggests that the methyl group deficiency observed with the higher amounts of GAA was not severe enough to substantially disrupt Met metabolism. Because the synthesis of creatine from GAA requires a methyl group from S-adenosylmethionine, we hypothesized that GAA supplementation might decrease Met availability to the heifers either by increasing Met consumption for this process or by increasing transsulfuration. The lack of effect of GAA on plasma Met concentrations might suggest that either Met consumption was not markedly increased by GAA provision or the heifers were able to compensate by remethylating homocysteine and preventing a net consumption of Met. Plasma taurine, a product of sulfur AA metabolism (P = 0.07) when Met was supplemented. Plasma valine (P < 0.05) was reduced by supplementation of Met, an effect that has been attributed in Met-deficiency models to increases in protein deposition  by GAA supplementation, particularly with the initial dose of GAA. Increases in plasma arginine in response to supplemental GAA are likely the result of an arginine-sparing effect of GAA supplementation, likely by inhibiting GAA synthesis in the kidney. Quadratic effects were observed for plasma ornithine (P = 0.03) and glutamate (P = 0.003) for heifers receiving GAA supplementation. The effect of GAA supplementation on plasma ornithine may relate to increases in arginine availability, which could cause more ornithine to be formed from arginine.The synthesis of GAA occurs through the conversion of arginine and glycine to GAA  by arginP ≤ 0.05) with GAA supplementation. In addition, histidine was linearly decreased (P = 0.05) and aspartic acid was linearly and quadratically decreased (P ≤ 0.01) by supplemental GAA. The causes and consequences of these changes are unknown.Plasma tryptophan, tyrosine, threonine, and asparagine linearly increased , there might be a concern about a methyl group deficiency in response to GAA, although this could be ameliorated by supplemental Met. Methyl group sources other than Met may also ensure against this problem, although we did not evaluate any methyl group sources besides Met. In addition to developing a useful model to assess methyl group utilization in cattle, we were also able to demonstrate for the first time that GAA is used by cattle as a precursor for synthesizing creatine. This research has direct implications for both the dairy and beef cattle industries, because GAA may be an economical means of improving creatine status of cattle.


Immunoglobulin G4-related disease (IgG4-RD) is a new disease entity of rare and complex immune-mediated fibroinflammatory conditions that can affect any organ. The concomitance of IgG4 sclerosing sialadenitis and dacryoadenitis with rhinosinusitis is extremely rare. We report a case of IgG4 sclerosing sialadenitis and dacryoadenitis (Mikulicz’s disease) diagnosed in a middle-aged African American man with a long-standing history of chronic rhinosinusitis who presented with progressively worsening bilateral salivary and lacrimal glands swelling. Imaging revealed pansinusitis, symmetric enlargement of the lacrimal glands, parotid glands, and submandibular glands. Serological IgG4 level was significantly elevated and the diagnosis of IgG4 sclerosing sialadenitis was confirmed by histopathology. A robust clinical response in the facial swelling and nasal manifestations was noted after the initiation of immunotherapy with corticosteroids. Immunoglobulin G4-related disease (IgG4-RD) is a recently recognized immune-mediated fibroinflammatory systemic condition that often mimics other disease processes, such as malignancy or granulomatous diseases. The most affected organ is the pancreas and it is the first organ to be recognized as an IgG4-related disease. In 2003, Kamisawa et al. detected a predominance of IgG4-positive plasma cells in association with CD4 and CD8 T lymphocytes in the tissue biopsy of patients with autoimmune idiopathic pancreatitis identifying IgG-associated pancreatitis as a new entity of diseases .In addition to the pancreas (Type 1 autoimmune pancreatitis), IgG4-related disease can potentially affect nearly any organ including biliary system, salivary glands, lacrimal glands, eyes , lymph nodes, retroperitoneum, large vessels, thyroid gland, lungs, pleura or kidneys. It can be multi-centric in its distribution or isolated to a single organ.In this article, we report a rare presentation of IgG4 sclerosing sialadenitis and dacryoadenitis causing a rapidly progressive swelling of the head and neck in a patient with a chronic history of chronic rhinosinusitis.A 46-year-old African American man was transferred to our facility for an ear-nose-throat (ENT) evaluation of a progressively worsening swelling of the head and neck. His symptoms started with painless enlargement of the right submandibular glands nine months ago, followed by swelling of the left submandibular gland and bilateral parotid glands six months later. Most recently, he noticed bilateral eyelid swelling associated with excessive lacrimation and diplopia for the past two months. The patient reported a 17-year history of rhinorrhea, nasal congestion, frontal headaches and hyposmia that is refractory to conservative management. A review of systems was remarkable for unintentional weight loss and intermittent pruritic erythematous maculopapular rash that mostly appears on his both arms and spontaneously disappears. He denied fever, arthralgias, dry mouth, dry eyes, nose crusting, epistaxis, change in the nose shape, wheezing, cough or shortness of breath.In the past year, he had two recent hospitalizations for acute bronchitis treated with bronchodilators and antibiotics. He denied smoking, alcohol consumption or illicit drug use. He denied any past surgical history and current medications. He has worked in the construction industry for the past 30 years and denied any family history of rheumatological conditions.On physical examination, he appeared comfortable with normal body temperature, normal heart rate, respiratory rate and blood pressure. The parotid, submandibular and lacrimal glands were firm, non-tender and symmetrically enlarged on both sides Figure . BilaterInitial laboratory workup was remarkable for an elevated erythrocyte sedimentation rate (ESR) (34 mm/hr) and eosinophilia (8%). C-reactive protein (CRP) was found to be within normal limits (0.4 mg/dL). Serological antibodies testing was negative for antinuclear antibody (ANA), rheumatoid factor, double-stranded DNA, anticentromere, anti-Ro (SSA) and anti-La (SSB), anti-Scl-70 and ribonucleoprotein (RNP). Additional serological testing included negative serum fungal antibodies and normal serum angiotensin-converting enzyme level. Serum immunoglobulin G class 3 and 4 were elevated at >227 mg/dL  and 1541 mg/dL , respectively.Computed tomography (CT) scan of the head with contrast revealed opacification of the bilateral maxillary, sphenoid, ethmoid and frontal sinuses, and enlargement of the bilateral lacrimal, parotid and submandibular glands Figure .Ultrasound (US) of the neck demonstrated bilateral submandibular gland soft tissue edema without gross evidence of drainable fluid collection Figure .The patient underwent a US-guided core biopsy of the left submandibular gland. Biopsy demonstrated atrophic salivary gland tissue with a chronic inflammatory infiltrate and extensive fibrosis. A CD138 stain highlighted several plasma cells and IgG4 highlighted a majority of these cells with an increase in the background staining  of all patients with ophthalmic IgG4-RD. Dacryoadenitis is commonly associated with salivary gland swelling, similar to what we experienced in our case ,6.2%-68% ofNasal obstruction and anosmia/hyposmia are the most commonly present nasal manifestations in IgG4-RD. It is more common in males than females with a ratio of 3:1. Imaging classically reveals bilateral thickening of the affected sinuses and nasal obstruction. Complications are not uncommon, and it may involve the orbit causing proptosis and eyelid swelling. Patients with IgG4-RD and concomitant chronic rhinosinusitis are more likely to have elevated serum IgG4 and eosinophilia which were present in our case -9.The diagnosis of IgG4-RD is challenging, and a high index of suspicion is necessary. The abundant infiltration of lymphocytes with plasma cells expressing IgG4 in immunohistostaining and storiform fibrosis is pathognomonic. However, neither histopathological findings nor a clinical picture alone is sufficient to make the diagnosis. The most accurate diagnostic tool is based on the clinical, serological and radiological findings altogether. Serum IgG4 level may be elevated with a sensitivity of 87.2% and a specificity of 82.6%. Imaging modalities using CT, CT with positron emission tomography (PET), or magnetic resonance (MR) imaging can be used to guide the management. When IgG4-RD is suspected a core or open biopsy is warranted to rule out malignancy and IgG4-RD mimickers. Fine needle aspiration alone is often inconclusive. The exclusion of other conditions that can mimic IgG4-RD clinical picture or histopathology is essential prior to reaching the diagnosis of IgG4-RD ,11.An international consensus guidance statement on the management and treatment of IgG4-RD recommends corticosteroids as first-line therapy. The initiation of a steroid-sparing agent is recommended to avoid the long-term side effects of corticosteroids and to prevent the risk of relapse. It is recommended for patients who are in remission to continue maintenance therapy which may consist of low-dose glucocorticoids or a steroid-sparing agent including rituximab, cyclophosphamide, azathioprine or mycophenolate. In asymptomatic patients who have limited disease, watchful waiting can be adopted .The prognosis of IgG4-RD is yet to be fully understood. However, it is considered to be relatively favorable with a 10-year overall survival rate of 89.0% -14. MoreThe IgG-related disease is a new entity of immune-mediated fibroinflammatory conditions that can be a rare etiology of any mass-like lesions. The diagnosis should be based on clinical findings, serological and pathological findings. The clinical manifestation usually responds drastically to treatment with corticosteroids. Other therapeutic options including rituximab can be used in refractory or recurrent disease.


A broken intramedullary nail is a well-known complication of non-union of femur shaft fractures. Numerous surgical techniques have been presented before in patients with non-union of long bone fractures. We report the surgical technique used to perform removal of the broken distal segment of a nail in a patient who achieved uneventful union after intramedullary nailing of closed femur shaft fracture. A ball-tipped guidewire was inserted through the broken segment of the femur nail. A pre-bend plain wire was then inserted. With the help of a vise-grip, both wires were twisted in order to make a secure handle between guidewires and a broken implant. With the help of a mallet upward-directed blows were applied to extract a broken segment of the nail. We found ball-tipped guidewire technique a useful and effective technique in removing the broken distal portion of the nail. A broken intramedullary nail is a well-known complication after non-union of long bone fractures. On other hand, the broken intramedullary nail is not frequently encountered once the union has been achieved . SeveralIn 2005, a 33-year-old male experienced a closed left femur mid-shaft fracture after a motor vehicle accident (MVA). The very same day, he underwent for the interlocking nail of the left femur. There was an uneventful postoperative period and the union was achieved after 12 weeks. After 15 years, he presented with pain over the left thigh area primarily localized to the distal screw site with prominent distal-most screw-on local examination. This discomfort was presumed to be due to implant-related, so a decision was made to remove the hardware. Pre-operatively broken distal segment of the nail was not anticipated as it was very difficult to pick that segment radiographically . The devRemoval of broken hardware is a challenging surgery even for an experienced trauma surgeon. Therefore, the surgeon must be aware of the simple and efficient techniques for removing broken hardware. A ball-tipped guide-wire technique is a simple and efficient technique for extracting a broken distal portion of nail and can be performed in a situation where it is not easy to access special equipment for extracting broken nail. Before our surgical technique, various methods for removing broken nail segments have also been implemented . Most teThe surgical technique we used in our case is recommended in situations where the union has already been achieved or the fracture site opening is not desirable . Our technique is also effective in cases where special surgical equipment for removing a broken portion of the nail is not readily available in operation theatre .The only limitation of our surgical technique is that it would not be used in a situation when small diameter nails were used to fix long bone fractures. We recommend using this method only when dealing with a nail of the diameter of 10 mm or above that allows easy placement of a ball-tipped wire and one or more plain guide wires. In our technique, cortical windows are not needed to remove distal broken nails, which is an added advantage .Previous literature supports the use of kuntscher nails, small diameter nails, small reamers, and extraction hooks in circumstances where nail diameter is small . The staIn order to remove damaged Kuntscher nails, Maini et al.  used theWe believe that the ball-tipped guide wire surgical technique is an easy and reliable approach for extracting the broken distal segment of the nail in a situation where special equipment for removing broken hardware is not readily available. This technique also avoids cortical window to prevent complications such as an iatrogenic fracture.The authors declare no conflict of interest.The authors declare that no funding was involved in this study.


Spliced Leader trans-splicing is an important mechanism for the maturation of mRNAs in several lineages of eukaryotes, including several groups of parasites of great medical and economic importance. Nevertheless, its study across the tree of life is severely hindered by the problem of identifying the SL sequences that are being trans-spliced.In this paper we present SLFinder, a four-step pipeline meant to identify de novo candidate SL sequences making very few assumptions regarding the SL sequence properties. The pipeline takes transcriptomic de novo assemblies and a reference genome as input and allows the user intervention on several points to account for unexpected features of the dataset. The strategy and its implementation were tested on real RNAseq data from species with and without SL Trans-Splicing.SLFinder is capable to identify SL candidates with good precision in a reasonable amount of time. It is especially suitable for species with unknown SL sequences, generating candidate sequences for further refining and experimental validation. Spliced Leader (SL) trans-splicing, that is, the incorporation of a short RNA (the spliced leader) on the 5′ end of a different transcript, is an important but poorly understood part of the mRNA maturation process of many eukaryotic lineages. SL genes are often encoded in tandem repeats measuring a few kilobases, close to 5S rRNA genes  but therCaenorhabditis elegans has two distinct types of SLs, one (SL-1) is incorporated at the start of the operon and the other (SL-2) is used to resolve the downstream coding sequences into different transcripts ) SLFinder offers a practical alternative for the discovery of novel SL sequences aside from homology searches or fortuitous identification. This modular pipeline was proved with freely available RNAseq data for organisms with and without reported Splice Leader sequences with very good results. Putative SLs found by SLFinder can be later refined regarding their exact length and confirmed through additional bioinformatics analyses and wet lab experiments. This software represents a step forward toward a more comprehensive understanding of the distribution of SL Trans-Splicing in the tree of life, its evolutionary history and importance.Additional file 1: Supplementary Table 1. Reported SL sequences for the species C. elegans, C. intestinalis, H. vulgaris and S. mansoni.Additional file 2: Supplementary Table 2. Basic descriptors of the analyzed datasets. Data Set, GenBank SRA ID, N° Reads, N° Trimmed Reads, Assembled Bases, Total Transcripts, N50, Median Contig Length, Average Contig Length, Genes predicted by Trinity (Trinity Genes) and GC content.Additional file 3: Supplementary Table 3. Hook hits obtained for each dataset .Additional file 4: Supplementary Table 4. Putative SLs loci identified by SLFinder in the C. elegans dataset. Previously reported SL-RNAs are also indicated.Additional file 5: Supplementary Figure 1. Common issues to consider when utilizing SLFinder.Additional file 6: Supplementary Figure 2. Non-functional SL loci found during SLFinder analyses.Additional file 7: Supplementary Table 5. Putative SL loci identified by SLFinder in the C. intestinalis dataset. Annotated lncRNA and protein-coding genes closer than 100 pb are also indicated.Additional file 8: Supplementary Table 6. Putative SL loci identified by SLFinder in the H. vulgaris dataset. Annotated protein-coding genes closer than 100 pb are also indicated.Additional file 9: Supplementary Figure 3. Sequences of putative SL identified in H. vulgaris dataset.Additional file 10: Supplementary Table 7. Putative SL loci identified by SLFinder in the S. mansoni dataset using Wormbase’s genome assembly as reference (WBPS). Annotated protein-coding genes closer than 100 bp are indicated.Additional file 11: Supplementary Table 8. Putative SL loci identified by SLFinder in the S. mansoni dataset using GeneDB’s genome assembly as reference. Annotated protein-coding genes closer than 100 bp are indicated.