Developing non-destructive methods (NDT) that can deliver faster and more accurate results is an objective pursued by many researchers. The purpose of this paper is to present a new approach in predicting the concrete compressive strength through means of ultrasonic testing for non-destructive determination of the dynamic and static modulus of elasticity. For this study, the dynamic Poisson’s coefficient was assigned values provided by technical literature. Using ultra-sonic pulse velocity (UPV) the apparent density and the dynamic modulus of elasticity were determined. The viability of the theoretical approach proposed by Salman, used for the air-dry density determination (predicted density), was experimentally confirmed (measured density). The calculated accuracy of the Salman method ranged between 98 and 99% for all the four groups of specimens used in the study. Furthermore, the static modulus of elasticity was deducted through a linear relationship between the two moduli of elasticity. Finally, the concrete compressive strength was mathematically determined by using the previously mentioned parameters. The accuracy of the proposed method for concrete compressive strength assessment ranged between 92 and 94%. The precision was established with respect to the destructive testing of concrete cores. For this research, the experimental part was performed on concrete cores extracted from different elements of different structures and divided into four distinct groups. The high rate of accuracy in predicting the concrete compressive strength, provided by this study, exceeds 90% with respect to the reference, and makes this method suitable for further investigations related to both the optimization of the procedure and = the domain of applicability . Non-destructive testing methods (NDT) are essential tools in estimating concrete properties . A comprehensive analysis of the mechanical properties is useful in the process of structural optimization, as well as in terms of budget efficiency.In the case of Reinforced Concrete (RC) structures, one of the key properties is the compressive strength. An investigation from this point of view can provide an overview of the structural integrity of a building. Such an analysis helps civil engineers in optimizing the process of structural intervention by deepening the understanding of how the building works from the structural point of view and also considering the concrete mix design and the associated physical, mechanical, and durability characteristics. Therefore, the interventions can be targeted on those elements that have a deficient behavior, which can induce negative effects into the structure ,4,5,6,7.The samples are prepared and tested in the laboratory;The samples are collected on the construction site during the concrete casting, followed by curing and testing in the laboratory;The concrete cores are extracted directly from the structure (specific elements) and, after specific processing and conditioning, they are tested in the laboratory.Traditionally, the concrete compressive strength is determined through destructive testing (DT) which is considered the most reliable testing, and thus been referred to as the reference method. In DT, there are identified three possible situations:In the first situation, this testing is performed in order to evaluate or calibrate different mixing sequences of concrete mixture ,9. The sKL1 = 1.35; CFKL2 = 1.20; and CFKL3 = 1.00). The confidence factor is used as a correction factor for incomplete knowledge and level of uncertainty [According to ,14 the aertainty . To reacertainty .NDT represents a possible, viable alternative, mainly in terms of cost efficiency and also as they are fast in delivering results. However, NDT techniques measure indicators that are sensitive to a specific concrete property. For example, the ultrasonic pulse velocity and rebound hammer are sensitive to mechanical properties such as the compressive strength and porosity of concrete . AnotherOne of the first and most used NDTs is the visual inspection (VI) . It focuThe rebound hammer (RHS) determines the surface hardness. The main advantages are the fact that is a simple to use method with low cost and energy. The method tests the concrete strength on a depth of 2–3 cm, this being the reason why it should be combined with other methods that tests the concrete elements in depth .The radiographic testing (RT) consists of a radioactive isotope source that transmits photons continuously through the concrete element, photons which are developed on a radiation sensitive film. It is mainly used for visualizing interior features of an element. There are many types of radiography that each have a specific application. In order to detect voids, cracks, or other interior defects, gamma-ray radiography was found to be useful . This meThe carbonatation testing (CT) is made by spraying an exposed surface of the concrete element with a solution containing 1% phenolphthalein. The calcium hydroxide reacts with the solution resulting in a pink color, while the carbonated area will remain uncolored .Infrared thermography (IT) is used in order to determine the internal voids, cracks, or delamination by measuring the time delay before the temperature changes .The electromagnetic and radar testing (E&RT) are the most used NDT methods for the identification of reinforcements position, diameter, and distance from the surface .The drilling resistance method (DRM) presumes estimating the concrete compressive strength by counting the time required to drill a certain distance in the concrete element with a constant force and rotation speed. Serkan et al.  proved tUltrasonic pulse velocity (UPV) is an NDT which has been extensively investigated for decades . The metOver the years, several NDT methods have been developed with the main purpose of estimating, as correctly as possible, the mechanical properties of materials and elements. A short overview of the NDT methods used on concrete structures is presented thus:The most used NDT methods, for estimating concrete compressive strength, are the Schmidt rebound hammer, the ultrasonic pulse velocity, and the sonic rebound (SonReb) which consists of a combination of the first two methods. Făcăoaru et al.  developeResearchers tried to develop various relationships between the measured indicators and the mechanical properties of concrete, by using different techniques, such as response surface (RS) ,32,33,34Breysse concluded  that a uThis paper aims to present a methodology in estimating the concrete compressive strength by using ultrasonic pulse velocity as the only on-site testing method and a series of mathematical relations connecting the UPV with the moduli of elasticity (dynamic and static) and finally with the compressive strength.car) is corrected by a series of coefficients described in Equation (1) provided by Romanian Norm NP 137 [is).is—equivalent concrete compressive strength (MPa); a—coefficient that takes into account the influence of the core diameter; b—coefficient that takes into account the height/diameter ratio; c—coefficient that takes into account the influence of the degraded layer; e—coefficient that takes into account the nature of the leveling layer; g—coefficient that takes into account the humidity of the concrete core; d—coefficient that takes into account the position and diameter of the reinforcement bars; and fcar—resulted compressive bearing capacity (MPa).This method is considered to deliver the most reliable results regarding the concrete compressive strength and, in this study, all results are reported to this method, considered to be the reference one. Consequently, the precision rate of the proposed method is also established with respect to the DT, as reference base of evaluation. DT consists of extracting concrete cores from the existing elements, cores which are then subjected to a series of laboratory processing and conditioning after which they are subjected to compression load until failure. The resulting compressive bearing capacity ; L—path length in concrete (mm); and T—transit time (µs).A calibration between compressive strength and ultrasonic pulse velocity for each concrete sample assures enough dependability for the two indicators . Naik etModulus of elasticity of concrete (E) is a property of concrete that estimates the potential deformation of a structural element under service conditions . The facs) is a fundamental parameter that is defined by the stress–strain diagram under static loads [The static modulus of elasticity (Eic loads  and it id), in comparison to Es, is defined by the ratio of stress–strain under vibratory conditions [d are resonance frequency or UPV [d. For this study, Ed was determined accordingly to Romanian Guide GE 039 [d—dynamic modulus of elasticity (MPa); Θd—dynamic Poisson’s ratio; γ—air dry density (kg/m3); g—gravitational acceleration (m/s2); and VL—ultrasonic pulse velocity (km/s).The dynamic modulus of elasticity .L—ultrasonic pulse velocity (km/s).Romanian Guide GE 039  presentsd = 0.25 the values of the function depending on the dynamic Poisson’s ratio becomes:Thereby, when considering the ΘInserting Equation (5) in Equation (3) results the dynamic modulus of elasticity has the following expression:3) and VL—ultrasonic pulse velocity (km/s).Regarding the air-dry density Salman  and Panze) with the mean values of the predicted ones using Equation (7) (γt), it was shown it reached a precision rate of 98%.In order to establish the accuracy of the proposed equation, the air-dry density of concrete was experimentally determined. The samples were weighed and measured with the purpose of determining the apparent volume. Comparing the mean values of air-dry density obtained experimentally  was calculated individually and then a direct link between them was established. Ed was determined via UPV (Equation (6)) and Es was determined via DT (Equation (8)).Romanian Guide GE 039  stipulatis) determined with Equation (1), using the mathematical relationship presented by Noguchi et al. [s—static modulus of elasticity (MPa); fc = fis—concrete strength (MPa); and γ = γt—concrete air-dry density determined via UPV (kg/m3).For determining the static modulus of elasticity, with the air-dry density determined with Equation (7) and compressive strength obtained destructively , using the ultrasonic pulse velocity. Comparing the values of the two moduli of elasticity, determined for each specimen separately, it was established a direct and linear link between them described in Equation (9).c). Extracting that parameter and rewriting Equation (8) results in a relationship (Equation (10)) where the compressive strength value depends only on parameters that can be determined via UPV.Using Equation (9), the static modulus of elasticity can now be determined only from the ultrasonic pulse velocity measurements and using Equation (7) the air-dry density can be obtained through the same measurements. Therefore, in Equation (8) the only unknown parameter remains concrete compressive strength  determined via the Destructive Method, as follows: is ranges from 15 to 20 Mpa;Group 1 [15–20) MPa: fis ranges from 20 to 25 Mpa;Group 2 [20–25) Mpa: fis ranges from 25 to 30 Mpa;Group 3 [25–30) Mpa: fis exceeds 30 MPa.Group 4 ≥ 30 MPa: fFor a more accessible interpretation of the results, the core specimens were divided into four groups. The considered division criterion is the value of compressive strength (fc).The theoretical methods for assessing the concrete air-dry density and compressive strength, for the four considered groups of specimens, were statistically evaluated with respect to the experimental and reference procedures, in terms of coefficient of variation (CoV), and also the accuracy (ACoV is defined by Everitt  by the mc—accuracy (%).The air-dry density and compressive strength (measured and predicted) were also analyzed in terms of accuracy, defined in accordance with ISO 5725-1  as the ris) can be noted, with a minimum of 15.8 MPa and a maximum of 38.9 MPa. Additionally, the corresponding values for the NDT testing (fc) range from 15.1 MPa to 37.0 MPa , with respect to the mean values for each of the four groups of core specimens, presented in Furthermore, when analyzing each group, it can be noticed that for groups 1, 2, and 4, the theoretical compressive strength, evaluated via the proposed method, tends to be a little overestimated with respect to the reference , while in the third group there is a clear match of the values.As presented in is) and predicted compressive strength (fc) is presented.2, which is a statistical indicator of the quality of the theoretical model, is in this case equal to 0.78.A good correlation is achieved between the two sets of values. The coefficient of determination rFor a better validation of the proposed method, the obtained results are compared to both the SonReb method and the DT. For the SonReb method, six structural elements (columns) were investigated on-site by using ultrasonic pulse velocity and Rebound Hammer Schmidt. Each element was tested in three sections, with five UPV and nine RHS measurements/section. In A graphical representation of the accuracy is presented in For the six elements investigated through both NDT methods, the precision rate in the case of the proposed method reaches up to 96%, while in the case of the SonReb method the precision reaches up to 82%. The aim of this paper is to present the results obtained by combining the on-site measurements of UPV and theoretical interpretation using a set of equations developed by different researchers linking the values of ultrasonic pulse velocity to the dynamic modulus of elasticity, static modulus of elasticity, and finally concrete compressive strength.Estimating concrete compressive strength through this method delivered results with high accuracy, which, in this case, ranged between 84 and 100%. It can be noticed that the high level of accuracy remains the same regardless of the range value of compressive strength, which in this study is between 15.8 and 38.9 MPa. Additionally, the coefficient of variation (CoV) shows reduced values, ranging along compact intervals, both for the dry-air density evaluation  and also for the compressive strength evaluation . Further investigations will also consider the methodology and statistical approach proposed by Breysse et al. .As this method relies only on UPV measurements, the on-site surface preparation and testing process must be performed with a high level of precision; otherwise, the results will have a higher level of uncertainty.In contrast to the SonReb method, the proposed one has the advantage, so far, that there is no requirement of information about the classical concrete mix design such as cement type and dosage, granulometry, and nature of aggregates. This information is often difficult to obtain, especially in the case of buildings where the concrete mix was produced on-site with no known recipe. In the SonReb method, not knowing these parameters correct can lead to errors up to ±30%. In this case, although the concrete mix design was known, hence all the coefficients were correctly assumed, the accuracy of the SonReb method had a lower value than the proposed method on each analyzed concrete column when compared to DT.The current results are clearly encouraging, offering new research perspective for method optimization and further confirmation to prove its viability, especially in terms of concrete mix design diversity, which has experienced an exponential growth in the last decades. The single and multiple additions in concrete compositions, waste, or by-products generated by the industry, may induce concrete hardened state changes which lead to complex investigation in terms of overall behavior, NDT included. A preliminary approach on this area represents the on-going study of the current research.


Immunoglobulin G4 (IgG4)-related disease is a chronic inflammatory disease that was recognized in 2011. Pleuritis associated with IgG4-related disease is rare and can be difficult to diagnose. Although there have been previous reports on pleuritis associated with IgG4-related disease by thoracoscopic findings, this is the first to observe pleuritis with IgG4-related disease from normal pleural thoracoscopic findings.A 70-year-old Japanese female treated for breast cancer 33 years ago was referred to our hospital complaining of dyspnea on exertion. Chest computed tomography (CT) revealed left pleural effusion that was exudative and predominant with lymphocytes, elevated adenosine deaminase (ADA) and Class III cytology . Subsequently, thoracoscopic pleural biopsy was performed for definitive diagnosis. Although pleural macroscopic findings appeared normal, we performed pleural biopsy at random sites. This patient was negative for mycobacterium tuberculosis, and neither granulomas nor malignant cells were found in the collected specimens. An infiltration of inflammatory cells, mainly plasma cells and lymphocytes, was observed. Immunostaining revealed the number of IgG4-positive plasma cells was 102/high power field (HPF), and the percentage of IgG4 positive/immunoglobulin G (IgG)-positive cells was 41.4%. Since IgG4 serum levels were high and IgG4-related submandibular sialadenitis was also observed, a definitive diagnose of pleuritis associated with IgG4-related disease was confirmed.We diagnosed pleuritis associated with IgG4-related disease by thoracoscopic pleural biopsy samples taken from a visually normal pleura. Although exudative pleural effusion with high ADA and lymphocyte predominance is a characteristic of tuberculous pleuritis, other diseases might be present. Since thoracoscopy can increase the diagnostic yield, pleural biopsy should be considered even if thoracoscopic pleural findings are deemed normal. Immunoglobulin G4 (IgG4)-related disease is a chronic inflammatory systemic disease that was recognized in 2011 [We report a case of pleuritis associated with IgG4-related disease confirmed by randomly selected pleural biopsy sites after normal thoracoscopic findings.A 70-year-old Japanese female complaining of dyspnea on exertion was admitted to our hospital after chest X-ray showed left pleural effusion. Her medical history included hypertension, dyslipidemia, paroxysmal atrial fibrillation, cerebral infarction, and left breast cancer. Her hypertension, dyslipidemia, and paroxysmal atrial fibrillation were well managed by medication, and she underwent total left mastectomy and remnant gauze removal surgery 33 and 13 years ago, respectively. She was a nonsmoker with no history of autoimmune diseases, multiple myeloma, tuberculosis, or exposure to asbestos. Her family history revealed that her father had liver cirrhosis, and her mother had been diagnosed with lung cancer. There was no family history of autoimmune disease or tuberculosis.On admission, she was lucid, with heart rate of 74 beats/minute, blood pressure of 138/68 mmHg, peripheral oxygen saturation of 96%, and body temperature of 35.8 °C. Heart sounds were normal but with decreased breath sounds in the left lung field. No spontaneous pain or tenderness was present in the chest. Her abdomen was flat and soft, with no abdominal tenderness or bowel sounds. No pitting edema was observed in either her forearms or legs; however, bilateral submandibular gland enlargement was observed. She had no motor or sensory deficits in her extremities and no arthralgia or morning stiffness associated with rheumatoid arthritis.Mycobacterium tuberculosis was not detected . Elevated IgG and IgG4 levels were observed in the pleural effusion . Class III cells were identified from the pleural effusion, suggesting the possibility of breast cancer dissemination. Mycobacterium cultures for sputum, pleural fluid, and pleural biopsy samples were negative.Inflammatory findings such as C reactive protein (CRP) and tumour makers were within the normal range . Autoantibodies were serologically negative. Serum immunoglobulin G (IgG) was 2001 mg/dL including 637 mg/dL for IgG4 . Chest X-ray and CT showed left pleural effusion without abnormal lung shadow and no pleural thickening  was negative, and no granulomas were found in the pleural specimens. There were no malignant epithelial lesions and immunostaining showed no deviation in IgG-k or IgG-λ chains. Epstein-Barr virus-encoded small Rino Nucleic Acid (RNA) and Congo red stain were also negative. IgG4-positive cells were 102/high power field (HPF), and the percentage of IgG4 positive/IgG positive cells was 41.4%, suggesting pleuritis associated IgG4-related disease Fig. a, b.Fig.d-glucose (FDG) positron emission tomography (PET)-CT for systemic retrieval analysis. FDG PET-CT showed FDG accumulation within the bilateral submandibular glands of the left pleura. Subsequently, under ultrasound guidance, we collected biopsy samples from the right side of the submandibular gland. An infiltration of plasmacytes and lymphocytes around the adeno-atrial tissue was present; however, no malignant cells or granulomas were noted. Immunostaining confirmed IgG4-positive cells were 96/HPF and the percentage of IgG4 positive/IgG positive cells was 55.2%, confirming pleuritis-associated IgG4-related disease.We then performed 2-deoxy-2- abnormal shadow on chest CT; (2) serum IgG4 level of > 135 mg/dL; (3) histopathologic features fulfilling the comprehensive diagnostic criteria; (4) presence of extrathoracic lesions . IgG4-rePleural effusion for heart and renal failures is often due to systemic diseases associated with leakage or bilateral pleural effusion. Pleural effusion due to IgG4-related disease is expected to be exudative pleural effusion due to inflammation and can take the form of either unilateral or bilateral pleural effusion patterns. Pleural effusion in pleuritis associated with IgG4-related disease has been reported to be lymphocyte-dominated exudative pleural effusion with high ADA . To dateHowever, Miyoshi et al. reported that only 1 of 32 cases of tuberculous pleuritis had a positive pleural fluid culture, and it is thought that the diagnosis based on pleural fluid examination alone is extremely difficult. Pleural biopsy, which was performed in this case, is said to be useful for diagnosis and has superior sensitivity and specificity compared to pleural fluid examination . In tubeOn the other hand, exudative pleural effusion with high ADA and lymphocyte-dominated pleural effusion is not a finding specific to tuberculous pleuritis since it is also found in primary effusion lymphoma, leukemia, pyothorax, rheumatic disease, and pleural effusion associated with malignant pleural mesothelioma. It has been reported that thoracoscopic pleural biopsy can provide a definitive diagnosis 90% of the time , 13. TheThus far, three patterns of thoracoscopic findings have been described for IgG4-associated pleuritis: (1) milky pleural plaques suggestive of hyalinized collagen fiber deposits; 2) diffuse inflammatory thickening of the pleura; (3) nodules on the parietal pleura [ diffuse Although thoracoscopic findings of the pleura appeared normal in this case, we performed random pleural biopsies to confirm a diagnosis of pleuritis associated with IgG4-related disease. We suggest that thoracoscopic pleural biopsy should be considered even when pleural findings on thoracoscopy appear normal.


The present study evaluated the effect of both methionine sources on metabolism and growth of Nile tilapia. A growth trial was performed using three isonitrogenous and isoenergetic diets, containing plant ingredients as protein sources: DLM and MHA diets were supplemented on equimolar levels of Met, while REF diet was not supplemented. Hepatic free Met and one-carbon metabolites were determined in fish fed for 57 d. Metabolism of dl-Met and MHA was analysed by an in vivo time-course trial using 14C-labelled tracers. Only dl-Met supplementation significantly increased final body weight and improved feed conversion and protein efficiency ratios compared with the REF diet. Our findings indicate that Met in DLM fed fish follows the transsulphuration pathway, while in fish fed MHA and REF diets it is remethylated. The in vivo trial revealed that 14C-dl-Met is absorbed faster and more retained than 14C-MHA, resulting in a greater availability of free Met in the tissues when fish is fed with DLM diet. Our study indicates that dietary dl-Met supplementation improves growth performance and N retention, and that Met absorption and utilisation are influenced by the dietary source in tilapia juveniles.Commercial diets for tilapia juveniles contain high levels of plant protein sources. Soybean meal has been utilised due to its high protein content; however, soy-based diets are limited in methionine (Met) and require its supplementation to fulfil fish requirements.  Nevertheless, there are still many challenges to overcome in order to farm tilapia under economic and environmentally sustainable principles. Part of the challenge is dependent on the development of sustainable feeds that will ensure an optimal nutritional status for the fish while promoting N and P retention, thus reducing nutrient excretion into the aquatic environment.Tilapia is the second most farmed fish worldwide, just after carp–4 due to its high protein content and relatively well-balanced amino acid profile. However, soy-based diets are limited in methionine (Met) and require its supplementation in order to fulfil fish requirements.In order to increase the sustainability of aquaculture feeds, inclusion levels of plant ingredients in fish diets have increased. Soybean meal has been successfully included in aquafeedsl-Met is converted into s-adenosylmethionine (SAM), a methyl donor for several reactions. Subsequently, SAM donates its methyl group and is transformed into s-adenosylhomocysteine (SAH), which is rapidly converted into homocysteine. Hepatic homocysteine can be remethylated back to Met by adding a methyl group from trimethylglycine or can irreversibly enter the transsulphuration pathway. The transsulphuration pathway converts homocysteine into cystathionine and then cysteine. Ultimately, cysteine can be incorporated into proteins, metabolised into glutathione or oxidised to form taurine.Met is an indispensable amino acid involved in protein synthesis, transmethylation reactions and antioxidant defence. Met metabolism occurs mainly in the liver where dl-Methionine (dl-Met) and methionine hydroxyl analogue  are synthetic sources of Met that are often supplemented to animal feeds. dl-Met is a racemic mixture of d- and l-isomers of Met, while MHA is a racemic mixture of d- and l-isomers of MHA. MHA chemical structure is similar to that of Met; however, it contains a hydroxyl group instead of the amino group. Recently, studies in rainbow trout (Oncorhynchus mykiss) suggested that dl-Met and MHA uptake in the gut apical surface is facilitated by Na-dependent transporters and mediated by proton-independent transporters across the basolateral membrane,10. Data comparing the intestinal flux rates of dl-Met and MHA suggest a faster intestinal transport of the former synthetic source,10,11.l-amino acids, d-isomers of dl-Met first need to be converted to a keto-Met intermediate  and then transaminated to l-Met before becoming available,12. On the other hand, both the d- and l-isomers of MHA need to be converted to KMB to become available,12. These differences are likely to be reflected as differences in absorption and metabolism and may result in different biological efficiencies. Numerous studies in terrestrial animals and fish advocate that MHA supplementation results in lower bioavailability of this compound compared with dl-Met–15. In rainbow trout, dietary MHA-Ca salt was found to have lower bioavailability than dl-Met, resulting in 69 % lower fish weight gain, 60 % lower growth rate and 73 % lower N retention in fish. Similarly, growth performance and feed utilisation indicators in common carp (Cyprinus carpio) fed dl-Met and MHA-Ca supplemented diets demonstrated that MHA-Ca was 41–50 % as available as dl-Met on weight-for-weight basis. In contrast, some authors report similar efficiencies among Met sources based on growth and feed efficiency in several aquatic animals–21. On the other hand, a study performed in channel catfish observed that dietary MHA-Ca supplementation resulted in improved body weight, weight gain, feed conversion ratio and protein efficiency ratio. Based on the data published, NRC concluded that it is reasonable to assume that the biological efficacy of MHA in fish is 75–80 % that of dl-Met on an equimolar basis (63–67 % on a weight basis). Generating data on growth, diet utilisation and Met metabolism in a commercial relevant species such as the Nile tilapia (Oreochromis niloticus) is of paramount importance for the aquaculture industry.Since animals can only metabolise In this context, the objective of the present study was to understand how the dietary source of Met affects Met metabolism and growth of Nile tilapia juveniles. Radiolabelled Met sources were used in a nutrient flux assay to evaluate their influence in the amino acid metabolic pathways.. No Met was supplemented to this diet. Experimental diets were the REF diet supplemented with 1·5 g/kg dl-Met (dlM diet) and 1·8 g/kg MHA-Ca . To minimise variability among diets, one common basal diet was formulated, and the respective supplemental Met source was added in DLM and MHA diets. Diets were formulated to meet the minimum requirements of amino acids on digestible basis for Nile tilapia juveniles, except for Met. Apparent digestibility coefficients of amino acids for the ingredients used were taken from published review data,25. Diets were supplemented with selected indispensable amino acids and di-calcium phosphate to avoid amino acid or mineral imbalances.Three experimental diets were formulated to be isonitrogenous and isoenergetic . Diet REUpon ingredient grinding with a hammer mill  and its mixing in a double-helixmixer, all diets (pellet sizes 1·2 and 2·0 mm) were manufactured using a twin-screw extruder  at SPAROS Lda. Upon cold extrusion, diets were dried in a vibrating fluid bed dryer . After cooling, the oils were added to the pellets by vacuum coating (model PG-10VCLAB). Throughout the duration of the trial, experimental feeds were stored at room temperature, in a cool and aerated storage room. Proximate composition and amino acid analysis were determined in all experimental diets, as reported in The experiment was carried out in compliance with the Guidelines of the European Union Council (Directive 2010/63/EU) and Portuguese legislation for the use of laboratory animals. Nile tilapia  juveniles were obtained from Til-Aqua International B.V. and the experiment was conducted at Centre of Marine Sciences of Algarve (CCMAR) facilities . CCMAR facilities and their staff are certified to house and conduct experiments with live animals . Upon arrival, fish were acclimatised to the new rearing facilities in a recirculating aquaculture system and were fed a commercial diet .sd 0·5)°C and dissolved oxygen in water was maintained above 85 % of saturation. Water quality parameters were monitored daily and adjusted when necessary: pH was maintained between 7·0 and 8·2 and the concentration of unionised ammonia and nitrites in water was 0 mg/l during the whole experimental period. Mortality was monitored daily.Juvenile Nile tilapia were reared in 100-litre cylindrical tanks in a recirculating aquaculture system equipped with a mechanical filter, a submerged biological filter and a UV steriliser. Photoperiod was natural (10 h light: 14 h dark), temperature averaged 25·9 (sd 0·4) g were allocated into nine tanks at an initial density of 1·1 kg/m3 (fifty fish per tank). Eight fish from the initial stock were sampled and the whole fish were stored at −20°C until analysis of proximate composition. Triplicate tanks were randomly assigned to one of the three dietary treatments . Fish were fed to visual satiety by hand, three times a day  and feed intake was recorded daily for 57 d.Fish with an initial mean body weight of 2·3 . Whole-body, liver and viscera weight of five individual fish were recorded for calculation of biometric indexes and liver samples were snap-frozen in liquid Ndl-Met and MHA. REF diet was not included in this trial since the aim was to assess putative differences in the metabolic flux of the Met supplemental sources and not of the intact protein. Random fish fed the DLM or MHA diet were transferred to the nutrient flux laboratory after being fasted for 24 h.At the end of the growth trial, to understand how different dietary Met sources are absorbed and metabolised in tilapia juveniles, a time-course metabolic trial was performed using radiolabelled dl-(1–14C)-Methionine  and (1–14C)-calcium bis-methionine hydroxyl analogue  (Campro Scientific GmbH) were used as tracers to radiolabel the experimental diets (DLM and MHA). The methodology for labelling the experimental diets was established in previous works of the authors–28. The tracers were diluted in freshwater Ringer solution and a known value of the tracer was dispensed using a micropipette on individual pellets of the correspondent experimental diet. The pellets were dried at 50°C for 1 h. Eight pellets per fish, corresponding to 0·3 % body weight, were loaded into a hollow plastic tube of 1·5 mm inner diameter and stored for subsequent tube-feeding. Prior to tube-feeding, the amount of radioactivity (DPM) of ten individual pellets from each experimental diet labelled with the corresponding tracer was determined in a TriCarb 2910TR low activity liquid scintillation analyser (Perkin Elmer) after adding the scintillation cocktail .in vivo method of tube-feeding used to perform the metabolic trials was adapted from Costas, which was a modification of the method first described by Rust and modified by Rønnestad. Fish were anaesthetised  and subsequently were taken out of the water using a fish net and placed onto a dry plastic tray. The previously loaded plastic tube containing the radiolabelled pellets was inserted into the fish mouth and the feed pellets were gently pushed directly into the oesophagus using a solid piece with a smaller diameter placed inside as a plunger. The diameter and length of the hollow plastic tube were previously tested to avoid injuring the fish oesophagus. This procedure lasted approximately 10 s. The metabolic fate of the tracer (14C-dl-Met or 14C-MHA) was considered to represent the fate of the tracee (dl-Met or MHA-Ca).The n 6–7 fish for each diet and incubation period). Fish was removed from the chamber and weighed.After tube-feeding, fish were placed into a tank with clean and aerated freshwater to eliminate any residual anaesthetic and were monitored for eventual pellet regurgitation. After this period, fish were transferred into individual incubation chambers containing 2 litres of freshwater at 26°C. Each chamber was hermetically sealed and supplied with a gentle oxygen flow. After the incubation period, oxygen flow was stopped and fish were euthanised inside the chambers by a lethal dose of anaesthetic (750 mg/l of MS-222 buffered with sodium bicarbonate). The incubation periods were 1, 2, 3, 4 or 6 h , spleen, pancreas and perivisceral fat and will be designated from hereafter as Viscera compartment; skin-on fillets (muscle with skin), as Muscle compartment and the rest of the fish  as Residual compartment. Viscera and Liver compartments were analysed as whole. Muscle and Residual compartments were minced using a coffee grinder until a homogeneous mixture was obtained and 0·5 g samples were taken for further analysis.Water samples were collected from each individual chamber to determine the amount of radioactivity (DPM) present in the incubation water. The radioactivity present in the incubation water resulted from evacuated (non-absorbed) and/or catabolised radiolabelled Met source as COFree fraction). In order to get a better insight of the metabolic flux of 14C-dl-Met and 14C-MHA, tissue samples from the 6 h incubation period were homogenised and underwent a series of extraction procedures to separate organic compounds such as protein, lipids and other metabolites as described previously by Rocha. Briefly, samples were transferred to a clean vial and further homogenised in distilled water using an Ultra-turrax homogeniser (IKA). Total lipids were extracted using a modified Bligh and Dyer method for small volumes, and total protein was extracted based on a TCA precipitation method. The supernatant containing non-extracted metabolites was collected for radioactivity determination. Protein pellet was resuspended in an appropriate volume of Solvable™ (Perkin Elmer) and kept at 50ºC until complete solubilisation was achieved. Lipids and other metabolites are designated as Others fraction, while protein as Protein fraction. Scintillation cocktail  was added to all samples and DPM were counted in a TriCarb 2910TR low activity liquid scintillation analyser (Perkin Elmer). All counts were corrected for quench and lumex. Radioactivity found in the Incubation Water and in the Free fractions of all compartments was normalised for fish or tissue weight and expressed as DPM/g. Radioactivity present 6 h after feeding the radiolabelled nutrients in the Protein and Others fractions of all compartments was expressed as DPM.All fish tissues were incubated at 4°C for 24 h with 6 % (w/v) TCA, with periodical stirrings. After this period, tissue samples were taken and the TCA samples collected for radioactivity determination  from 1 to 6 h was calculated as follows:t is the time point, DPMi is the amount of radioactivity found at ti and n is the total number of measures. AUC was expressed as a percentage of total cumulated (1–6 h) radioactivity (DPM) per dietary treatment.where ®, Evonik Nutrition & Care) before diet formulation.Raw materials  were analysed for DM, crude protein and amino acid content using NIR  by wet chemistry  using the combustion/Dumas method; crude fat by petroleum ether extraction using a Soxtherm Multistat/SX PC (Gerhardt); gross energy by combustion in an adiabatic bomb calorimeter  calibrated with benzoic acid and P content by digestion at 230°C in a Kjeldatherm block digestion unit followed by digestion at 75°C in a water bath and absorbance determination at 820 nm (adapted from AFNOR V 04-406).Chemical analysis followed standard procedures of the Association of Official Analytical Chemists®, Evonik Nutrition & Care) using ion exchange chromatography. Hepatic free amino acids and SAM and SAH contents were determined after homogenisation of freeze-dried samples in 0·1 M HCl on ice, centrifugation at 1500 × g at 4°C for 15 min and deproteinisation of the supernatant by centrifugal ultrafiltration . All samples were pre-column derivatised with Waters AccQ Fluor Reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) using the AccQ Tag method (Waters), except samples for SAM and SAH analysis, which were not derivatised. All analyses were performed by ultra-high-performance liquid chromatography on a Waters Reversed-Phase Amino Acid Analysis System, using norvaline as an internal standard. Amino acids were identified by retention times of standard mixtures (Waters) and pure standards (Sigma-Aldrich). Instrument control, data acquisition and processing were achieved by the use of Waters Empower software.Amino acid content in diets and whole-body fish samples were analysed by wet chemistry  = 100 × (apparent feed intake/ABM/d), where ABM is average body mass = /2.Feed conversion ratio = apparent feed intake/wet weight gain.Protein efficiency ratio = wet weight gain/crude protein intake.Hepatosomatic index (%) = 100 × .Viscerosomatic index (%) = 100 × .Protein or energy retention (% intake) = 100 × /(protein or energy intake)).Daily N intake (mg N/kg/d) = N intake/ABM/d.Daily N gain (mg N/kg/d) = /ABM/d.Daily N loss (mg N/kg/d) = daily N intake − daily N gain.. All data were checked for normal distribution and homogeneity of variances. Differences among groups were identified by one-way ANOVA followed by Tukey’s multiple-comparison test or, when the assumptions for the ANOVA failed, by Kruskal–Wallis one-way ANOVA by ranks followed by Dunn’s multiple-comparison tests. Data from the time-course metabolic trials were subjected to linear regression analysis to understand the relationship between the tracer (14C-dl-Met or 14C-MHA) and incubation time in each compartment . Additionally, one-way ANOVA followed by planned contrasts test was performed to compare the differences between 14C-dl-Met and 14C-MHA fed fish at each time point. At 6 h time point, Mann–Whitney U test was additionally used to identify differences in the Protein and Others fractions of the several body compartments. All statistical differences were considered significant at P < 0·05. Statistical analyses were performed using the open source software R version 3.6.1.Sample size was determined based on preliminary power analysis to ensure a probability of at least 80 % in the detection of treatment effects. Data are presented as means and standard deviations. Data expressed as a percentage were arcsine square root transformed previously to the statistical analysissd 4·7) g) than fish fed the REF (24·1 (sd 3·0) g) and the MHA (24·8 (sd 2·7) g) diets (P < 0·05). Fish biomass gain differed marginally with the different dietary treatments (P = 0·08). Feed intake was similar among experimental groups. Fish fed the DLM diet presented a significantly lower feed conversion ratio than fish fed the REF diet (P < 0·05). Protein efficiency ratio was significantly higher in fish fed the DLM (3·4 (sd 0·1)) than the REF (3·1 (sd 0·1)) diet (P < 0·05). Dietary treatments did not affect significantly hepatosomatic and viscerosomatic indexes (P > 0·05).At the end of the growth trial, survival was 100 % in all treatments and fish had a 9–10-fold increase in body weight . Fish feP < 0·05; P > 0·05). Additionally, whole-body total Met content was significantly higher in DLM fed fish (3·86 (sd 0·01) g/kg) than in fish from the REF (3·54 (sd 0·11) g/kg) or MHA (3·50 (sd 0·14) g/kg) dietary treatments (P < 0·05). No significant differences were found for the other amino acids among experimental groups. Protein and energy retention were significantly higher for fish fed the DLM diet than for fish fed the other diets  mg N/kg/d), significantly different from REF (657 (sd 8) mg N/kg/d) or MHA (660(sd 22) mg N/kg/d) fed fish (P < 0·05). Moreover, fish from the DLM group presented the lowest daily N loss (667 (sd 43) mg N/kg/d), which was significantly different from the REF group . SAM (1·25–1·35 mg SAM/g dry weight (DW) liver), SAH (0·23–0·27 mg SAH/g DW liver) or SAM/SAH ratio (4·79–5·48) were not affected by the dietary treatments (P > 0·05). Homocysteine levels (P < 0·05). Cystathionine was significantly lower in fish fed the supplemented diets than in fish fed the REF diet . Fish fed the DLM diet had more hepatic taurine content than fish fed REF and MHA diets (P < 0·05). In addition, there were no significant differences in taurine content between fish fed REF and MHA diets  mg TMG/g DW liver) than the REF (0·06 (sd 0·00) mg TMG/g DW liver) diet (P < 0·05). No significant differences in hepatic trimethylglycine content were found for fish fed the MHA diet compared with the other groups (P > 0·05).Free Met (sd 2·90) mg/g DW liver) than in liver of fish fed the REF (86·48 (sd 3·29) mg/g DW liver) and the MHA (86·08 (sd 1·81) mg/g DW liver) diets (P < 0·05). Similarly, the sum of free indispensable and dispensable amino acids was significantly higher in liver of fish fed the DLM diet than in liver of fish fed the other diets . Also, at the end of the time course (6 h) a higher amount of 14C-MHA was present in the Incubation Water compartment than of 14C-dl-Met  at 2 and 3 h after diet ingestion. The AUC for the Residual and Muscle Free fractions was 1·3 and 1·9-fold higher in DLM fed fish than in fish fed MHA diet, respectively, indicating a higher bioavailability of the Met source in the fish fed the DLM diet (The presence of DLM diet .Viscera and Muscle Protein fractions from DLM fed fish (P < 0·05). No significant differences between treatments were detected in Liver and Residual Protein fractions (P > 0·05). Regarding the Others fraction (lipids and other metabolites), there were no significant differences between treatments in all compartments (P > 0·05). The amount of tracer present in the Liver Others fraction was the lowest  and the highest in the Residual Others fraction .At the end of the incubation period (6 h), a significantly higher amount of tracer was determined in the in vivo method using radiolabelled tracers (14C-dl-Met and 14C-MHA) was utilised to gain a deeper understanding on how different Met sources may affect the dietary Met utilisation by Nile tilapia juveniles. The amount of tracer found in the Incubation Water increased with time in both treatments; moreover, the analysis of the AUC from 1 to 6 h after ingestion revealed a higher amount of 14C-MHA than of 14C-dl-Met indicating a greater evacuation of unabsorbed MHA and/or higher catabolism of this Met source compared with dl-Met. The solubility of MHA-Ca in water is higher than that of dl-Met; however, since the labelled pellets were placed directly into the fish oesophagus and were not in contact with water, the radioactivity present in the Incubation Water was the result of the evacuation of unabsorbed tracer. Similarly, in broiler chicks fed 14C-dl-Met or 14C-MHA a higher amount of tracer was present in the excrements of 14C-MHA fed birds than in excrements of birds fed the 14C-dl-Met,40. dl-Met and MHA uptake is mediated by Na-dependent transporters across the brush border membrane,10; however, MHA is also partly transported by diffusion. In rainbow trout, it has been demonstrated that MHA presents a slower intestinal absorption rate than dl-Met. This indicates that MHA stays longer in the intestine being more prone to bacterial degradation that would lead to a further decrease in the intestinal absorption of MHA and its evacuation. Combined, these two factors may contribute to the increased amount of unabsorbed 14C-MHA relative to 14C-dl-Met in the Incubation Water.The Viscera and Liver Free fractions of DLM and MHA fed fish presented a similar cumulative bioavailability of 14C-dl-Met and 14C-MHA for the 6 h period. However, a comparison between the two dietary treatments revealed different patterns of tracer flux in these compartments throughout the in vivo trial. Fish fed the DLM diet presented a peak in Liver Free fraction 1 h after feeding, while no peak was observed during the 6 h time course for MHA fed fish. The d-isomer of dl-Met and l- and d-isomers of MHA need to be converted to l-Met to become available to the animal via a two-step process,43; however, the l-isomer present in dl-Met is readily available. Therefore, the results from the in vivo metabolic trial suggest a faster availability of dl-Met to metabolic processes than of MHA.In our study, Residual Free fraction  faster than in MHA fed fish. Moreover, for the 6 h trial, the cumulative Met source bioavailability in Residual compartment was higher in DLM than in MHA fed fish. Although the liver is the major site of conversion of d-Met and of d- and l-MHA to l-Met, this process also occurs in the kidney,39,44. This may explain the high amount of tracer found in the Residual Free fraction, as this compartment includes the kidney among other tissues. Other studies have reported higher amounts of 14C-dl-Met than of 14C-MHA recovered in the kidney of broilers,40. In a study where chicken kidney homogenates were incubated with 14C-labelled l-Met, dl-Met or MHA, dl-Met was found to be the substrate most readily converted into the intermediate KMB, revealing that the majority of the KMB produced in the kidney would result from the oxidation of the d- and not the l-isomer of dl-Met. Further studies are necessary to confirm if in fish the kidney plays an important role in the conversion of d- to l-Met.In fish fed the DLM diet, the tracer was available to the Muscle Free fraction of DLM fish, implying that dietary Met is available earlier for utilisation when fish are fed this source. Also, cumulative Met bioavailability in Muscle Free fraction was higher in DLM than in MHA fed fish. Therefore, one can consider that Met is readily available for protein synthesis faster and for a longer period in DLM fed fish than in fish fed the MHA diet. In fact, 6 h after feeding, the amount of tracer found in the Protein fractions of all compartments of DLM fed fish was higher than in MHA fed fish, with significant differences detected in Viscera and Muscle compartments. This indicates that DLM fish exhibited a faster and more effective incorporation of Met into muscle protein. Although the amount of tracer present in the Liver Protein fraction of DLM fish was higher than in fish fed the MHA diet, the difference was not significant due to a high variability within treatments. The vital physiological functions of the liver in detoxification, protein synthesis and digestion, constantly producing metabolites, may account for this variability.After being converted, the Met originating from the different dietary Met sources is transported to the rest of the body to be utilised by the fish. As a consequence of the faster absorption and hepatic metabolism, 2 h after feeding a higher amount of tracer was observed in the Others fraction, was very low. Metabolic studies in gilthead seabream,45 and Senegalese sole using a 14C-amino acid mixture have previously demonstrated that absorbed amino acids were preferentially used for protein synthesis and only a small proportion were converted into lipids.In both dietary treatments, the amount of tracer retained in the lipid fraction, the major component of the  and Atlantic salmon ,49 were unable to observe a similar effect of dietary supplementation in hepatic Met levels, possibly due to the fact that these studies report postprandial results unlike the present study where fish were sampled 24 h after feeding, hence the result of basal metabolism. The increase in free Met levels in liver indicates a higher availability for protein synthesis, transmethylation reactions and antioxidant defence in fish fed supplemented diets (DLM and MHA diets) compared with fish fed the non-supplemented diet (REF diet). In the present study, DLM and MHA fed fish presented similar levels of hepatic Met, SAM and SAH, while homocysteine was significantly lower in fish fed the DLM diet in comparison with the other dietary treatments. This suggests that the different Met sources follow different metabolic pathways. In neonatal pigs, to meet Met requirements, protein synthesis is favoured over Met transmethylation and the Met pool is conserved by increasing homocysteine production and suppressing transsulphuration. In the present study, the hepatic free amino acid analysis indicates that fish fed MHA and REF diets probably remethylate homocysteine back to Met, with the addition of a methyl group from trimethylglycine. On the contrary, DLM fed fish seem to divert Met to the transsulphuration pathway, resulting in a significantly higher hepatic taurine content in DLM compared with MHA fed fish. Similarly, feeding Atlantic salmon with soy-based diets supplemented with dl-Met also resulted in an increase in the transsulphuration pathway and consequently higher hepatic taurine content. These results indicate a stimulation of the transsulphuration pathway in fish fed the DLM diet.At the end of the growth trial, the content of hepatic free amino acids and one-carbon metabolites was determined in fish fed control and supplemented diets. The content of total hepatic free amino acids in fish fed the DLM diet was significantly higher than in fish fed the REF or the MHA diets, due to an increase in both indispensable and dispensable amino acids. Since these are not postprandial results, it indicates a higher bioavailability of amino acids for metabolic purposes in Nile tilapia fed diets supplemented with DLM. Met supplementation caused an increment of free Met in liver, independently of the source. Met dietary supplementation studies in Nile tilapia,52, lipid digestion, osmoregulation and antioxidant defence–56 and it also increases amino acid retention,57. In Nile tilapia, it has been demonstrated that dietary taurine supplementation improves growth performance. In the present work, taurine status in DLM fed fish might have partially contributed to the improvement in growth performance observed in the growth trial. Diet supplementation with dl-Met increased fish body weight when compared with REF and MHA fed fish. Relative to the REF diet, DLM diet produced more 214 g in biomass gain, whereas MHA diet produced only more 124 g (on tank basis). This indicates that on equimolar basis MHA is only 58 % as efficient as dl-Met in terms of biomass gain. dl-Met supplementation improved feed conversion and protein efficiency ratios when compared with the basal diet (REF), while MHA fed fish presented intermediate results. These results are in agreement with previous studies in fish,16,17,59, demonstrating that dl-Met supplementation improves growth and promotes protein accretion more efficiently in Nile tilapia.DLM and MHA fed fish had a similar intake of dietary Met, cysteine and taurine. The experimental diets were soy-based, hence low in taurine. In fact, taurine content in all diets was below 0·1 g/kg. Taurine is an end product of Met metabolism and of the transsulphuration pathway. Therefore, although Met and cysteine hepatic levels were similar in fish fed DLM and MHA diets, a higher hepatic taurine content was found in the former, probably due to a higher availability of Met, as indicated by the metabolic trials. Higher hepatic taurine content is beneficial for the fish as taurine is involved in numerous physiological functions. In fish, taurine plays important roles in bile salt formationResidual and Muscle Free fractions shown higher availability of Met in DLM than in MHA fed fish and 6 h after feeding a greater amount of tracer in the Muscle Protein fraction was found in the former. These differences were also reflected in the long term. At the end of the experimental period, protein retention in DLM fed fish was higher than in fish fed the MHA diet. Protein and total Met whole-body content were also higher in fish fed the DLM diet than in fish from the MHA group. The differences found between DLM and MHA fed fish in total Met content and availability explain the differences in protein content, reinforcing that Nile tilapia ultimately utilises dl-Met more efficiently for protein deposition than MHA.In the metabolic trial, it was established that the distinct Met sources are utilised differently by Nile tilapia juveniles in the short term. , where the relative bioavailability of MHA was compared with dl-Met by dose–response trials regarding growth performance and nutrient retention. Lower N losses are related to higher protein digestibility and/or lower catabolism, resulting in lower N release to the environment. The in vivo metabolic trial revealed that the amount of tracer present in the Incubation Water increased with time and was lower in fish fed the DLM diet than in MHA fed fish. Consequently, in the long-term trial this is reflected in the N balance, indicating that dietary dl-Met supplementation contributes to a reduction in the environmental impact of Nile tilapia farming.The augmented protein retention was ultimately reflected in the N balance. All diets were isonitrogenous and feed intake was similar in all treatments, resulting in similar N intake among treatments. However, DLM fed fish presented the highest N gain and the lowest N losses, indicating that these fish were more efficient in retaining N than MHA fed fish. Similar results have been reported in studies performed with rainbow trout juvenilesin vivo study indicated that dl-Met is more retained than MHA probably due to a faster absorption rate as well as a greater availability of free Met in the tissues to be utilised by Nile tilapia. Additionally, Met from the different sources appears to follow distinct metabolic pathways; while Met from the DLM seems to be transsulphurated, Met from MHA and REF diets is probably remethylated to Met to maintain the free Met pool. In the long term, dietary dl-Met supplementation of soy-based diets improved growth performance and N retention in Nile tilapia, reducing the environmental impact and contributing towards a more sustainable industry.In conclusion, dietary Met sources influence Met absorption and utilisation in Nile tilapia juveniles. The


Neuropeptides are small proteins produced and released by neurons. Neuropeptides are key mediators in the communication between neurons. Urocortin 3 is an important neuropeptide that is involved in stress-related behaviors. In this study, our aim was to determine in which regions of the lamprey brain urocortin 3 is expressed. The study of the distribution of neuroregulatory peptides in lamprey species helps in understanding their early evolution of neuropeptidergic systems in vertebrates because lampreys are one of the most ancient vertebrate groups. The sea lamprey life cycle lasts between five and nine years, and it includes a long larval stage and a transformation to the adult stage. We analyzed the organization of urocortin 3-expressing neuronal populations in the brain of larval and adult sea lampreys to establish whether this neuropeptide is expressed differentially in these two widely different phases of the sea lamprey life cycle. Our results revealed important differences between larval and adult stages. Moreover, comparing our results with the distribution of urocortin 3 reported in other vertebrate groups also revealed a poor evolutionary conservation of urocortin 3 expression in vertebrates. Our work sheds light on the evolution and role of urocortin 3 in vertebrates.In this study, we analyzed the organization of urocortin 3 (Ucn3)-expressing neuronal populations in the brain of the adult sea lamprey by means of in situ hybridization. We also studied the brain of larval sea lampreys to establish whether this prosocial neuropeptide is expressed differentially in two widely different phases of the sea lamprey life cycle. In adult sea lampreys, Ucn3 transcript expression was observed in neurons of the striatum, prethalamus, nucleus of the medial longitudinal fascicle, torus semicircularis, isthmic reticular formation, interpeduncular nucleus, posterior rhombencephalic reticular formation and nucleus of the solitary tract. Interestingly, in larval sea lampreys, only three regions showed Ucn3 expression, namely the prethalamus, the nucleus of the medial longitudinal fascicle and the posterior rhombencephalic reticular formation. A comparison with distributions of Ucn3 in other vertebrates revealed poor conservation of Ucn3 expression during vertebrate evolution. The large qualitative differences in Ucn3 expression observed between larval and adult phases suggest that the maturation of neuroregulatory circuits in the striatum, torus semicircularis and hindbrain chemosensory systems is closely related to profound life-style changes occurring after the transformation from larval to adult life. A 41-amino-acid corticotropin-releasing factor or hormone  purified from the hypothalamus was characterized by its ability to stimulate the release of corticotropin from pituitary cells . More rePetromyzon marinus and Lethenteron camtschaticum; [The distribution of CRH-type peptides and receptors was studied in the brain of mammals and some other vertebrates with immunohistochemistry, in situ hybridization and biochemical methods. In the rat brain, the distribution of these peptides was observed in distinct neuronal populations for each of the peptides: distributed in hypothalamic populations , in thehaticum; ). HowevePetromyzon marinus) in two very different life phases, larval  and adult , that show important differences in some brain centers [The study of the distribution of neuroregulatory peptides in lamprey species may help in understanding their early evolution in vertebrates because lampreys (jawless vertebrates) are the extant outgroup of the other (jawed) vertebrates. Ucn3 is particularly interesting because in the mouse, it is expressed in brain regions involved in stress-related behaviors  ,28,29,30 centers .n = 11), downstream young adults  and upstream  migrating adults of the sea lamprey, Petromyzon marinus L., were used in this study. Larvae and downstream migrating young adults were collected from the River Ulla  with permission from the Xunta de Galicia. Upstream migrating adults were acquired from local suppliers. Before all experiments, animals were deeply anesthetized with 0.1% tricaine methanesulfonate  in fresh water and killed by decapitation. All experiments were approved by the Bioethics Committee of the University of Santiago de Compostela and the Xunta de Galicia and were performed in accordance with European Union and Spanish guidelines on animal care and experimentation.Larvae . For polymerase chain reaction (PCR) cloning, specific oligonucleotide primers  were designed based on the P. marinus Ucn3 transcript sequence deposited in GenBank with accession number KX446867.1 [Templates for in vitro transcription were prepared by PCR amplification of the cloned Ucn3 cDNA fragment using the primers mentioned above. In this case, the reverse primers included the sequence of the universal T7 promoter (TAAGCTTTAATACGACTCACTATAGGGAGA). For the generation of sense probes, the sequence of the T7 promoter was included in the forward primers. Digoxigenin (DIG)-labeled riboprobes were synthesized using the amplified fragments as templates and following standard protocols using a T7 polymerase . In situ hybridization (ISH) experiments with the sea lamprey Ucn3 DIG-labeled riboprobe (1 µg/mL) were performed as previously described for other neuropeptide riboprobes in the sea lamprey ,33,34 usPhotomicrographs of representative sections were taken with an Olympus photomicroscope  equipped with a digital camera . Representative schematics, drawings and plates of photomicrographs were generated with CorelDRAW 12 .The expression of the Ucn3 mRNA in the sea lamprey brain was studied by in situ hybridization in upstream migrating adults, young post-metamorphic adults (juveniles) and larvae. In adults, expression was found in more regions than in larvae, extending from the telencephalon to the medulla . These nIn the telencephalon, a prominent population expressing Ucn3 was observed in the striatum forming an arch parallel to the ventricle a. In theIn the midbrain, a large population of Ucn3 positive cells was observed in the caudal torus semicircularis as a bilateral population that joins caudally in the dorsal midline (horseshoe-shaped) d–f. CaudIn the rhombencephalon, four populations of Ucn3 positive cells were observed. The most rostral population was comprised of scattered Ucn3 positive cells located in the isthmic region named as dorsal isthmic gray or isthmic reticular formation, near the tract of the anterior octavomotor nucleus g. A sparIn the larval brain, the Ucn3 positive populations are restricted to the diencephalon and the rhombencephalon. Unlike in adults, no positive populations were observed in the telencephalon. In the diencephalon, Ucn3 positive neurons were observed in the prethalamus (1 to 3 positive cells per hemisection) a,a’ and In the larval rhombencephalon, Ucn3 positive neurons were only observed in the medial region of the rhombencephalic reticular nucleus caudally to the octaval nerve entrance (posterior reticular formation). There were a few cells located close to the midline near the medial longitudinal fasciculus c,c’ or oHere, we report the first study on the distribution of Ucn3 mRNA in the brain of an agnathan, the sea lamprey. Ucn3 expression was analyzed at two very different life stages, adult  and larval. A comparison between adults and larvae revealed important developmental changes in Ucn3 expression, with new positive populations appearing in the striatum, torus semicircularis, isthmus and nucleus of the solitary tract after the metamorphosis. Transformation in the sea lamprey is complex, and in the nervous system, it largely affects the visual system including the eye and optic tectum ,39,40,41In the lamprey telencephalon, a conspicuous group of Ucn3-expressing cells was found in the striatum. The sea lamprey striatum mostly consists of GABAergic neurons ,46,47, aRecent studies in lampreys stressed the great similarity of circuits and the neuronal activity of the striatal system with those of mammals . PresentThe lack of Ucn3 expression in cells in the preoptic region and hypothalamus of the lamprey is notable and suggests that Ucn3 is not related directly with the hypophysis, which is in contrast with the expression of Ucn3 in the preoptic and/or hypothalamic nuclei of teleosts ,18. HypoIn the diencephalon of both larvae and adults, two populations of Ucn3 positive neurons were observed, namely in the prethalamus and in the nucleus of the medial longitudinal fascicle. These regions contain neurons that project to the medial and posterior rhombencephalic reticular nuclei, intervening as premotor centers , and recLampetra fluviatilis in their studies) or not. Cells in the caudoventral region of the tectum express the dopamine receptor D2 and project to the putative substantia nigra pars compacta [Xenopus laevis [The toral population of Ucn3-expressing neurons of the adult sea lamprey is located rostrally to the mid-hindbrain boundary and the trochlear nucleus. Recently, a roughly similar region ventrocaudal to the optic tectum was identified as the putative substantia nigra pars reticulata homologue ,50, althcompacta . Judgingcompacta , in a was laevis . A studys laevis , indicats laevis . A recens laevis . With res laevis ,37,67, is laevis . These ps laevis  and a cis laevis , which is laevis  and mamms laevis ,16, and In the rat hindbrain, Ucn3 positive cells were detected mainly in the superior paraolivary nucleus  and, in This in situ hybridization study of the Ucn3-expressing neuronal populations in the brain of the sea lamprey reveals that this neuropeptide is expressed differentially in two widely different phases of the lamprey life cycle. In adult sea lampreys, but not in larvae, Ucn3 is expressed in the striatum, torus semicircularis, isthmic reticular formation, interpeduncular nucleus and nucleus of the solitary tract. These important differences in Ucn3 expression between larval and adult phases suggest that the neurochemical maturation of higher neuroregulatory circuits is closely related to life-style changes occurring after transformation. With regards to larval sea lampreys, the only nuclei showing Ucn3 expression, namely the prethalamus, the nucleus of the medial longitudinal fascicle and the posterior rhombencephalic reticular formation, are shared with adults and might be related to motor control. A comparison with the distribution of Ucn3 reported in other vertebrate groups also revealed a poor evolutionary conservation of Ucn3 expression in vertebrates.


Diabetic foot infection (DFI) is a serious complication of diabetes mellitus and identification of the causative bacteria is an essential step in selecting the appropriate antibiotic therapy. This study aimed to evaluate the bacterial pattern and antibiotic susceptibility of the bacteria causing DFI in Lampung Province in Indonesia.This study is a retrospective study reviewing the medical records of DFI patients admitted to the Dr Hi Abdul Moeloek Regional General Hospital in 2017–2019. DFI patients with complete medical record data were included in this study. Demographic, clinical, laboratory, wound culture and antibiotic susceptibility data were collected from the medical records using a short structural chart. The data obtained then reviewed.Enterobacter spp. as the predominant bacteria. Gram-positive bacteria were found in 19 (14.5%) subjects with Staphylococcus spp. as the predominant bacteria. Gram-negative bacteria found in this study showed high susceptibility to amikacin, meropenem and sulbactam/cefoperazone. Meanwhile, the Gram-positive bacteria showed high susceptibility to meropenem, sulbactam/cefoperazone and amikacin.In this study, 131 DFI patients met the study criteria and were included. Based on the wound culture results, Gram-negative bacteria were obtained in 112 (85.5%) subjects with Enterobacter spp. as the most predominant bacteria causing DFI in the studied population. The highest antibiotic susceptibility was seen for amikacin, meropenem and sulbactam/cefoperazone.The findings of the study revealed  Diabetes mellitus is one of the leading causes of morbidity and mortality in the world. To this day, an estimated 463 million adults live with diabetes worldwide. This figure is projected to grow to up to 700 million by 2045 . The 201Diabetes mellitus is an endocrine disorder that can result in various complications, including diabetic foot infection (DFI). The lifetime risk of Type 1 and Type 2 diabetes patients with DFI is 34%  have consistently shown that the most common pathogens in DFI are aerobic Gram-positive cocci, particularly Staphylococcus aureus and coagulase-negative Streptococcus and Staphylococcus. Recent studies of DFI from tropical/subtropical regions  have shown that the main cause of DFI is aerobic Gram-negative bacilli, either alone or in combination with Gram-positive cocci. Obligate anaerobic bacteria can also play a role in DFI, especially in ischaemic limbs and DFI with abscesses  recommend treating clinically infected diabetic foot ulcers with empirical antibiotics until microbiological culture results are available. Empirical antibiotics should be chosen in accordance with the severity of infection, clinical presentation and the prevalence of microorganisms in the local area and their antibiotic susceptibility. For DFI that occurs in diabetes patients in tropical/subtropical climates, IDSA recommends an empirical antibiotic regimen covering frequently isolated Gram-positive and Gram-negative pathogens as well as obligate anaerobes in moderate to severe DFI. The selection of subsequent antibiotic regimens must be carried out based on the clinical response, culture and antibiotic susceptibility results .The variations in the bacteriological profile of DFI that depends on geographic, cultural and climatic factors could make the international clinical guidelines for the selection of appropriate empirical antibiotic therapy difficult to apply in developing countries , 7. ThisThis is a retrospective study reviewing the medical records of diabetic patients with DFI treated at the Dr Hi Abdul Moeloek Regional General Hospital in 2017–2019. This hospital is a provincial hospital and the main referral hospital of Lampung Province. Patients with complications of diabetes are usually referred to this hospital. The study population was defined as the total number of patients with diabetes treated at the Dr Hi Abdul Moeloek Regional General Hospital. Sample sizes were calculated using the single proportion formula. Using the global prevalence of diabetic ulcers among diabetics (6.3%)  and takiDFI is defined clinically as the manifestation of an inflammatory process due to the invasion and multiplication of microorganisms in any tissue in a diabetic patient’s foot, the anatomical area under the malleoli. This condition usually results from disruption of the protective skin envelope at a site of trauma or ulceration in a patient with neuropathy or peripheral artery disease . We hospThe collection of medical records of diabetic patients with DFI was carried out through the Dr Hi Abdul Moeloek Regional General Hospital medical record database according to the 10th International Classification of Disease (ICD-10) diagnosis. The diagnosis of diabetes mellitus was defined as defective code E11 on the diagnosis of ICD-10. The diagnosis of DFI was confirmed by the presence of a diagnosis in the form of a handwritten medical record: ‘diabetic foot’, ‘diabetic foot ulcer’, ‘diabetic foot infection’ or ‘diabetic foot gangrene’. The data were then collected based on a questionnaire structured in several sections to collect various aspects of information from the medical record, including demographic data, history of diabetes, laboratory parameters during hospital stay, DFI bacteriological profile, antibiotic susceptibility profile, empirical antibiotic regimens given before the availability of microbiological culture results and susceptibility data, and observed outcomes in the form of mortality and length of hospital stay. The standard identification of the causative microorganisms in our hospital was carried out using standard microbiological methods with samples taken from the patient’s foot ulcer and antibiotic susceptibility testing was carried out using the disc diffusion method.t-test or Mann-Whitney U test for continuous variables. The results were considered significant if the P-value was less than 0.05.The data obtained from this study are summarised and then presented in diagrams, tables and bar charts as appropriate. Data analysis was performed using Statistical Package for Social Science software . Continuous variables are reported as mean (standard deviation [SD]) or median (interquartile range [IQR]) and categorical variables are reported as proportions. Distribution of microorganisms isolated in wound culture was reported as frequency (%), while antibiotic resistance and susceptibility were reported as proportion (%). Comparisons were conducted via Pearson’s chi-squared test or Fisher’s exact test for categorical variables and independent From 2017 to 2019, 131 patients were treated with DFI and met the inclusion criteria of this study. Among them, 57 (43.5%) were males and 74 (56.5%) were females. The mean age of the study subjects was 53.9 (9.2) years old. The mean length of stay of the subjects was 10.7 (5.9) days, with a median of 10 (IQR 8) days and 18 (13.7%) subjects died during hospital stay. The characteristics of the research subjects are shown in 3 and 422133 (170205)/mm3, respectively. The mean random blood glucose of the patient at admission was 265 (123.2) mg/dL.The patients’ mean haemoglobin was 9.3 (2.0) g/dL, with only 11 (8.4%) DFI patients without anaemia reported. The mean white blood count and platelet count were 19084.4 (7821.3)/mmThe most widely used empirical antibiotic regimen for DFI treatment before antibiotic susceptibility data can be obtained in this study was a regimen consisting of ceftriaxone and metronidazole (56.5%), followed by ceftriaxone alone (15.3%), a regimen consisting of ciprofloxacin and metronidazole (14.5%), and other regimens (13.7%).Enterobacter spp. (25.2%) as the predominant bacteria, followed by Klebsiella spp., Proteus spp., Pseudomonas spp., Alcaligenes spp., Morganella spp., Sphingomonas spp., Escherecia spp., Acinetobacter spp., Citrobacter spp. and Yersinia spp. Gram-positive cocci were also found with Staphylococcus spp. (10.7%) as the predmominant bacteria, followed by Streptococcus spp. These results are shown in Based on the results of the wound culture, 13 pathogens were identified. Polymicrobial infection occurred among 10 (7.6%) subjects, while 111 (84.7%) others had a monomicrobial infection. The most common pathogens found were Gram-negative bacilli, with P = 0.204). Furthermore, mortality was revealed to not be associated with the nature of infection (P = 0.643) , meropenem (92.6%) and sulbactam/cefoperazone (90.6%). Meanwhile, Gram-positive bacteria were mostly susceptible to meropenem (92.9%), sulbactam/cefoperazone (86.7%) and amikacin (80%). These results are shown in .5%, meroEnterobacter spp., Klebsiella spp. and Pseudomonas sp. as amikacin and for Proteus spp. as meropenem. Meanwhile, it was revealed that the most sensitive antibiotic for Staphylococcus spp. was sulbactam/cefoperazone , tetracycline (22.6%), cefixime (13.9%), ampicillin (13.4%), penicillin (11.1%), erythromycin (10.7%), amoxicillin (10.7%) and cefadroxil (10.6%). Meanwhile, Gram-positive bacteria showed the lowest susceptibility to chloramphenicol (30.8%), amoxicillin (23.1%), cefadroxil (21.4%), cefotaxime (11.1%), cefoperazone (10%), tetracycline (6.7%), cefixime (0%), clindamycin (0%), erythromycin (0%), ampicillin (0%) and penicillin (0%).The individual components of empirical antibiotic regimens used in the Dr Hi Abdul Moeloek Regional General Hospital intended to cover aerobes bacteria that showed low susceptibility against Gram-negative or Gram-positive pathogens. Ceftriaxone showed only 34.1% and 50% susceptibility against Gram-negative and Gram-positive pathogens, respectively. Meanwhile, ciprofloxacin showed 52.2% and 37.5% susceptibility against Gram-negative and Gram-positive pathogens, respectively.DFI is one of the main causes of morbidity in diabetic patients. It is more common in the older age group than in the younger groups. In this study, it was found that the mean age of patients with DFI was 53.9 years old. This is in accordance with the results of several other local studies that reported the average age of DFI patients to be over 50 years old . The majVarious types of bacteria can cause DFI and determining the specific bacteria causing this infection is an important step for a clinician to avoid using excessive and prolonged usage of broad-spectrum antibiotics. This aims to prevent various drug-related side effects, financial burdens, and antibiotic resistance .Our study reported a predominance of monomicrobial infection in subjects of DFI, similar to several studies , 16–18. Enterobacter spp. and followed by Klebsiella spp., Proteus spp. and Pseudomonas spp. The present study confirmed Gram-negative bacilli as the most common bacteria causing DFI, as supported by several previous studies. Research by Pemayun and Naibaho (Klebsiella pneumonia (33.3%) as the predominant bacteria finding, followed by Escherichia coli (24.2%) and Acinetobacter baumannii (12.15%). Similar findings have previously been reported by various studies from other Southeast Asian countries  as independent risk factors for increased length of hospital stay and in-hospital mortality. However, our study did not address other factors that may contribute to the length of hospital stay and in-hospital mortality, such as severity of illness, transfer from another acute care hospital, surgical site infection or prior antibiotic use.Our findings reported no association between the nature of infection and length of hospital stay or in-hospital mortality. This finding contrasts with a study by Lipsky et al. , which rDiabetic patients with DFI have several factors that cause a high risk of carrying multidrug-resistant microorganisms (MDR), such as inappropriate previous administration of antibiotics, chronic wounds and frequent hospitalisations. The causative pathogen and antimicrobial susceptibility profile should be considered when selecting an antibiotic regimen .Escherichia coli, Pseudomonas aeruginosa, Enterobacteria, Serratia, Proteus, Acinetobacter and Klebsiella. Amikacin also shows antibacterial activity against Staphylococcus (This study found that Gram-negative bacteria were mostly susceptible to amikacin (95.5%). Amikacin is a semisynthetic kanamycin derivative from an aminoglycoside class antibiotic. Like other aminoglycosides, amikacin works by binding to the 30S subunit ribosomal protein, causing mRNA reading error. This, in turn, inhibits protein synthesis or causes defective protein production, which leads to the death of microorganisms. Amikacin is unique among aminoglycosides due to its resistance to aminoglycoside-inactivating enzymes, enabling its usage for various microorganisms that are usually resistant to other aminoglycosides. Amikacin and other aminoglycosides have an antibacterial spectrum covering a wide range of bacteria, including lococcus –30. The lococcus . Apart flococcus , which rStaphylococcus aureus and other methicillin-susceptible Staphylococci and most Streptococcus . Meropenem is an antibiotic from the carbapenem class with broad antibacterial activity against Gram-positive, Gram-negative and even anaerobic bacteria. Like other betalactam antibiotics, meropenem works by inhibiting the synthesis of bacterial cell walls, causing bacterial death. This antibiotic has excellent activity against tococcus . Gram-poStaphylococcus aureus, showed high resistance to commonly used antibiotics, such as penicillin and tetracyclines.In this study, the bacteria causing DFI showed resistance to commonly used antibiotics. Gram-negative bacteria showed the lowest susceptibility to chloramphenicol, tetracycline, cefixime, ampicillin, penicillin, erythromycin, amoxicillin and cefadroxil. Gram-positive bacteria showed the lowest susceptibility to chloramphenicol, amoxicillin, cefadroxil, cefotaxime, cefoperazone, tetracycline, cefixime, clindamycin, erythromycin, ampicillin and penicillin. Xie et al.  reportedIndividual components of empirical regimens given prior to the culture intended to cover aerobes in DFI were found to have low susceptibility against both Gram-negative and Gram-positive bacteria. The high rate of resistance to both the individual component of empirical antibiotic regimens given prior to the availability of causative pathogen identification and susceptibility data in the present study may be due to several factors, including hospitalisation history, recent use of broad-spectrum antibiotics, history of surgery and chronic wounds, irrational use of antibiotics and horizontal transfer of antibiotic resistance genes .In the clinical setting, empirical antibiotic regimens are normally commenced to treat patients with DFI before the availability of microbiological culture results. They are intended to cover possible causative pathogens. The choice of empirical antibiotic regimens is often based on the patient’s clinical presentation and the bacteriologic profile of the local setting . The finLimitations of this study include the inability to generalise the study to the whole of Indonesia, as it was mainly a single-centre study. Due to the retrospective study design, pathogenic bacteria’s bacteriological profile differentiation based on the severity and grading of the DFI was not possible due to a lack of documented information in medical records. The lack of a specific culture for anaerobic bacteria is another limitation of this study. Further prospective studies are required to evaluate the clinical features and response to antibiotic treatment in DFI.Enterobacter spp. being the predominant pathogen in the studied population. The bacteria found in DFI have varying resistance to commonly used antibiotics. This study’s findings provided valuable information concerning DFI in Lampung Province, Indonesia and might be used as a consideration for empirical antibiotic regimen selection to treat DFI in a local setting. Further studies with a larger sample size of the bacteria isolated from DFI in other areas in Indonesia are justified.This study showed that the most common organisms found in DFI were aerobic Gram-negative bacteria, with