L-proline (PLP) motifs. Our recent work demonstrated that both nuclear localization and PLP-binding are required for tumor growth inhibition by Pfn1, and this is at least partially due to Pfn1 association with the PLP-containing ENL protein in the Super Elongation Complex (SEC) and the transcriptional inhibition of pro-cancer genes. In this paper, by identifying a phosphorylation event of Pfn1 at Ser71 capable of inhibiting its actin-binding and nuclear export, we provide in vitro and in vivo evidence that chemotherapy-induced apoptotic sensitization by Pfn1 requires its cytoplasmic localization and actin-binding. With regard to tumor growth inhibition byPfn1, our data indicate a requirement for dynamic actin association and dissociation rendered by reversible Ser71phosphorylation and dephosphorylation. Furthermore, genetic and pharmacological experiments showed that Ser71 of Pfn1 can be phosphorylated by protein kinase A (PKA). Taken together, our data provide novel mechanistic insights into the multifaceted anticancer activities of Pfn1 and how they are spatially-defined in the cell and differentially regulated by ligand-binding.The essential actin-binding factor profilin-1 (Pfn1) is a non-classical tumor suppressor with the abilities toboth inhibit cellular proliferation and augment chemotherapy-induced apoptosis. Besides actin, Pfn1 interacts with proteins harboring the poly- L-proline (PLP)-containing proteins many of which are actin-regulatory factors and cooperate with Pfn1 to control actin polymerization -dependent phosphorylation site in Pfn1 at SerUntagged, Myc-tagged, and HA-tagged Pfn1 in pcDNA3, His-tagged Pfn1 in pRK172, untagged Pfn1 in pLenti-CMV/TO-Neo-DEST, and YFP-Pfn1 with and without NES or NLS tag in pFLRu-FH vector have been described previously . Point m71-Pfn1 antibody (F5675), a synthetic phospho-Pfn1 peptide harboring pSer71 [Ac-CLGGQKC(pS)VIRDSL-amide] was conjugated to keyhole limpet hemocyanin and used to immunize rabbits. Antiserum was subjected to double affinity purification using both the antigenic phospho-peptide and the same peptide without the phosphate on Ser71 .Primary antibodies used for Western blots are as follows: mouse anti-HA-tag , mouse anti-Myc-tag , mouse anti-β-actin , mouse anti-α-tubulin , mouse GAPDH , rabbit anti-Pfn1 , rabbit anti-VASP , rabbit anti-cleaved caspase-7 , rabbit anti-cleaved PARP , rabbit anti-GFP . To raise the polyclonal pSerAll cell lines were purchased from ATCC with the exception of MDA-MB-231 cells stably expressing a tri-modal reporter fusion used to inject NOD/SCID mice as previously described . All celTo study Pfn1 interaction with actin and VASP, HEK293T cells grown in 6-well dishes were transfected with Myc-tagged or HA-tagged Pfn1 constructs (WT and mutants), lysed by RIPA buffer, and subjected to immunoprecipitation using antibodies against Myc or HA tags as described previously . To affiFor drug treatment in 2D cultures, MDA-MB-231 or BT-549 stable cells were seeded at 1,000 cells per well in 96-well plates or 500 cells per well in 24-well plates, and treated on the next day with vehicle or paclitaxel in quadruplicate wells. Viable cells were quantified 5–7 days later by the Alamar Blue assay . Briefly3, they were randomly divided and treated with 0.9% sodium chloride or paclitaxel (10 mg/kg) by weekly intraperitoneal injection for two consecutive weeks. Clinical grade paclitaxel (6 mg/ml) was purchased from Siteman Cancer Center pharmacy at Washington University School of Medicine. Primary tumors were measured by Caliper on a weekly (for NOD/SCID) and semiweekly (NU/NU) basis until tumor resection and euthanasia of the mice.The animal experiment was carried out in strict accordance with the guidelines recommended for care and use of laboratory animals by the National Institutes of Health. The Animal Studies Committee at Washington University  approved all animal protocols. Five-week old female NOD/SCID and NU/NU mice were purchased from Charles River and kept under standard institutional care. Experimental details for orthotopically inoculating MDA-MB-231 stable cells were previously described . For pact-test was used to determine the statistical significance of the differences in cell growth rates, tumor sizes and weights between control and experimental groups. All statistical analyses were performed using GraphPad Prism 7.0. P-values < 0.05 were considered significant.Unpaired two-tailed student 71 appears to be a bona fide phosphorylation site as preventing (S71A) and mimicking (S71D) phosphorylation had opposite effects on actin-binding of Pfn1. While Pfn1(S71A) binds more actin than Pfn1(WT), Pfn1(S71D) completely fails to bind actin , we observed by Western blot little detection of Pfn1(WT) and the phospho-resistant Pfn1(S71A) but robust detection of the phosphomimetic Pfn1(S71D) by F5675  (71 is invariably preceded at −2 position by a basic residue (most commonly Arg), suggesting that it could be phosphorylated by the AGC kinase family ] is a possibility. The preferred amino acids at +1 and +2 positions for PKA substrates are hydrophobic  while those for +5 and +6 positions are leucine. The Ser71-containing motif in Pfn1 meets all these requirements in particular a perfect match at +5/+6 positions. To test whether PKA is indeed the kinase for Ser71, we co-transfected HEK293T cells with GFP or GFP-tagged alpha catalytic subunit of PKA with HA-Pfn1, and examined the level of Ser71 phosphorylation by Western blot after anti-HA immunoprecipitation. While equal amount of HA-Pfn1 was pulled down, co-transfection with GPKA significantly increased its pSer71 level detected by the F5675 antibody  by the pSer71-Pfn1 antibody in contrast to HA-Pfn1(WT)  did not affect the ability of GPKA to increase the detection of HA-Pfn1 by the F5675 antibody   sequencesh Pfn1) . Ser71 ie family . A closeantibody . We next HA-Pfn1 . To confPfn1(WT) . As a seantibody . Taken tin vitro and in vivo  showed anti-proliferative effect as observed previously, both Pfn1(S71A) and Pfn1(S71D) mutants were inactive  vs. Pfn1(S71D) in the mammary fat pads of female nude mice. Tumors formed by both cell lines grew at similar rates as observed in the NOD/SCID mice. When the average tumor volumes in both groups reached ∼70 mm3, mice were randomly divided into two subgroups (n = 5 per group) which were treated with vehicle or paclitaxel  for 2 weeks. Semiweekly Caliper measurement showed an obvious tumor-regressing effect of paclitaxel in the Pfn1(S71A) group, with the tumor volume difference between the vehicle and paclitaxel groups being statistically significant at the last two time points  and Pfn1(S71A) were predominantly localized in the cytoplasm. However, YFP-Pfn1(S71D) was diffusely present within cytoplasm and nucleus  or nuclear localization sequence (NLS) as previously described  levels in breast cancer cell lines . We foun71 is a bona fide phosphorylation site in Pfn1 selectively inhibiting its actin-binding with little effect on its PLP-binding. This is consistent with the fact that Ser71 is located within the actin-binding site of Pfn1 and distal to its PLP-binding pocket , despite increasing Ser71 phosphorylation levels. This could be due to several possible reasons. First, PKA-mediated Ser71 phosphorylation may occur to a small fraction of total cellular Pfn1 either transiently or at specific subcellular locations. Given the high cellular abundance of Pfn1 (>50 μM in most tissues)  , the nettissues) . Therefoin vitro and in vivo studies. Our recent work suggested that tumor growth inhibition by Pfn1 is mediated at least in part by its nuclear function in repressing SEC-dependent transcription of pro-cancer genes including c-MYC (137 phosphorylation and dephosphorylation (mimicked by S137D and S137A) which, respectively, blocks and enables Pfn1 binding to PLPs that are present in the SEC component ENL (71-resistant and mimetic mutants (S71A and S71D) in MDA-MB-231 cells, we made the unexpected observation that both cannot inhibit tumor growth. This indicates that, rather than functioning as an on/off switch, reversible phosphorylation and dephosphorylation at Ser71, causing dynamic actin dissociation and rebinding, are required for Pfn1 to inhibit tumor growth. Given that actin-binding promotes nuclear export of Pfn1  in the nucleus (71 phosphorylation. Given that Ser71 phosphorylation prevents nuclear Pfn1 export, we speculate that it may inhibit the apoptosis-sensitizing effect of Pfn1 via a two-pronged mechanism by abolishing its actin-binding and reducing its cytoplasmic levels (71 is a bona fide phosphorylation site of Pfn1 capable of inhibiting its actin-binding ability, preventing its nuclear export, and influencing its tumor-inhibitory functions.In addition to suppressing tumor growth, Pfn1 also sensitizes cancer cells to drug-induced apoptosis. This has been best demonstrated in breast cancer cells treated with cytotoxic agents many of which are common cancer chemotherapies including paclitaxel . Althoug nucleus  and deac nucleus . Thus, Pc levels . Taken tThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by The Animal Studies Committee at Washington University in St. Louis.JS conceived of the project and wrote the manuscript with input from all authors. JS, FW, CZ, and S-JK performed the experiments. JS, FW, CZ, SC, and S-JK performed the experiments and analyzed the data. AB and MB provided technical expertise and intellectual input.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Detailed information about concrete behavior in real structures is an important issue in controlling its performance during its service life, and the use of embedded sensors to obtain desired information such as temperature, onset of the cracking process and evolution of strains, has gained the attention of the building concrete industry. Data obtained using this technology can provide valuable information for decision making about the need for corrective interventions that can ensure the integrity and safety of concrete structures for long period of time. This paper presents a review of the current state-of-the-art of embedded fiber optic sensors used to assess concrete information of a wide range of aspects, comprising: existing alternative technologies, characteristics and advantages, practical applications and future developments. Complementarily, the work presents preliminary results of the use of fiber optic sensors to automatically and continuously perform expansion readings of AAR in concrete elements that facilitate both the storage—with elimination of the usual interruptions for manual readings—and the availability of continuous results of expansion data that are not possible to obtain with usual AAR tests code reading recommendations. Concrete is one of the main structural materials, widely used worldwide in several types of work and construction projects. Durability and performance to withstand mechanical loads are some reasons for its widespread use. As a heterogeneous material, made with constituents from different origins, concrete structures may exhibit pathologies during their useful life, due to both the applied loads and its interaction with the environment. Over time, significant scientific comprehension of the nature of this material has been developed, including the perception that certain problems may cause changes in its micro- and macro-structure with the possibility of generating an early deterioration process, and even the failure of the material ,2,3. TheStructural health monitoring (SHM), with a focus on concrete structures, has emerged along with the need to increase the safety of structures and to provide guidelines for efficient and cost-effective maintenance plans. Currently, for the SHM approach, several point sensors are used, but due to the complexity of concrete structures, it is often necessary to use a large number of sensors. Fiber optic sensors (FOS) present great advantages in terms of multiplexing capabilities, chemical stability, resistance to electromagnetic field, compactness, etc. These advantages make FOS a safe technology for a large range of applications, namely, industrial, medical and structural health monitoring ,6,7,8. FTo evaluate the behavior of concrete structures under normal loading conditions and during their use, it is essential to use safe monitoring strategies that allow material quality indicators to be obtained, in order to take decisions on possible intervention processes before the damage process increases. Over time, new technologies have been investigated, with the use of fiber optic sensors being one of the most promising ,15,16,17The use of embedded FOS for reading expansions due to alkali-aggregate reaction (AAR) in concrete elements is a fairly new line of scientific investigation, because those readings are typically associated with the manual operations of removing the specimen from the environment in which it was placed with the subsequent several manual works that introduce undesirable errors in the measurements. The work presents preliminary results regarding the use of FOS to automatically and continuously perform expansion readings of AAR in concrete elements that facilitate both the storage—with elimination of the usual interruptions for manual readings—and the availability of continuous results of expansion data, which are not possible to obtain with usual AAR tests code reading recommendations.The occurrence of cracks in concrete directly affects the concrete’s service life and durability. Structural health monitoring (SHM) is becoming increasingly necessary for concrete structures as a complement to the visual inspections usually performed. Monitoring concrete structures is extremely important to identify problems early and to adopt the most appropriate recovery measure. In recent years, several SHM techniques have been developed for concrete structures using fiber optic sensors, piezoelectric materials and radioactive materials (X-rays and γ-rays) ,19,20,21The use of fiber optic sensors is shown to be increasingly promising because the sensors exhibit a higher sensitivity, durability, and stability, when compared to traditional sensors, and also are a smaller size. In addition, the sensors can withstand high temperatures and environmental aggressions, which make the FOS ideal for SHM of large concrete structures ,23,24,25Fan et al.  developeLiao et al.  developeWaeystens et al.  used fibScott et al.  developeTan and Bao  developeFiber optic sensors offer a number of advantages over conventional electrical measuring gauges and transducers to calculate strains and displacements, to determine the onset of the cracking process and the crack opening widths, and to identify localized damage and other important information, in order to monitor concrete structures performance.Surface or embedded FOS have also been used in many newly constructed civil structures—bridges, buildings, dams, pavements, pile cap blocks—providing technical information about static and dynamic behavior, temperature, wind or water pressure, and structural health, allowing for continuous real-time monitoring of structural behavior ,32. EvenThe main characteristics that make FOS potential candidates for SHM of concrete structures are the following: (1) FOS consist of an electrically nonconductive material; (2) FOS can be safely used in an explosive atmosphere; (3) FOS are not subject to electromagnetic interference; (4) FOS can be chemically inert; (5) FOS have a very wide range of operating temperatures; and (6) the sensors can be multiplexing.On the other hand, practical application in a field environment demands the observation of some special technical issues such as fragility, ways of installation (surface or embedded), deterioration and connectors; factors that should be carefully investigated before the selection of a specific FOS package to use. These issues are very important if one intends to use FOS in in situ and real-time monitoring of expansions in concrete elements due to internal swelling reactions (ISR).2 and beams of 175 cm in length and with a cross section of 35 × 55 cm2 were used. Fiber optic sensors were inserted in the cylinders (one in each) and in the beams (two in each). The FOS sensors used were the embedded fiber optics (e-FOS), temperature-compensated and strain gauge for cylinder and beam testing, respectively. The sensors are based on Fabry–Perot interferometry.The work developed by Yehia et al.  used optTo identify temperature variations during the hydration of the concrete, thermocouples were installed in the middle and on the surface of the beams, in order to detect temperatures between 40 °C and 100 °C. All sensors were connected to a data acquisition system right after molding to record strains due to the heat of hydration and also the shrinkage of the concrete. The same procedure was used for both beams and cylindrical specimens. Monitoring was performed continuously for 7 days.The results, provided by the sensors, for each test performed on the concrete samples, as a function of time, showed an instantaneous response to any changes in loading as well as temperature changes, which indicates the sensitivity and the ability of the FOS to capture responses of the concrete specimens due to loading or during hydration.3 with a 16 mm diameter steel bar inside, located in the middle of the cross section. The optical fibers were placed along the length of the element in six different places. After processing the raw data, the authors defined measurement bases in post-processing. The length of the bases taken was 10 mm and the distance between successive bases was 5 mm. This means that the sensor bases overlapped by half their lengths.Another research that used FOS to read concrete strains was developed by Sienko et al. . In thisThe experimental results showed strains of the fibers individually, according to each measurement base defined by the authors , before In another work, Horch et al.  went beyReference measurements were made when the structure was 18 weeks old. Follow-up further readings were made when the structure was 26 weeks and 34 weeks old.Temperature measurements were made using sensitive fibers able to read strain values close to 10 µm/m. This was performed in order to allow the levels of thermal expansion of the concrete to be compensated.The temperature results obtained exhibited large ranges of oscillation (noise). This fact was reported by the authors as a non-consistent behavior that was assigned to some alteration coming from the cables or the machine. On the other hand, the temperature values oscillated around a constant average value throughout the foundation slab. The aim of the work developed by Horch et al.  was to dYuan et al.  developeLi et al.  developeKesavan et al.  investigThe authors also reported that the embedded optical sensors kept working with no visible damage or degradation, even after attaining a strain value close to 2000 με. For flexural behavior, the authors reported a quite efficient performance, although some strain underestimations had been observed, when compared with the results from usual strain gauge measurements. Despite this fact, the 7% of variation that was reported is usually accepted for SHM of concrete structures. The same behavior was observed in fatigue, in high stress levels and in low cycle loadings.Bassil et al.  studied Bao et al.  investigFiber optic sensors can be used to measure strains in concrete elements. It is well-known that internal swelling reactions cause expansions due to the stresses generated by the formation of hydration products . The use of FOS specifically for the analysis of structures that present these types of pathology (AAR or DEF) is an open field to scientific research and qualified information about this subject is still very scarce.One of the works that used FOS to assess internal expansion in concrete was developed by Dunant and Scrivener . The autIn this work , three sRocha et al.  developeThe results of the tight buffer data were not reliable after 10 days of measurement, while the cable, which was a more protected element, showed consistent results up to the 15th day of measurements. After these periods of time in each case, the information obtained was reported as not relevant by the authors. However, the authors also reported that the expansion detected by the system, in the time interval in which it worked, results obtained were consistent with the measurements of the external pins. The values obtained were smaller than the external pin readings, as it was expected, due to the gradient of deformation from the outer surface to the center of the sample. However, an important observation that should be highlighted was the FOS were capable of detecting the evolution of AAR expansions.Rocha et al.  also repFinally, although no studies have been found using fiber optic sensors only for evaluation and monitoring of expansion by DEF, it is important to consider that although the origin of AAR and DEF are different, the effects on concrete structures in a macroscopic view are quite similar, i.e., both cause expansion in concrete elements and often occur simultaneously. From the studies presented, it is possible to state that regardless of the internal reactions existing in the structure, the expansions can be adequately detected using FOS.The heat generated by cement hydration reactions usually imposes thermal strains in concrete elements that may lead to an early cracking process, which decreases its durability. The durability of concrete depends on the material quality and the exposure conditions, which, in turn, depends on the temperature, on the physical-chemical aggressiveness of the surrounding environment and, also, depends on the mechanical loading.To evaluate the feasibility of using embedded sensors to assess temperatures and strains in concrete elements in early ages, mainly those arising from internal swelling reaction due to AAR, an experimental procedure in concrete specimens was developed. To create a favorable environment for the development of internal swelling reactions in the concrete, in addition to the effects of the hydration heat, the strategy prescribed in  was used3 were prepared, two of them were kept in the aggressive environment as prescribed in [In this very first phase of the research, three concrete prisms with dimensions of 75 × 75 × 285 mmribed in , and theribed in . A thermFiber optics and temperature sensors were embedded in the concrete specimens during concrete casting. It is important to highlight that early-age strain measurements in concrete using fiber optic sensors are highly influenced by the time the sensor is placed in contact with the material and by the dependence on the emitting sensor signals during the hardening of the concrete, mainly the temperature level. Some discussions regarding these issues were made in previous sections of this paper, for the type of fiber optic sensor used in the research.Fiber Bragg grating sensors inscribed in optical fiber were used to obtain strain measurements inside concrete samples. The acquisition of the Bragg wavelengths was made with an HBM FS22 DI sensor, referred to later as Braggmeter or FS22. This device relies on a broadband laser source (1500 nm to 1600 nm) and continuous swept laser scanning to obtain real-time measurements of the Bragg wavelengths, with an acquisition rate ranging from 50 Hz to 1000 Hz. Up to four optical fibers, each one with several embedded FBG sensors, can be monitored simultaneously. A custom data acquisition program was developed to enable the acquisition and storage of Bragg wavelengths measured by the FS22 over the whole experiment. This was necessary because the native program that came with the device had some internal settings that did not meet the test demands. In particular, the minimum acquisition rate of 50 Hz would generate a large quantity of measurement data over several weeks, with only a fraction of it really being needed. In order to facilitate data analysis, the custom program was designed to obtain 10 measurements of the Bragg wavelengths over a few seconds, and to then select the median value, which would be used during data analysis. Once this was carried out for each FBG, the program would wait 10 min before repeating the process. The advantages of this method are twofold: it limits the amount of data collected, and at the same time limits the impact of a possible disturbance, thanks to the repetition of measurements over a few seconds. Moreover, because collected data are timestamped, this means that in case of an unfortunate event such as a power loss, it is possible to know precisely how much time has passed without measurement.The FBG sensors used were inscribed in silica core-cladding fibers with a polyimide coating by a research partner, with a phase mask technique and a 240 nm UV laser. Bragg wavelengths range between 1530 and 1550 nm, reflectivity of the FBG is around 30% to 40%, with a full width at half maximum (FWHM) of reflected wavelengths around 0.15 nm. Just before concrete sample casting, a temperature sensitivity characterization was performed to ascertain precisely the response of the four fibers with five FBG on each. The reference temperature (ambient air) was accurately measured with a PT100 temperature probe, whose accuracy was previously verified using a calibration furnace. The temperature sensitivity characterization was performed in two ways, either by putting the stress-free fiber in cold water with temperature monitored by a PT100 temperature probe, or in a furnace with temperature also being monitored with a PT100. The wavelength variation observed by the Braggmeter and the temperature variation detected by the PT100 ensured that the sensitivity of the FBG sensors was the same as that reported in the literature: about 10 pm/°C. These measurements have also revealed the very high sensitivity of FBG sensors to spurious disturbances. To measure the evolution of strains and stresses during the setting phase of concrete specimens, an instrumentation based on fiber Bragg grating sensors was developed. The choice of such a type of sensor is explained by their low intrusivity , as well as their sensitivity to the physical parameters whose evolution is to be measured.Each of the three concrete prism specimens described in According to the response of an FBG sensors, the total strain is given by Equation (1):KT and Kε being fiber-dependent constants of 7.8 × 10−6/°C and 0.78 × 10−6/(μm/m), respectively. However, in view of the significant temperature variations expected in concrete during the setting phase, the effects of thermal expansion cannot be neglected, which leads to Equation (2).With As shown in If one compares the behavior of the three prisms investigated—one placed at ambient conditions  and the At the time when the prims 2 and 3 were plane into the tank full of aggressive water, from the third day of the test, an accelerated and important increase in both total strains and temperatures was observed . This was caused by the acceleration of expansive reactions, which was generated by the sodium hydroxide content added to the water used to make the concrete, and also to the water in which the prisms were immersed.It is also important to highlight that the FOS and the scheme of the tests developed worked well in capturing the early strains in the concrete prisms investigated. The lack of strain measures after the fourth day of the test with the prisms placed inside the tank  was due FOS technology has been successfully used for monitoring several types of concrete structures. The application of this type of technology has been increasingly frequent and promising. These are complex studies that need to be planned and performed in a conscious way for the results to be valid.Most of the existing tests with FOS are carried out in a laboratory setting; however, there are already tests on real structures, mainly on bridges and viaducts, and some tests on buildings. The studies discussed in this paper highlight the potential for monitoring the performance of reinforced concrete structures with the use of FOS. On the other hand, there are important challenges to be overcome, especially the aspects related to data processing and the issues associated with the degradation of the sensors, mainly if one intends to use embedded FOS to assess expansions from AAR.Moreover, the information about the equipment used and a justification for choosing the same ones is not always well-detailed in the existing works. This fact greatly limits the possibility of reproducing the same tests in a laboratory, or using this approach in real concrete works.From the bibliographical review performed, one can say that there are currently several types of fiber optic sensors available on the technical market. These are usually sensors with multiple purposes, and they have been effective in several works investigated, especially in the study of deformations in concrete elements.Internal expansion in concrete may come from different internal swelling reactions and the investigation of FOS to measure such expansion is an open field of research; few studies have been found on this topic, although its use is promising. Most studies were conducted to measure strains due, almost exclusively, to mechanical loads imposed on concrete specimens. In the specific case of internal expansions due to AAR, the measurement scheme with FOS proved to be functional and operational and it presents several advantages when compared with the current methods of expansion measurements, usually prescribed in AAR test code reading recommendations. Among these advantages, the research performed in this paper shows that it is possible to have an automatic and continuous availability of results of expansion data and, at the same time, assure improvements in the storage conditions and operations to read expansions from AAR in concrete samples.


Autoimmune pancreatitis is a chronic and benign disease of autoimmune etiology that can occur isolated or constitute a manifestation of immunoglobulin G4 (IgG4)-related disease . It is a pathological condition that can mimic pancreatic cancer by presenting as a mass in imaging studies and provoking symptoms such as obstructive jaundice and dramatic weight-loss. The inflammatory infiltrates in the pancreas can also produce endocrine dysfunction leading to diabetes.The authors report the case of a 68-year-old man that presented with unexplained weight loss and poorly controlled diabetes despite progressive pharmacological adjustments, with a later onset of obstructive jaundice, for which he underwent pancreaticoduodenectomy with the pre-operative diagnosis of pancreatic malignant neoplasm, which was posteriorly identified as type 1 autoimmune pancreatitis. In these cases, the differential diagnosis might be particularly challenging, requiring a high level of suspicion to avoid unnecessary procedures.Corticosteroid therapy can lead to the resolution of symptoms as well as glycemic control, and it is the cornerstone of IgG4-related disease treatment. However, corticosteroid-sparing agents may be of interest to achieve clinical suppression. Immunoglobulin G4-related disease (IgG4-RD) is a systemic immune-mediated condition characterized by inflammatory cell deposits in various organs leading to fibrosis and dysfunction, associated with increased serum IgG4 levels. Patients can present with subacute development of a mass in the affected organ, even though 60% to 90% of these patients have multiple organ involvement . The preThere are two types of autoimmune pancreatitis (AIP). The IgG4-RD associated pancreatitis is known as type 1 AIP. IgG4-related endocrinopathies are rare; however, in type 1 AIP, endocrine and exocrine dysfunction develop commonly, manifesting with hyperglycemia and steatorrhea . AIP-relThe authors report a case of autoimmune pancreatitis subjected to aggressive surgical treatment due to pseudotumoral presentation and uncontrolled diabetes resolved after corticosteroid therapy.A 68-year-old male with a medical history of diabetes mellitus under treatment with metformin presented with unintentional weight loss of 18 Kg over the last 10 months (6 Kg in the previous month), corresponding to 25.7% of his initial body weight. Laboratory evaluation revealed an increased erythrocyte sedimentation rate of 55 mm/h, glycated hemoglobin of 10.2%, and an elevated cancer antigen 19-9 (CA 19-9) of 50.8 U/mL. Even though the latter isn’t validated as a screening or diagnostic test, it was solicited amongst other parameters for clinical orientation. Liver and kidney function, as well as the other blood tests, were normal. His family practitioner decided to add gliclazide for metabolic control. The patient also underwent an upper gastrointestinal endoscopy and a colonoscopy that showed a non-bleeding angiodysplasia in the caecal top. A thoracic, abdominal, and pelvic computed tomography (CT) scan was obtained, which showed a roughly spherical appearance of the cephalic pancreatic region with 4.3 cm and 3.5 cm diameters  was executed with alterations suggesting pancreatic neoplasm with biliary invasion. A plastic stent was placed in situ with good biliary drainage. The patient was then referred to a hepatobiliary surgical center, where he was admitted for a pancreaticoduodenectomy that was performed without reportable complications. The histology evaluation of the surgical specimen revealed pancreatic parenchyma with an inflammatory infiltrate intensely rich in plasmocytes that expressed IgG4 involving adipose tissue, duodenum, stomach, bile duct, and various reactive ganglia with the final histological diagnosis of autoimmune pancreatitis type 1. After the procedure, the plasmatic subclasses of IgG were measured without significant alterations, and the patient remained without specific treatment with good weight recuperation and resolution of symptoms.However, despite the previously therapeutic adjustment of antidiabetic medication, metabolic control was not achieved with the need for progressive pharmacological adjustments, including insulin. A subsequent investigation revealed an IgG level of 1872 mg/dL (reference < 1822 mg/dL) and an IgG4 of 8530 mg/L (reference < 1400 mg/L). The patient was then started on corticosteroid therapy and methotrexate with an excellent clinical response.There are a variety of pancreatic diseases that can form solid masses and mimic neoplasms. About 5%-10% of performed pancreatectomies with the preoperative clinical diagnosis of pancreatic carcinoma will be considered non-neoplastic lesions after anatomopathological examination (pseudotumors) . ExampleAutoimmune pancreatitis (AIP) is a nosological entity characterized by an autoimmune inflammatory process in which there is a prominent lymphocytic infiltrate leading to pancreas fibrosis and causing organ dysfunction. There are two types of AIP: type 1, which is a systemic disease associated with elevated serum IgG4 and tissue infiltration by IgG4+ plasma cells, and type 2, which is a specific pancreatic disorder not related to IgG4 . These fThe clinical features of autoimmune pancreatitis are nonspecific and resemble other pancreatic diseases, including mimicking pancreatic cancer. Obstructive jaundice is the most common form of presentation . AIP is IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition that affects multiple organs, mainly the pancreas, liver and bile ducts, salivary glands, kidneys, retroperitoneum, thyroid, aorta, and lymph nodes . It is cThe present case report illustrates the evolution of an unknown IgG4-RD evolving to autoimmune pancreatitis that led to difficult metabolic control. The presentation as a pseudotumor highlights the need for clinical awareness of these conditions since invasive and potentially life-threatening procedures such as surgery should and could be avoided. Furthermore, the authors emphasize the use of methotrexate as a corticoid-sparing agent with a good clinical outcome.


This paper proposes the use of X-ray computed tomography  measurements together with finite element method (FEM) numerical modelling to assess bond failures mechanism of fiber-reinforced fine-grain concrete. Fiber-reinforced concrete is becoming popular for application in civil engineering structures. A dynamically developing topic related to concretes is the determination of bond characteristics. Nowadays, modern technologies allow inspecting the inside of the element without the need to damage its structure. This paper discusses the application of computed tomography in order to identify damage occurring in the structure of fiber-reinforced fine-grain concrete during bond failure tests. The publication is part of a larger study to determine the bonding properties of Ukrainian steel fibers in fine-grain concrete. The authors focused on the visual evaluation of sections obtained from tomographic data. Separately, the results of volumetric analysis were presented to quantitatively assess the changes occurring in the matrix structure. Finite element analysis is an addition to the substantive part and allows us to compare real damage areas with theoretical stress concentration areas. The result of the work is the identification of a path that allows verification of the locations where matrix destruction occurs. Recycling has become an everyday habit, and newer and newer recovery technologies try to ensure minimal waste. New trends are forming, such as n mining . Properlobserved . This trobserved ,4.Construction and demolition waste, commonly referred to as CDW, is increasingly being used in the construction industry . SourcesElectrical Resistance Tomography, used to determine slurry flow measurements .Ultrasound tomography, which can be used to visualize the internal behavior of a concrete structure ,19.X-ray microtomography , covering the entire spectrum of materials ,21; applComputed tomography is typically associated with medical applications. The first CT scanner was developed in 1973 and its authors—Godfrey Newbold Houns-field and Allan MacLeod Cormack—received the Nobel Prize in 1979. Almost half a century of technology development has made modern tomographic examinations highly automated, safer, and faster . ComputeX-ray tomographs used in industry are based on X-ray spectroscopy. A detailed description of the principle of operation was published in . The devThe next step is the reconstruction of the 3D volume. During a full rotation, the device generates thousands of images in high grayscale . The most common is the implementation of complex algorithms at the reconstruction stage, resulting in correction of images such as beam hardening correction, automatic geometry calibration, and geometry optimization ,24. The The current software offers a number of built-in analysis types that are helpful when working with CT data. The leading analyses are porosity, geometric deviation, and material orientation analysis. The software also allows for FEM calculations and flow simulations. . SourcesAn extensive review of the analysis of concrete and asphalt construction materials is presented in . Accordi3, water 240 l/m3, sand 1644.43 kg/m3. The w/c ratio was kept at 0.54.The essence of the planned strength tests was to determine the forces required to pull out the fibers of the concrete cubes. Hooked-end fibers were placed in the concrete cubes  with a microfocus lamp using a cone beam. The warm-up and centering procedure was carried out on the instrument. In order to test irregularly shaped specimens, the samples were mounted on a low-absorbent foam for stable mounting on a manipulation table. The specimen was positioned to focus the viewing range on the fiber/concrete interface—the Region of Interest (ROI) option was selected. The ROI focuses the scope of the test on a portion of the specimen allowing us to select the section of interest. Each specimen was scanned twice, before and after the strength test, with the same scanning parameters—130 kV at 250 µA. A copper filter of constant thickness was used during the test. The measurement was performed with an accuracy at which the voxel dimension was equivalent to 30 µm. A calibration procedure was performed according to the manufacturer’s recommendations. One examination assumed the taking of 2700 images. A single run lasted approximately 50 min. During the examination, the detector’s shift module was activated to exclude the effect of defect of a single detector pixel and the auto-sco function, which allows for geometry optimization. For safety reasons, the radiation level was monitored during and after the examination.The first task was to reconstruct separate 3D solids based on data from separate scans. The program provided by the manufacturer of the tomograph , datos|x, was used for this purpose. During the reconstruction, the beam hardening correction, automatic geometry calibration, and geometry optimization algorithms were used. The reconstructed 3D geometry allows for visual inspection and further analysis.Afterwards, a registration of two scans  was performed. Scanning a component using ROI leads to one fundamental difficulty: there is no complete image of the outer edges, making it difficult to bring the two scans into a common coordinate system. In order to establish a common position, an internal algorithm was developed. The procedure required determining the porosity of two samples. Then, the coordinates of the 11 largest pores and the coordinates of the centers of gravity of the two samples were entered as input to the script. The transition between coordinate systems was determined. In successive steps, the coordinates of progressively smaller pores were compared. The 3D rotation angles and the displacement vector of one of the samples were output. Fitting was assessed visually. After 10 shift-rotation iterations, a correct match between the two data sets was found. A diagram of data workflow is shown in Porosity determination is widely used in concrete testing ,44 and dThe development of tomographic analysis allows us not only to evaluate data inside a given CT software, but also to further transform the obtained data and perform strength analyses. The dedicated software has a built-in Finite element method (FEM) calculation module; however, it is only designed for basic simulations . The autThe authors decided to develop models of fibers that could be numerically extracted from a three-dimensional (3D) concrete block. The CT data allow us to reconstruct the geometry in two ways: develop a surface consisting of triangular planes or define the geometry by using simple solids . The authors’ experience shows the wide applicability of the mesh method when working with samples with complex geometries. Fine triangles very accurately described the surface of, for example, a transverse flute, the model of which was used to simulate airflow. The high variability of the geometry justified the use of a complex meshing method, which in further steps, needed powerful computational software and high computing power. The second approach involves replacing the geometry of a given element by fitting basic 3D solids. The solids are matched in a semiautomated way. The approach enables fast and fairly accurate reproduction of the actual geometry of uncomplicated shaped elements such as simple rods, cubes, or rings. The approach allows for optimal use of computing power and a better representation of the geometry than the popular 3D extrude. Due to the variable shape of the fibers, it was deemed necessary to develop an accurate model containing a large number of triangles.The purpose of the analysis was to determine the stresses occurring at the contact between the steel fiber and the concrete. The assumed numerical verification would allow us to determine where the failure of the concrete structure may occur. In the course of the work, the focus was on accurate mapping of individual fibers extracted from concrete specimens. The fiber defined by the triangles was subtracted from the concrete cube. Visually, in The boundary conditions assigned to the models were full fixation of the lower nodes of the concrete mesh and static load applied to the upper edge of the fiber. The assigned force was determined according to MTS press pullout tests and was taken as the series average value. The material properties for the concrete and fiber were assigned according to tests that are the basis for another article. The data loaded into the numerical software  (Midas GTS NX) are shown in Tests were conducted by pulling the fiber out of the concrete. The test was performed on an MTS Model 685.53 machine. The displacement and force values were recorded during the tests. The exact force values are shown in The purpose of the visual inspection was to determine the potential damage occurring in the concrete structure due to the pullout force. By analyzing the scans of the samples before mechanical action with 10 mm anchorage, the contact surface between the fiber and matrix of good quality can be observed. In these specimens, no additional spaces or pores of large volume were observed at the reinforcement–matrix interface. The test did not reveal additional cracks. The aggregate was distributed homogeneously throughout the concrete. Pulling out the metal element caused visible changes within the direct contact area between the two materials. There are visible cracks in the concrete at the edge of the sample. Part of the loose material was removed during the test, reducing the volume of the concrete element. The changes are particularly visible at the top edge of the specimen and at the height of the metal bend. Visually, the changes are interpreted as a result of tensile and shear stresses acting on the matrix.The reconstructed specimen with an anchorage length of 15 mm revealed a higher overall porosity of the concrete. Visually, pores are assessed that they have a larger diameter. In visual assessment, the difference from the specimen with anchorage length of 10 mm is found to be significant. Air spaces between fiber and concrete are locally observed. No cracks are seen in the concrete structure. Similar to the previous specimen, no aggregate sedimentation occurred. The pullout of the fiber led to the destruction of the concrete in the area where the bending of the steel element occurred. Some of the loose material was removed during the test, reducing the volume of the specimen.The cross-section of the specimen with an anchorage length of 25 mm indicated a situation similar to the first specimens. Small air pores, uniformly distributed, are visible in the matrix. Locally, the fiber is surrounded by air pores. Cross-section analysis indicated fine cracks in the concrete around the upper contact zone. The pullout test led to crack propagation, but the material was not pulled out together with the steel fiber. The cross-sectional images are shown in The primary tool used to describe the volume changes is porosity analysis. 3 to over 10 mm3. Thus, there was more than a doubling of the local air space in the bend zone of the tested fiber. The increased pore volume is due to concrete deterioration and pullout of the loose material. The destruction occurred at the locations where the air pores between the fiber and concrete were noted.At a depth of 5 mm, the most significant change in structure occurred. The pore volume changed from 4 mmWhen analyzing the sample with an anchorage length of 15 mm , additioThe results for the sample with an anchorage length of 25 mm  indicateThe variable geometry of the fibers intuitively suggests that the stresses from the pullout test will not be consistent along the length of the element. The advantage of numerical methods over analytical approaches is the ability to input a geometrically and materially complex system. The models developed on the basis of tomographic investigations, assuming appropriate materials, closely represent the real stress concentrations in the tested specimens. The results are summarized in The highest shear stress values were obtained for the model anchored at 10 mm b. For thThe last test containing specimens anchored at a depth of 25 mm globally indicated another reduction in stresses. As in the previous cases, the stress concentrations occur at the bottom part of the course change and just below the top surface. By identifying low stresses, the FEM analysis indicates that potential failure may occur in areas where stresses are induced by random factors such as porosity or material weakening related to the setting of the concrete itself.The shear stresses are found locally just below the surface of the concrete and are characteristic of all three experimental models. The authors identify the lower left side of the bend as the second such location Data processing should be carried out in two ways, and each analysis requires a visual assessment. When using the results of the analysis, it is necessary to remember the assumptions made , which is verified visually. Obtained images present critical areas from the point of view of bond. Industrial computed tomography allows to assess the interior of the material without interfering in its structure. The obtained cross-sectional images make it possible to unequivocally determine the areas where the stresses occurring in the concrete exceeded its strength and changes in the material occurred.(2)Numerical models can be developed from CT data. The authors processed the data to generate models on which finite element simulations were performed. The simulation results identified the stress concentration locations. Both volumetric porosity analysis and numerical simulation indicated the same locations where damage can occur.(3)In the course of the work, contact characteristics between materials were derived from the authors’ assumptions. The combination of tomography and strength tests is a complex issue. The study can be deepened by taking into account the randomness of the pore distribution in the matrix.In this paper, the authors attempted to present the possibilities of tomographic image analysis. Modern approaches to research require the knowledge of modern instruments from researchers, both in terms of measurements and for further analysis. Current technology of tomographic imaging and dedicated software allows for complex defect analysis. The research itself requires experience of the operator and the ability to select parameters in such a way that the reconstructed solid is a set of high-quality data. The research can be summarized by the following conclusions:Based on the tomographic data, it is possible to determine a statistical model of the pore distribution and implement it into the model using, for example, Voronoi tessellation , or by r


B. To investigate, we compared the cardiac morphology and quantitative measurements of sl/sl rat to those of the control group.HSCR, a colonic neurocristopathy affecting 1/5000 births, is suggested to associate with cardiac septal defects and conotruncal malformations. However, we question subtle cardiac changes maybe more commonly present due to multi-regulations by HSCR candidate genes, in this instance, ETB+/−) crossbreeding. Age and bodyweight were recorded at time of sacrifice. Diffusion-staining protocols with 1.5% iodine solution was completed prior to micro-CT scanning. All rats were scanned using an in vivo micro-CT scanner, Caliper Quantum FX, followed by two quality-control scans using a custom-built ex vivo micro-CT system. All scans were reviewed for gross cardiac dysmorphology. Micro-CT data were segmented semi-automatically post-NLM filtering for: whole-heart, LV, RV, LA, RA, and aortic arch. Measurements were taken with Drishti. Following image analysis, PCR genotyping of rats was performed: five sl/sl rats, three wildtype, and three heterozygotes. Statistical comparisons on organ volume, growth rate, and organ volume/bodyweight ratios were made between sl/sl and the control group.Eleven neonatal rats were generated from heterozygote ; LV , RV , LA , and RA . Consistent trend was observed in growth rate (~ 20%) and organ-volume/bodyweight ratios (~ 25%). On the contrary, measurements on aortic arch demonstrated no significant difference among the two groups.Cardiac morphology and constituents were preserved. However, significant volumetric reductions were recorded in sl/sl rat, supporting the likely association of cardiac anomalies with HSCR, at least in ETB−/− subtype. Structural reduction was likely due to a combination of failure to thrive from enteric dysfunction, alterations to CaNCC colonization, and importantly coronary hypoperfusion from elevated ET-1/ETA-mediated hypervasoconstriction. Little correlation was detected between aortic arch development and sl/sl rat, supporting minor ETB role in large vessels. Although further clinical study is warranted, HSCR patients may likely require cardiac assessment in view of potential congenital cardiac defects.Despite the presence of normal morphology, significant cardiac growth retardation was detected in The online version contains supplementary material available at 10.1186/s12872-021-02281-2. B−/− HSCR model was associated with body-growth impairment; neonatal sl/sl rat has 16% less bodyweight than the control group.ETsl/sl rat has a normal cardiac morphology.sl/sl rats: 40% in whole-heart volume, 20% in growth rate, and 25% in whole heart/bodyweight ratios. Similar trend was observed in the measurements of LA, LV, RA, and RVSignificant reductions were detected in cardiac structures of B genotype and aortic arch sizes.No consistent correlation was observed between ETThese findings supported HSCR patients may have various degrees of neonatal cardiac anomalies; wholistic post-operative care should be considered, including screening for cardiac risk factors.B genotype and risks of hypertension, ischemic heart disease, exercise capacity, and heart failure.In consideration of the result from this paper, genotyping study of patients with and without heart failure would be an interesting step study to evaluate possible correlation between ETThe online version contains supplementary material available at 10.1186/s12872-021-02281-2. B) is a G-protein-coupled heptahelical receptor sharing the same class as endothelin A receptor (ETA). Both receptors are widely expressed with interconnected functions, particularly in the cardiovascular system. Both ETA and ETB initiate downstream signalling through binding to endothelin. Endothelin are 21 amino-acids peptides categorized into three subclasses: ET-1, ET-2, and ET-3 [B to ensure normal intestinal development whereas ET-1 and ET-2 exert vascular functions through ETA and ETB binding.Endothelin B receptor (ETB mutation is known to cause Hirschsprung’s disease (HSCR), a well-described aganglionic intestinal disease affecting 1/5000 births globally but with a incidence up to 1/1370 births regionally [B regulatory signalling suggests such treatments may not be sufficiently comprehensive. Recent studies suggested that multiple organ systems maybe be affected in syndromic HSCR patients [Homozygous ETgionally . HSCR ispatients ; up to 3patients .B mutation may partially stem from the migration failure of cardiac neural crest cell (CaNCC), a process dictated predominantly by ET-1/ETA signalling based on current literature [A mutation have been known to have high neonatal mortality with severe craniofacial and cardiac malformations [B mutation is not known to affect CaNCC directly, it has been reported to alter ET-1/ETA signalling [B−/− mutants.The pathogenesis of heart defects associated with HSCR and ETterature . Animalsrmations , 11. Altgnalling . ConsequB−/− mutant to suffer to structural changes during development. ETB’s cardiovascular effects are bimodal, mediating both vasodilation and vasoconstriction through its binding with ET-1 [B pathway yields endothelial vasodilation via nitric oxide (NO), prostacyclin, and endothelium-relaxing factor (EDRF) thus balancing the vasoconstriction mediated by ET-1/ETA in vascular smooth muscle cells (VSMC). Consequently, ETB is likely to possess a beneficial role in myocardial circulation and thus end organ perfusion [B−/− mutation to have impaired cardiovascular development and increased risks for hypertension and coronary artery disease due to the uncontrolled ET-1/ETA stimulation [Vascular dysfunction may also predispose ETith ET-1 –15, the ith ET-1 . Severalerfusion –19. By tmulation .B receptor, to the best of our knowledge, no macroscopic analyses have been completed to evaluate its effect on cardiac anatomy [sl/sl) rat, a naturally occurring ETB−/− animal model of WS-IV, with the phenotype of HSCR, hearing deficits, and white coat colour [Although several microscopic and physiologic studies have been conducted to determine the functions and distribution of ET anatomy , 15, 21.t colour .In order to acquire detailed yet structurally preserved anatomical information, we adopted X-ray micro-computed tomography (micro-CT) with modified tissue staining techniques . Micro-CB−/− mutant may suffer minor body growth impairment in early age.ETB−/− mutant.Gross cardiac morphology may be preserved in ETB gene may be associated with reduced heart size, growth rate, and heart volume/bodyweight ratio.However, loss of functional ETB dose dependent.Cardiac growth may be ETB has little effect on aortic arch growth.Absence of functional ETIn this study, we hypothesize the following:All tissues and animals used in this study were handled with care and strict compliance to ACT Health Human Research Ethics Committee (ACTH-HREC) and Australian National University Animal Experimentation Ethics Committee (ANU-AEEC), ethic approval project number A2011/67.B+/−) parents. This breeding colony was originally derived from a naturally occurring mutation and has been maintained in Australian National University (ANU) over the past 15 years.All rat specimens used in this study were generated from crossbreeding between the heterozygous  series: 20%, 50%, 70%, and 90% for 1 day each. Finally, tissue staining was completed in 1.5% iodine in 90% EtOH for a minimum of seven days prior to micro-CT scanning.In this study, all rat samples have been scanned using a commercial in vivo micro-CT scanner, Caliper Quantum FX. Additional quality assurance micro-CT scans were acquired using a custom-built ex vivo micro-CT system by ANU Applied Mathematical Department. The maximal resolution achievable by Caliper Quantum FX was 10 µm/voxel with an efficiency of 4.5 min per scan. The average dataset size was 256 MB. The resultant images were stored as DICOM series and visualized using Drishti, an open-source software . The ex , we found image quality of post-processed in vivo micro-CT scans satisfactory for the purposes of this study.Due to the limited access to the ex vivo micro-CT scanners, all image data acquired by Caliper Quantum FX were filtered with non-local means (NLM) algorithms to improve image quality . Two setAcquired micro-CT data were first denoised using NLM algorithm to improve signal-to-noise ratios and therefore image clarity . This coB−/− mutants. The luminal width of AA was measured at the aortic orifice in axial view for standardize comparison. Three-dimensional (3D) volumetric measurements were completed following sub-segmentation of each structure through volume rendering.Following image filtering, micro-CT data were segmented semi-automatically through individual CT slices for selected organs using Drishti segmentation algorithms . To ensuTo standardize comparison, the following anatomical definitions were adopted. The pulmonary circulatory inflow was defined by the superior and inferior vena cava orifices to right atrium; the outflow was defined by the pulmonary valve. The systemic circulatory inflow was defined by pulmonary vein orifices to the left atrium whereas the outflow was defined by the aortic valve. Both selections of left and right ventricles have included interventricular septal wall for better definition of organ boundary to standardize comparison. Lastly, for the comparison of AA, the boundary of AA was defined as arterial vessel between the aortic valves to the first branching point, brachiocephalic artery.H&E light microscopy was completed for two of eleven rats following micro-CT scanning to assess cardiac anatomy presented in micro-CT scans. The following steps were performed. The iodine-stained hearts were sectioned longitudinally into tissue blocks of 4 mm in thickness and placed in cassettes. Contrast washout and dehydration were performed in 90% EtOH for 48 h prior to paraffin embedment at 60 °C. These tissue blocks were then sliced into 4 µm thick tissue-sheets with a microtome. Tissue-sheets were then laid in water-bath of 5–6 °C while being positioned onto labelled-glass slides. These slides were dried overnight at 37 °C.Progressive H&E staining was completed by placing the slides in alum-hematoxylin solutions until the appearance of dark red colour. Washing and ‘bluing’ with lithium carbonate solution were then performed. Lastly, washing and counter-staining with 0.5% eosin alcoholic solution were completed.All H&E slides were reviewed with an Olympus IX71 microscope at 4× magnification.B/++), three heterozygous (ETB/−+), and five homozygous spotting lethal  rats were identified. The average ages of wild-type, heterozygous, and homozygous mutant rats were 90.7 h, 96 h, and 83.2 h respectively.After the completion of quantitative measurements, genotyping was completed as described in the following section. Three homozygous wild-type , Primer PS7 , Primer PS 15  and DNA polymerase [sl/sl rat) was run for one hour under the voltage setting of 100 V and current setting of 55 mA, with MassRuler  reference by the side. The resultant electrophoresis gel was visualized with Gel Documentation System DOC-Print VX5 (Vilber Lourmat).PCR genotyping was completed through the following protocols. Five-millimetres tail tips of the eleven rats were lysed using Proteinase K in lysis buffer consisting of 100 mM Tris pH 8, 5 mM EDTA, 0.2% SDS, and 200 mM NaCl in distilled water. The DNA was extracted via vortex heating and centrifuging to separate the DNA-containing supernatants from undigested materials. The supernatants were further vortexed and centrifuged to isolate the DNA pellets. The DNA pellets were then washed with 70% EtOH and dried. The DNA was then suspended and quantified using spectrophotometry. Next, PCR was completed with “Master-Mix” reagent, which included: 10*PCR buffer Qiagen-contained MgCllymerase . Afterwap value = 0.001) and high genetic penetrance (> 95% exhibits HSCR) of sl/sl rats, statistical comparison using the two-tail t-test was made between the sl/sl (ETB−/−) and the control group (ETB+/+ and ETB+/−) to determine the effect of ETB on heart growth. The comparison was made in the following parameters: organ size, organ growth rate, and organ volume/bodyweight ratios. Albeit small, the latter two were made to further standardize comparisons by accounting age and body size variations of individual rat. Additionally, these parameters provided estimating metrics for structural changes upon developmental maturation.Based on the segregation analysis of rat colony data showing strong autosomal recessive inheritance  were compared to explore potential regional-dependent effect. Lastly, data of respective wild-types (ETB+/+) and heterozygotes (ETB+/−) were provided in the supplementary figures to illustrate of potential gene-dose-dependent relationship.The difference (%) between the control and www.graphpad.com.All statistical analysis was completed one month after the secondary verification review of image segmentation: GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, B mutation was associated with minor changes in body size and body growth rate, at least at the age of eighty hours. As shown by Fig. sl/sl (ETB−/−) rats have 16.32% smaller bodyweight of than that of control group, p value = 0.03. On the other hand, when standardized to rats’ age, this difference was reduced to 3.53%, p value = 0.577, as shown by Fig. B+/+) and heterozygotes (ETB+/−), suggesting the absence of ETB dose-dependent relationship.Based on our measurements of neonatal rats, ETsl/sl heart contained all the essential components of a normal heart: aorta, aortic semilunar valve, right and left atrium, right and left ventricles, intact interatrial and interventricular septum, and patent pulmonary vessels. Expectedly, cardiac anatomy of wild-type and heterozygous rats shared the same features. This preliminary finding suggested ETB may have little impact on CaNCC migration.Previous studies have suggested disruptions in the endothelin system may lead to CaNCC migration failure and cause cardiac outflow tract defects . HoweverB mutation was associated with significant volumetric reductions in the heart and constituents.Following the morphological examinations, volumetric measurements were performed for quantitative comparison. Our data demonstrated homozygous ETsl/sl rat has a significantly smaller heart than the control group, up to 38.7% reduction, p value = 0.02. Similar changes were observed in the following constituents: LV , RV , LA , and RA . Volumetric measurement of AA also showed 22.00% reduction in sl/sl rats with respect to the control; however, this finding did not reach statistical significance, p value = 0.25.As demonstrated by Fig. B gene copies. As shown by Additional file sl/sl rat in the last. Concordantly, LA, RA, LV and RV measurements of wild-types were 42.76–48.09% larger than those of sl/sl rats, whereas those of heterozygotes were only 34.71–44.71% larger.Although not reported by current literature, cardiac size may be dose-dependent to ETsl/sl rats having approximately 40% smaller hearts with respect to the control group. These differences may widen with age until rats reach maturity.Overall, we demonstrated neonatal B mutation has a negative impact on cardiac growth rate. As shown by Fig. sl/sl rat was 23.70% lower than that of control group, p value = 0.05. Significant reductions were also observed in the growth rates of LV  and RV . Although not reaching the statistical power, lower growth rates of LA , RA  and AA  were also detected in the sl/sl rats.ETB gene. As shown by Additional file sl/sl rats, whereas those of wild-types were 29.43–35.91% higher. On the other hand, analyses on AA growth rates demonstrated no consistent correlations with ETB dose.Further analysis showed stepwise decreases in organ growth rates with respect to functional copies of ETsl/sl rats. This growth retardation may persist until age of maturation. Interestingly, this reduction was disproportionally larger than the 3.53% reduction in bodyweight growth rate, suggesting an intrinsic effect of ETB to the developing heart.Overall, an approximately 25% decrease in the growth rates of all heart constituents could be appreciated in neonatal B mutation on heart structure was disproportionally larger than its effect on body size. Figure sl/sl rat has approximately 20% smaller organ volume/bodyweight ratios than those of the control group in the following: whole heart , RA , LV , and RV . Although not achieving statistical significance, LA volume/bodyweight ratio of sl/sl rat also has a reduction of 25.75%, p value = 0.13. Overall, homozygous ETB mutation was associated with a disproportionally larger reduction in cardiac sizes with respect to changes in global body size. This was further supported by a stepwise decreasing pattern with reducing copies of functional ETB gene, as illustrated by Additional file sl/sl rat having 5.17% larger AA than that of the control group, p value = 0.70; this observation was consistent with ETB’s minor regulatory role on large vessel.Cardiac organ volume/bodyweight ratios demonstrated that effect of ETsl/sl rat. The differences in cardiac constituent/whole heart ratios were relatively uniform between sl/sl rat and the control group, as showed by Fig. B mutation on the heart was likely global. Overall, these differences between the two groups were insignificant albeit a 5.81% reduction was detected in the RV/whole heart ratio of sl/sl rat.Subtle variations in the structural changes of different heart components were detected in B−/− HSCR model exhibits significant cardiac structural impairment. This was consistently illustrated through three parameters taken in sl/sl rat: up to 40% volumetric reductions in heart structures, 20% reduction in heart growth rate, and 25% reduction in organ volume/bodyweight indices. The causes for these cardiac growth restrictions likely included the following: global growth impairments due to enteric dysfunction; alterations in CaNCC development; vasodysregulation caused by the absence of ETB.In this study, we demonstrated neonatal ETsl/sl rat despite preservation of normal organ morphology. Our findings suggested HSCR patients, at least in ETB−/− variant, are likely to share subtle cardiac and neurological impairments. This also suggested that CHD incidence in HSCR is likely underestimated due to under-reporting of subtle cardiac anomalies.Concurrent developmental anomalies occurred in up to 30% of HSCR patients, five to eight percent of whom suffer congenital heart diseases (CHD) . While CB knock-out mutation (ETB−/−) causes WS-IV syndrome with prominent HSCR phenotypes. As previously mentioned, aberrant mutation in ET-3/ETB signalling causes ENCC migration failure and thus ENS maldevelopment, which impairs the nutritional absorption in affected individuals [sl/sl rat, Fig. Homozygous ETividuals . Consistsl/sl rat. Although limited literature is available to comment on the presence of direct linkage between ET-3/ETB signalling on CaNCC migration, indirect alteration in ET-1/ETA signalling through elevated ET-1 levels in ETB−/− mutants has been suggested [A signalling share features of velocardiofacial syndrome like those of CaNCC migration failure. On the other hand, other than significant size shrinkage, sl/sl rat seemed to exhibit a grossly normal heart; neither large vessel nor cardiac septal anomaly was detected. Although prior lineage-tracing studies did not reveal the participation of CaNCC in the developments of myocardium and epicardium [Plxna2, fate-mapping, and the finding of thin ventricular myocardium as a result of CaNCC gene knock-out studies (e.g. BMPR1A and PAX3) suggested CaNCC contributes to epicardial developments and ventricular myocardium [sl/sl rat, supporting subtle alterations in CaNCC pathway from ET-1/ETA hyperstimulation may have partially contributed to the myocardium maldevelopment.Disruption in CaNCC migration may have contributed to the structural changes of the heart in uggested . CaNCC huggested . Consequuggested . Furtheruggested  while ituggested . Importauggested  demonstricardium , biomarkocardium –35. ConcB’s vascular control may have also contributed to the detrimental effect of cardiac development in sl/sl rat. ETB is predominantly expressed in the vascular endothelium where it initiates vasodilation through binding with ET-1, thus triggering decreased clearance of NO, prostacyclin, and EDRF. On the other hand, subtle ETB presence has also been detected in VSMC, where activation by ET-1 causes vascular constriction; albeit this effect is minor in normal physiological state [B’s functional duality through stronger recordings of vasoconstrictive response in endothelium-denuded porcine coronary artery following stimulation by Sarafotoxin 6c. This showed endothelial ETB partially regulates baseline vasodilation and basal coronary perfusion, which are vital to the developing heart. Adding insult to injury, the absence of ETB markedly reduces the clearance of ET-1 and thus increases ET-1 level by up to 6-folds in sl/sl rats [A stimulation and subsequent hyper-vasoconstriction of coronary artery. Consequently, coronary hypoperfusion worsens growth retardation of the heart, as shown by Figs. B copies, as shown by Additional files Disruption in ETal state , 18, 36.al state  illustra/sl rats . This leB-dependent relationship, as shown by Figs. sl/sl rat, as noted by Additional file A signalling. Additionally, this finding was consistent with prior reports that contractile control in large vessels is predominantly ETA-mediated [Lastly, three-dimensional analyses made on aortic arch demonstrated no conclusive ETmediated .sl/sl and the control groups. The effect of ETB on cardiogenesis was likely multifactorial rather than a pure manifestation of HSCR’s poor growth [B dysfunction leading to the cardiac reduction, in conjunction with prior studies, hypoperfusion from vascular dysregulation seems likely. Nevertheless, we acknowledged the possibility of growth impairment from enteric dysfunction and changes in CaNCC colonization to the developing heart, albeit these effects were likely minor if presented. Importantly, our finding provided a clue to the suspected cardiac impairment associated with HSCR, a traditionally thought surgical disease. Indeed, if this quantitative finding is translatable to human, HSCR patients are likely to suffer a significant cardiac structural reduction, ranging from 20 to 40%, and thereby increases risk for development of cardiac failure, at least in the ETB−/− subtype. Consequently, a wholistic management may be warranted.Overall, our result showed distinctive difference in cardiac growth between r growth . While wB−/− subtype. In conjunction to the known vascular dysfunction related to ETB−/− mutation, significant structural compromise of the heart in animal model suggested a potential risk for cardiac dysfunction. Thus, a structural review using cardiac magnetic resonance imaging (MRI) followed by a baseline echocardiogram may be beneficial. Additionally, clinical uses of endothelin receptor antagonists should be with increasing caution given the potential adverse effect of ETB inhibition.Our findings suggested early screening for cardiovascular risk factors in individuals with HSCR may be beneficial, especially in those with ETB−/− HSCR animals, further studies are required. Firstly, we acknowledge the statistical power can be improved by higher sampling; this can be achieved once a more efficient image segmentation platform becomes available. Secondly, despite image analysis was completed on a pre-set computing algorithm to minimize potential bias, future study should consider adopting additional operators and various software for verification of findings. Furthermore, functional assessments with echocardiograms may be beneficial in establishing the direct correlations between cardiac dysfunction and structural impairments. Lastly, clinical study using cardiac MRI can be beneficial in confirming cardiac defects in HSCR patients and should be considered in future studies.Although we have demonstrated significant structural defects in ETB function and cardiac size in the sl/sl rat. The exact mechanism of action is unclear at this point but may well be due to an effect on coronary artery tone. Preserved cardiac morphology and aortic arch dimensions suggest impaired CaNCC migration failure may play little role in HSCR. Nevertheless, the finding of significant cardiac size and growth impairment in HSCR model is consistent with an elevated risk of congenital structural heart disease in HSCR patients. It also raises the possibility that ETB gene may have a role in the development of heart failure, coronary artery disease, and hypertension in the general population.This study demonstrated a correlation between ETAdditional file 1: Figure 1. Stepwise structural shrinkages were associated with decreasing functional ETB. Stepwise reduction in cardiac size was associated with decreasing ETB copies, with wild-type having largest heart and constituents, followed by heterozygotes in the middle, and sl/sl having the smallest structures: whole heart , LV, RV , LA , and RA . The difference between wild-type and heterozygotes was relatively small whereas tissue shrinkages in sl/sl rat were markedly larger. On the other hand, aortic arch measurements did not suggest gene-dose-dependency: wild-type (1.39 mm3), heterozygotes (1.74 mm3), and sl/sl (1.29 mm3). RA = Right Atrium; RV = Right Ventricle; LA = Left Atrium; LV = Left Ventricle. *: statistically significant in comparison to ETB−/−group, p ≤ 0.05.Additional file 2: Figure 2. Stepwise growth rate reduction was associated with decreasing functional ETB. Stepwise decrease in cardiac growth rates corresponded to reducing ETB copies. As shown, wild-type has highest growth rates, followed by heterozygotes in the middle, and lowest in sl/sl rat: whole heart , LV, RV , LA , and RA . Wild-type and heterozygotes have relatively small growth rate variations when comparing their respective differences to sl/sl rat. No consistent correlations with genotype can be deduced from aortic arch measurements: wild-type (0.015 mm3/Hr), heterozygotes (0.018 mm3/Hr), and sl/sl (0.015 mm3/Hr). RA = Right Atrium; RV = Right Ventricle; LA = Left Atrium; LV = Left Ventricle. *: statistically significant in comparison to ETB−/−group, p ≤ 0.05.Additional file 3: Figure 3. stepwise reduction in organ/bodyweight ratio associated with decreasing copies of functional ETB. Positive correlations between cardiac organ/bodyweight indices and ETB gene copy was demonstrated. Wild-type has the highest ratio, followed by heterozygotes in the middle, and sl/sl having the lowest: whole heart , LV, RV , LA , and RA . Minor difference was presented between wild-type and heterozygote when comparing to the reductions observed in sl/sl rats. In conjunction with Figure 1, this supported ETB mutation has intrinsic effect on cardiac development. No consistent trend associating with genotype can be deduced from aortic arch measurements: wild-type (0.11 mm3/g), heterozygotes (0.13 mm3/g), and sl/sl (0.11 mm3/g). RA = Right Atrium; RV = Right Ventricle; LA = Left Atrium; LV = Left Ventricle. *: statistically significant in comparison to ETB−/−group, p ≤ 0.05.Additional file 4. Supplementary Table 1: sl/sl rat has reduced luminal width of aortic arch when comparing to the control groups. Two-dimensional measurement at the entry of aortic valve showed sl/sl rat having smaller intravascular width comparing to the control group. This trend persisted when respective age and bodyweight were standardized, as shown by the comparison of width growth-rate and width/bodyweight ratio, suggesting possible vasoconstriction.


Antibiotic overuse is linked to increased risk of antimicrobial resistance. Long-term antibiotics are commonly used for treating acne and prophylaxis of urinary tract infection. Their contribution to the overall burden of antibiotic use is relatively unknown.To describe the volume of commonly prescribed long-term (≥28 days) antibiotic prescriptions in adolescents and young adults, trends over time, and comparisons with acute prescriptions.A retrospective cohort study using UK electronic primary care records.Patients born between 1979 and 1996 and with data in the Care and Health Information Analytics database were included. The main outcome measures were antibiotic prescription rates per 1000 person-years and antibiotic prescription days per person-year between the ages of 11 and 21.In total, 320 722 participants received 710 803 antibiotic prescriptions between the ages of 11 and 21 years from 1998 to 2017. Of these 710 803 prescriptions, 191 443 (26.93%) were for long-term antibiotics (≥28 days and ≤6 months in duration). Long-term antibiotics accounted for more than two-thirds (72.48%) of total antibiotic exposure (days per person-year). Total long-term antibiotic prescribing peaked in 2013 at just under 6 days per person-year and declined to around 4 days in 2017.Among adolescents and young adults, exposure to long-term antibiotics (primarily lymecycline used for acne) was much greater than for acute antibiotics and is likely to make an important contribution to antimicrobial resistance. Urgent action is needed to reduce unnecessary exposure to long-term antibiotics in this group. Increasing the use of, and adherence to, effective non-antibiotic treatments for acne is key to achieving this. Treatment of acne with antibiotics is associated with an increased risk of common infections.14–15 Acne affects over 90% of teenagers.11 There are approximately eight consultations per 100 person-years for acne in 12- to 18-yearolds in the UK and oral antibiotics are the most common acne-related medication prescribed.16 Dermatologists prescribe more oral antibiotic courses per clinician than any other specialty, and many of these courses of antibiotics are prescribed for several months in duration.17 Non-antibiotic treatments, particularly topical treatments, are more appropriate for the majority of acne.12Long-term antibiotics are commonly used for the treatment of acne9 and the number of prescriptions issued rather than the number of units or prescription days. Describing antibiotic prescriptions linked to specific conditions or indications may underestimate total prescribing.9 Other work has focused on the identification of inappropriate antibiotic use but did not consider long-term antibiotic prescriptions, the contribution of which to the overall burden of antibiotic use is relatively unknown.10 This study therefore determined the rates and trends, and overall burden, of long-term antibiotic prescriptions in an adolescent cohort in primary care in Hampshire, UK.Previous work investigating antibiotic prescribing in primary care has focused on antibiotic choice18 Data were accessed via the CHIA governance team, and programming and extraction carried out by a member of the CHIA team. Extracted data included all antibiotic prescriptions. Data were not restricted based on consultations or Read codes . Analyses were performed by year and denominators were calculated using all participants registered with a practice, excluding those who were deceased or had moved out of the area. Only oral antibiotic prescriptions for tablets/capsules were included in the analysis.A retrospective longitudinal population-based cohort study was conducted using data from the Care and Health Information Analytics database (CHIA) for the period January 1998 to August 2017. The CHIA database is an anonymised analytical database with data from around 130 general practices across Hampshire containing information on over 1.24 million residents.15 The quantity of medication prescribed for each antibiotic was identified. Antibiotic prescriptions where the quantity prescribed was inferred (by dosing regimens) to be greater than 6 months were excluded as these were thought likely to be errors (issuing prescriptions for longer durations is not recommended). In England, for medicines commonly prescribed for long-term conditions, 93% of the total volume in 2019 was for 3 months or less. Long-term antibiotics were selected based on clinical indications listed in the BNF for acne and infection prophylaxis, and some key short-term antibiotics for comparison. This accounted for 84.0% of the extracted prescriptions. Prescriptions for the remaining short-term antibiotics prescribed for >28 days were excluded. This exclusion of these prescriptions together with those for more than 6 months’ supply excluded 1.49% of the prescriptions.CHIA was used to extract prescription data for patients born between 1979 and 1996 who attended one of the included GP surgeries at least once during adolescence. Antibiotic classes and specific antibiotics were selected using British National Formulary  codes, and both generic and brand names of antibiotics were included in the analysis for the antibiotics listed in Supplementary Table S1. Long-term antibiotic prescriptions were defined as prescriptions for 28 days’ or more supply of antibiotics. This definition has been applied elsewhere for acne prescriptions.Dosing regimens were inferred from the specific antibiotic and quantity supplied (Supplementary Table S1). Prescriptions for 56 oxytetracycline tablets or more in the long-term analysis were included as it was decided that there were no other likely indications. All analyses were undertaken in Stata statistical software version 14.0. The use of CHIA data within this study was approved by the Care and Health Information Governance Group.A patient and public involvement representative provided input into this work and commented that the work was important, particularly in terms of ‘informing future antibiotic prescribing guidelines in adolescents in order to weigh up the risk associated with prescribing prolonged courses’.There were 320 722 participants who attended one of the included GP surgeries at least once during adolescence. A total of 1 703 786 antibiotic prescriptions issued between 1 January 1998 and 9 August 2017 at age 11 onwards were identified. There were 122 571 prescriptions that did not contain unit values for the quantity of medication issued. There were 102 523 liquid/solution prescriptions further excluded (as the duration could not accurately be inferred from quantity supplied). The analysis was limited to the 11 antibiotics listed in Supplementary Table S1 (excluding 164 014 prescriptions). It was necessary to further exclude 19 548 prescriptions with suspected invalid quantities >28 days for short-term antibiotics and >168 days for long-term antibiotics. The analysis was further restricted to prescriptions issued for patients between ages 11 and 21 (584 327 exclusions). The following analysis includes a total of 710 803 prescriptions. The data selection/exclusion process is depicted in This large community-based study demonstrates high rates of antibiotic exposure related to long-term antibiotic prescribing for adolescents and young adults primarily in the second decade of life. Long-term antibiotic prescribing accounted for more than two-thirds (72.48%) of total antibiotic exposure days. A significant proportion of the cohort (11.01%) were prescribed at least one long-term antibiotic prescription, most commonly lymecycline, which is commonly used in the treatment of acne.The frequency of antibiotic dosing was inferred from the quantity supplied and this could affect the accuracy of the results, although dosing regimens for the most commonly prescribed antibiotics including lymecycline and amoxicillin are likely to be correctly inferred. Medication adherence was unknown for both acute and long-term antibiotic prescriptions. The study did not attempt to determine the indication for the antibiotic prescriptions and prescriptions that were not linked with Read codes were included. There was a significant number of prescriptions with missing quantity data (7.19%) and liquid preparations were not included in the analysis. The proportion of scripts for each individual antibiotic was similar in the missing data group and those included in the analysis.19 and, although Hampshire has lower antibiotic prescribing rates than the national average,20 the CHIA prescription rates are comparable with large national databases such as THIN (discussed below). Further research using other healthcare databases could help confirm the current findings.The CHIA encompasses extensive coverage of approximately 75% of the resident Hampshire population at the time of data extraction. Although not all local practices participate, those that are missing are dispersed across the catchment area, with varied rural/urban classification, socioeconomic deprivation, and patient composition. The authors are not aware of any systematic differences to those practices whose data are present,et al using the THIN database including similar total antibiotic prescription rates (413 per 1000 compared with 580 per 1000 person-years in patients aged under 19 in 2013).9 A study using the Clinical Practice Research Datalink (CPRD) database determined there were 489 prescriptions per 1000 person-years in 2017 (without age restrictions)21 compared with 306 prescriptions in the currently presented cohort. The difference could be because of higher proportions of prescriptions for older adults and very young patients included in the CPRD data. UK national data yielded an equivalent of 5 antibiotic days per person-year in 2017 for the entire population compared with 5.66 in this cohort in 2017.22 National data confirm an overall increase in antibiotic consumption in primary care, which peaked in 2014 and could have been driven by an increase in antimicrobial resistance.22 National data have also confirmed a fall since the 2014 peak because of the introduction of national targets implemented for general practice with financial incentives, education tools for prescribers, and patient leaflets.22In this analysis of routinely collected UK general practice data in Hampshire, relatively similar prescribing rates of individual antibiotics were found to those obtained by Dolk 16 Tetracyclines comprised 7.9% of prescriptions in those aged under 19 years and 14.0% in those aged 19–65 in the Dolk et al9 study compared with 19.4%  in 2014 in this study. In national data, lymecycline prescribing comprised 12.37% of all general practice antibiotic prescription days per person-year in 2017.22In the present study, 11.01% of participants aged 11–21 were prescribed at least one course of long-term antibiotics. Tetracyclines were the most commonly prescribed long-term antibiotics and this trend was observed in another study using the CPRD database.16 where prescriptions were extracted for patients who consulted with any acne Read codes (11.8 items per 1000 person-years in 2013), but prescriptions may happen without a consultation code being recorded. As the current study did not link prescriptions with Read codes or consultations, it probably includes a more complete assessment of relevant prescriptions. There were relatively low rates of prescribing of antibiotics for urinary tract infections in this cohort as indicated in Lower rates of lymecycline prescribing were found using CPRD data,Escherichia coli in asymptomatic young people found that resistance to many primary care-prescribed antibiotics is common and tetracycline resistance rates were high.23 Furthermore, healthy children carry bacteria resistant to antibiotics to which they are not usually exposed and resistance to tetracyclines could be acquired from family members or other children.24The number of antibiotic prescription days per person-year for long-term antibiotics was much greater than for commonly prescribed acute antibiotics and reflects considerable antibiotic exposure. In 2017, long-term antibiotics were prescribed just under 4 days per person-year compared with 1.7 days for acute prescriptions. These data suggest that long-term antibiotics  and particularly lymecycline prescriptions comprise a major burden of antibiotic exposure in adolescents and young adults in the UK. It is feasible that this level of exposure could contribute significantly to AMR. A review and meta-analysis of faecal carriage of antibiotic-resistant 12 Recent research indicated that one in four patients with acne are prescribed oral antibiotics during a new acne consultation.16 Action is required to increase use of, and adherence to, alternative and effective topical therapies and for timely review of response to antibiotic therapy.Lymecycline is primarily prescribed for acne in adolescents and young adults, and recent National Institute for Health, and Care Excellence guidance for acne has published recommendations that include non-antibiotic treatment choices for acne of any severity.14–15 In a retrospective cohort study of 84 977 individuals with acne treated with a topical antibiotic, oral antibiotic, or both, the odds ratio of developing an upper respiratory tract infection diagnosed by a GP was 2.15 times higher compared with patients not treated with antibiotics (P<0.001).14 These findings were supported by a subsequent cross-sectional study, in which self-reported pharyngitis was nearly twice as common in those exposed to oral antibiotics (66.7% compared with 36.2%).15 A potential explanation for this finding may be the depletion of natural oral flora supported by the finding that recolonisation therapy with a streptococcal spray has been shown to protect against recurrent infections.25Treatment of acne with antibiotics is associated with an increased risk of common infections.26 and it is possible that long-term antibiotics may cause more profound and persistent changes. Long-term antibiotic use has been associated with a range of adverse outcomes including increased risk of colorectal adenoma,27 an increased risk of future cardiovascular events among older females at usual risk,28 and weight gain.29 Tetracyclines are also used as growth promoters in livestock to promote host lipid metabolism, energy harvest, and weight gain.30 Tetracycline usage during the fourth decade of life was, however, associated with reduced odds of obesity at enrolment to the Sister study.29 Other work using retrospective cohort data has suggested that long-term antibiotic use in healthy adolescents with acne was not associated with weight gain.31Antibiotics have been shown to have profound and sometimes persisting effects on the intestinal microbiota, characterised by diminished abundance of beneficial commensals and increased abundance of potentially detrimental microorganisms that can persist for years,Importantly, this study has shown that, in this cohort of adolescents and young adults, population exposure to long-term antibiotics is larger than that for acute antibiotics. Repeat use of long-duration antibiotics is associated with greater risk of antimicrobial resistance, and cross-resistance can occur where exposure to a specific antibiotic can induce resistance to other classes. Urgent action is needed by policymakers to curtail the use of long-term antibiotics, primarily lymecycline, for acne and promote the use of, and adherence to, alternative management strategies.