Wearable devices that allow continuous monitoring of peripheral oxygen saturation (SpO2), such as the Garmin Fēnix® 5X Plus (GAR), might provide early detection to prevent hypoxia-induced diseases. We therefore aimed to validate GAR-derived SpO2 readings at 4559 m. SpO2 was measured with GAR and the medically certified Covidien Nellcor SpO2 monitor (COV) at six time points in 13 healthy lowlanders after a rapid ascent from 1130 m to 4559 m. Arterial blood gas (ABG) analysis served as the criterion measure and was conducted at four of the six time points with the Radiometer ABL 90 Flex. Validity was assessed by intraclass correlation coefficients (ICCs), mean absolute percentage error (MAPE), and Bland–Altman plots. Mean (±SD) SO2, including all time points at 4559 m, was 85.2 ± 6.2% with GAR, 81.0 ± 9.4% with COV, and 75.0 ± 9.5% with ABG. Validity of GAR was low, as indicated by the ICC (0.549), the MAPE (9.77%), the mean SO2 difference (7.0%), and the wide limits of agreement  vs. ABG. Validity of COV was good, as indicated by the ICC (0.883), the MAPE (6.15%), and the mean SO2 difference (0.1%) vs. ABG. The GAR device demonstrated poor validity and cannot be recommended for monitoring SpO2 at high altitude.Decreased oxygen saturation (SO Wearable devices are increasingly used to monitor physiological biomarkers . If cert2) is reduced ) × Σ/[actual data value]) × 100. MAPE does not have a standardized threshold for determining the validity of measurements; however, Fokkema et al. considered a difference of ±5% as practically relevant for wearable sensor data [2 testing in patients with chronic obstructive pulmonary disease showed intraday fluctuations of 1.6% [2 MAPE threshold ranging ±3.2% ) was assumed as a criterion of acceptable validity in our study. Furthermore, Bland–Altman analysis, including mean difference and limits of agreement, was conducted to plot the difference between the scores of two measurements against the mean SO2 values derived from the measurements [t-tests, with a p-value of 0.05 set as the threshold for significant differences.The following statistical procedures were applied to determine the validity of the results. A two-way mixed effects, absolute agreement, multiple raters/measurements intraclass correlation coefficient (ICC)  was calcfference . The measor data . Since r of 1.6% , an SpO2urements . FinallyContinuous data are given as arithmetic mean ± SD and categorical data as percentages. All statistical analyses were performed using SPSS 27 for Windows .2 measurements at all time points at high altitude. Overall, SO2 values obtained with GAR were highest (85.2% ± 6.2), compared to 81.0% ± 9.4 for those obtained with COV (p = 0.011) and 75.0% ± 9.5 for those obtained with ABG (p ≤ 0.001). Mean SO2 differences between GAR vs. ABG were 7.0%, compared to 0.1% between COV vs. ABG.The ICC for GAR vs. ABG was low (0.549), whereas the ICC for COV vs. ABG was good (0.883). According to the predefined MAPE acceptable validity cut-off of <3.2%, neither GAR (9.77 %) nor COV (6.15 %) fulfilled this criterion when compared to ABG . p < 0.001; R2 = 0.735) and a weak correlation for GAR vs. ABG . The median of the individual Pearson’s r correlation coefficients between GAR and COV was 0.534, ranging from −0.5 to 0.909. Correlational analysis did not reveal a significant magnitude dependence of the difference between ABG and GAR and the average between ABG and GAR . 2 ≤ 70%), GAR did not report any SpO2 readings.The Pearson’s r indicated a strong correlation between COV vs. ABG  with wide limits of agreement , whereas the validity of COV was good (mean difference compared to ABG: 0.1%) despite wide limits of agreement  . The overall incidence of AMS was 77% (10/13) and the incidence of HAPE was 54% (7/13). The Pearson’s r showed a strong correlation between ABG and the AMS severity assessed by the Lake Louise Score (LLS) and a weak correlation between GAR and AMS severity . ® 5X Plus-derived SpO2 readings at 4559 m. The main result was the poor validity of GAR, indicated by an ICC of 0.549, an MAPE of 9.77, a mean SO2 difference of 7.0%, and wide limits of agreement  vs. ABG. The aim of this study was to evaluate the validity of the Garmin Fēnix2 data continuously and to improve health and safety during altitude activities through the early detection of lower SpO2 levels than expected at any given altitude. However, only a few validity studies of wearable SpO2 sensors exist. All of them were performed only at simulated altitude, using medical transcutaneous oximeters for criterion measurement with conflicting results. Lauterbach et al. evaluated the accuracy of SpO2 readings derived from the same Garmin device as used in the present study at simulated altitudes up to 3660 m. The authors concluded that GAR exhibits minimal overestimation  of SpO2 and that the device may be a viable method to monitor SpO2 at high altitude [2 sensor in 10 healthy participants at simulated altitudes from 3000–5500 m [2 values, yielding an ICC of less than 0.280 over all the altitudes studied. Our study is the first to investigate the validity of GAR-derived SpO2 measurements in a field setting at 4559 m, comparing them with the standard criterion of arterial blood gas analysis, in addition to measurements obtained with a medically certified transcutaneous oximeter, and applying comprehensive statistical analyses. Compared to measurements taken at simulated altitudes in a hypoxic hypobaric chamber, SpO2 values at high altitude resemble real-life conditions, since they include the impact of environmental variables, such as cold and light, but also physiological variables, such as hyperventilation and periodic breathing, which may interfere with data stability [Wearable wrist-worn devices have the potential to serve as a convenient method to collect SpOaltitude . Howevertability . Our mul2 threshold of 84% was reported for predicting the development of severe AMS with satisfactory specificity and sensitivity between 3600 to 3700 m using a 24-h data memory fingertip-type medical oximeter [2 levels compared to ABG, with poor agreement indicated by wide limits of agreement in our Bland–Altman analysis  and a low coefficient of determination (R2 = 0.109), which precludes using GAR to reliably categorize mountaineers with increased risk for AMS using the abovementioned cutoff value. Furthermore, in our study, GAR showed the lowest predictive value for assessing the severity of AMS (R2 = 0.007), whereas COV performed better (R2 = 0.278) and ABG yielded the best prediction (R2 = 0.644). There is increasing evidence that blood oxygenation is lower in individuals who suffer from AMS. Recently, an SpOoximeter . Based o2 levels due to impaired alveolar gas exchange at high altitude [p = 0.625), GAR tended to overestimate SpO2 especially when blood oxygenation was low. This is indicated by the relatively flat regression line of ABG vs. GAR in comparison to the line of identity (2 values when ABG measurements were lowest. Added to the fact that taking measures to prevent altitude sickness is particularly important when O2 saturation is low, the overestimation of SpO2 by GAR at high altitude may falsely suggest that no risk is present. This does not only limit the usefulness of GAR but also means that reliance on its results could be potentially life-endangering. This notion is also in line with Luks and Swenson [2 levels might also be an indicator for COVID-19-related pneumonia and adverse clinical outcome [2 analysis, e.g., via video processing, might add even more convenient methods to self-monitor SpO2 levels at high altitude [Mountaineers suffering from HAPE often present with very low SpOaltitude . Althougidentity . In addi Swenson , who ana outcome . Easy toaltitude , provide2 readings from wrist-worn devices might be influenced by skin tone, and this factor was not assessed in our study. However, there was no effect of skin tone on any SpO2 variables in the study of Hermand et al. [2 measurements, and thus affect the conclusions of this study. We performed the measurements in a small sample of participants with a history of HAPE; a larger sample would have increased statistical power. Besides the logistical difficulties associated with high-altitude studies, the validity data obtained in this study should also be applicable to subjects without HAPE susceptibility, given the wide range of SpO2 reported here. Our study has some limitations worth mentioning. SpOd et al. . Also, f2 data obtained by the Garmin Fēnix® 5X Plus at 4559 m do not meet acceptable validity criteria. Systematic overestimation of SpO2 levels at high altitude increases the probability that mountaineers misinterpret the risk of high-altitude illness, which has the potential to lead to life-threatening situations. Therefore, we cannot recommend GAR for monitoring SpO2 with the aim of acclimatization management or predictive health monitoring.In conclusion, SpO


Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Enterobacter cloacae were examined. Caffeine and pentoxifylline directly interact with ciprofloxacin and tetracycline, with neighborhood association constant values of 15.8–45.6 M−1 and enthalpy change values up to −4 kJ·M−1. Caffeine, used in mixtures with tested antibiotics, enhanced their antibacterial activity in most pathogens tested. However, antagonistic effects of caffeine were also observed, but only with ciprofloxacin toward Gram-positive pathogens. Xanthines interact with aromatic antibiotics at the molecular and in vitro antibacterial activity level. Given considerable exposure to caffeine and pentoxifylline, these interactions might be relevant for the effectiveness of antibacterial pharmacotherapy, and may help to identify optimal treatment regimens in the era of multidrug resistance.Antimicrobial resistance is a major healthcare threat globally. Xanthines, including caffeine and pentoxifylline, are attractive candidates for drug repurposing, given their well-established safety and pharmacological profiles. This study aimed to analyze potential interactions between xanthines and aromatic antibiotics , and their impact on antibiotic antibacterial activity. UV-vis spectroscopy, statistical-thermodynamical modeling, and isothermal titration calorimetry were used to quantitatively evaluate xanthine-antibiotic interactions. The antibacterial profiles of xanthines, and xanthine-antibiotic mixtures, towards important human pathogens  Escherichia coli and methicillin-resistant Staphylococcus aureus represent the most frequent and deadly causes of infection [Increasing antimicrobial resistance (AMR) is a major healthcare threat globally. According to recent estimates in the European Union, AMR contributes to more than 670,000 infections and 33,000 deaths annually . Among dnfection . The burnfection . In the nfection .Most infections with antibiotic-resistant bacteria are associated with healthcare institutions . TherefoThe pipeline for new antimicrobials, especially those to treat multidrug-resistant bacteria, is narrow . RemarkaOne of the approaches to overcome drug resistance, or to reactivate the target for the antibiotic, is to use additional compounds, such as natural biologically active substances, or even existing drugs, as adjuvants of antibiotics. As evidence, amphotericin C was approved to treat visceral leishmaniasis  and doxyXanthines, including caffeine and pentoxifylline, have well-established safety and pharmacological profiles, making them attractive candidates as drug adjuvants. Caffeine, a component of popular beverages, foods, dietary supplements, and drugs, is the most abundantly consumed pharmacologically-active substance worldwide. The estimated mean daily caffeine intake is 165 mg, and approximately 105 mg is related to drinking coffee . The aveCaffeine and pentoxifylline were also shown to diminish the activity of a broad range of small aromatic compounds  interact non-covalently with caffeine and pentoxifylline. As such sequestration might affect biological activity of antibiotics captured in the hetero-complexes, we also evaluated the impact of xanthines on the in vitro activity of antibiotics toward a panel of seven Gram-positive and Gram-negative human pathogens. Given both considerable exposure to xanthines  and lack of effective treatment options for several emerging bacterial infections, knowledge on possible modulatory action of xanthines, toward antibiotics commonly used in clinical practice, appears relevant.To investigate the direct interactions between antibiotics and xanthines in aqueous solutions, we performed UV-vis spectroscopic titrations of the antibiotic solution with caffeine or pentoxifylline. All absorption spectra were analyzed at wavelengths >320 nm, for which xanthine absorption is negligible. Absorptions spectra normalized to the concentration of the absorbing ligand (antibiotic) for antibiotic-caffeine titrations are shown in In the analyzed range of concentrations, tetracycline and ciprofloxacin are present as monomers (no dimerization or higher-order aggregation was recorded). Observed spectral changes (represented most prominently by the hypochromic shift) can therefore be attributed to a new component that emerged upon the addition of xanthine solution . The presence of an isosbestic point at 372 nm for tetracycline-caffeine mixtures (and at 339.5 nm for ciprofloxacin-caffeine mixtures) indicates that only two absorbing entities are present in the mixture . Corresponding spectral changes were observed for tetracycline-pentoxifylline and ciprofloxacin-pentoxifylline mixtures, thus demonstrating ligand hetero-complexation.Once spectra of the antibiotic, complexed with xanthine, were calculated, based on the law of spectra additivity, it was possible to estimate the molar fraction of free and complexed antibiotic in each mixture during spectrophotometric titration. Examples of such two-component spectra decomposition for selected ciprofloxacin-caffeine and tetracycline-caffeine mixtures are shown in AC for antibiotic-xanthine interactions, calculated with the model, were in the range of 10 M−1  of antibiotic-xanthine hetero-aggregation, the net heat of interaction per mole of titrant added was extrapolated for antibiotic concentration → zero . However, in Gram-positive bacteria, only tetracycline antibacterial activity increased upon caffeine addition, while ciprofloxacin activity was inhibited. Meanwhile, the effects of pentoxifylline on the antibacterial potential of ciprofloxacin and tetracycline were less pronounced than those of caffeine. Meaningful potentiation of antibiotic activity by pentoxifylline was observed only for A. baumannii. Slight inhibitory effects of pentoxifylline were reported for ciprofloxacin in S. aureus and E. cloacae, as well as for tetracycline in P. aeruginosa. The MIC values for antibiotics, alone and in combination, with the highest sub-inhibitory concentration of caffeine and pentoxifylline are listed in The profile of antibiotic activity modulation is distinct for caffeine and pentoxifylline. Caffeine potentiates the antibacterial activity of both ciprofloxacin and tetracycline in all Gram-negative pathogens evaluated, with mostly additive effects, as determined by FICI values  had increased caffeine sensitivity with growth inhibition present at 2.5 mg/mL or lower [E. coli, E. cloacae, K. pneumoniae, and A. baumannii. Meanwhile, pentoxifylline was previously shown to lack antibacterial activity against E. coli with MIC values >1 mg/mL [Being the vital component of the broadly available beverages with psychostimulatory properties , caffeine is consumed on a daily basis. The single doses of xanthines, administered as a part of everyday diet (caffeine), or in pharmacotherapy (pentoxifylline), reach levels of up to 100–400 mg  , and pea cloacae . Likewispathogen . Similar7 strain . Finallyor lower . In our S. aureus or Streptococcus mutans; however, the intrinsic antibacterial activity of caffeine was weak [E. coli [E. coli and Bacillus anthracis. No possible explanation of such caffeine action was given [Prior studies showed that extracts of roasted coffee exhibited antibacterial activity against human pathogens like was weak . Yet, thwas weak . Indeed,[E. coli . The autas given . As an aas given .ACK) and enthalpy changes (ΔH) were lower than those determined for xanthines and model mutagens, heterocyclic foodborne carcinogens, or anticancer drugs  [S. aureus and E. faecium).In our study, using UV-Vis spectroscopy, combined with statistical-thermodynamical modeling and ITC, we provided evidence for non-covalent complex formation between xanthines (caffeine and pentoxifylline) and two aromatic antibiotics, ciprofloxacin and tetracycline. The neighborhood association constants ( kJ M−1) ,30,43. TE. coli [S. aureus, P. aeruginosa, K. pneumoniae, and A. baumannii [The observed reduction of ciprofloxacin activity in the presence of xanthines is in line with reports by Kang et al., who showed antagonistic activity of caffeine toward ciprofloxacin in E. coli , and by aumannii . Our finaumannii ,26,30.P. aeruginosa caffeine concentrations of >1 mg/mL, although the bacteria were resistant to caffeine alone at concentrations up to 16 mg/mL. In contrast, pentoxifylline lacked antibacterial activity at concentrations up to 16 mg/mL, which likely explains its lack of influence on antibiotic activity for most studied organisms.In contrast to Gram-positive bacteria, caffeine potentiated the antibacterial activity of both tetracycline and ciprofloxacin in Gram-negative pathogens. It seems plausible that the attenuation of ciprofloxacin by caffeine, observed in Gram-positive bacteria, even if present in Gram-negative pathogens, is surpassed by another mechanism of caffeine action, which is specific for Gram-negative bacteria. It could be speculated that heterocomplexation of an antibiotic with xanthine increases antibiotic solubility and/or membrane permeability (as xanthines penetrate into membranes easily) , which cOverall, the activity of caffeine, both alone and in combination with ciprofloxacin and tetracycline, towards Gram-negative pathogens appears promising. The elucidation of its mechanism of action is appealing, especially considering there is a shortage of effective pharmacological treatment options particularly for Gram-negative-associated infections . In addiIn conclusion, xanthines, such as caffeine and pentoxifylline, interact with aromatic antibiotics at the molecular and in vitro antibacterial activity level. Given the considerable exposure to caffeine and pentoxifylline, these interactions might be relevant for the effectiveness of antibacterial pharmacotherapy. According to our findings, caffeine potentiates the antibacterial activity of selected antibiotics in Gram-negative pathogens. Therefore, as a compound with a well-established safety profile, caffeine and its potential to enhance antibacterial action of known antibiotics may be worth further investigation when searching for optimal treatment regimens in the era of multidrug resistance.2HPO4 and NaH2PO4  was used in UV-Vis spectroscopy and ITC measurements. The buffer was filtered through a 0.2 μm pore Millex Millipore filter and degassed before experiments. Caffeine and pentoxifylline stock solutions were prepared by dissolving their weight amounts in a sodium phosphate buffer (pH 6.8) or deionized water at concentrations of approximately 10−1 M, and stored at 4 °C. Antibiotic stock solutions were prepared by dissolving their weight amounts in a sodium phosphate buffer (pH 6.8), or deionized water immediately before the experiments. The concentrations of antibiotic solutions were assessed by UV-Vis spectroscopy using determined molar absorption coefficients (ελ), ε358 = 14,900 M−1 cm−1 and ε322 = 12,380 M−1 cm−1 for tetracycline and ciprofloxacin, respectively.All chemicals, including xanthines caffeine  and pentoxifylline xanthine), and antibiotics tetracycline hydrochloride and ciprofloxacin hydrochloride, were purchased from Sigma-Aldrich . Structures of the above mentioned compounds are shown in λ, M−1 cm−1).The 2 mL aliquots containing the antibiotic were placed in a quartz cuvette (1 cm light path) and titrated with 5–150 μL of caffeine or pentoxifylline solution. Example of changes in concentrations of all mixture constituents during titration are presented in TAC/TCC → 0 . The spectra of mixtures containing the antibiotic and the xanthine were decomposed into a weighted sum of components (antibiotic in its free form and antibiotic in hetero-complexed with xanthine) by non-linear regression analysis. This allowed estimation of the concentration of free antibiotic and antibiotic complexed with xanthine for all mixtures analyzed spectroscopically.To reflect changes only in a structure of antibiotics, all the UV-Vis spectra were analyzed in the range of wavelengths above 320 nm, for which light absorption of xanthines is negligible. The spectrum of the antibiotic in complex with the xanthine was calculated by extrapolation of molar extinction coefficient (for each wavelength) to ACK) for antibiotic-caffeine and antibiotic-pentoxifylline complexation along with the concentrations of all mixture components were determined with statistical thermodynamics of mixed aggregation based on the Zdunek et al. model [C (xanthine) is capable of both homo- and hetero-complexation, and the other, A , is only capable of hetero-complexation with xanthine. To determine neighborhood ACK (hetero-neighborhood) and CCK (homo-neighborhood) equilibrium constants with the model, a weight function for each oligomer needs to be calculated. CCK equilibrium constant values were determined using constant values of caffeine homo-complexation reported previously [TAC and TCC are total concentrations of antibiotic and xanthine, respectively, AC and CC are concentrations of free antibiotic and xanthine molecules, respectively, ACC is the concentration of antibiotic-xanthine hetero-neighborhoods, while CCC is the concentration of xanthine homo-neighborhoods. The calculations were performed using SigmaPlot 11 , Microsoft Office Excel (Microsoft), and Mathcad Prime 6  software.Mixed association constant values  experiments were conducted in a 0.1 M sodium phosphate buffer (pH 6.8) at 25 °C using an AutoITC isothermal titration calorimeter , with 1.4491 mL of sample and reference cells. The cell containing deionized water was used as the reference. All solutions were degassed before titrations. The experiment consisted of injecting 10.02 μL  of buffer solution of the appropriate antibiotic (1 mM) into the reaction cell initially containing xanthine in the buffer solution (15 mM). The titrant was injected in 5 min intervals to ensure that the titration peak returns to the baseline prior to the next injection. Each injection lasted 20 s. Background titrations were run using identical titrant with the pure buffer solution placed in the sample cell. To account for the heat of dilution, the result of a background titration was subtracted from each experimental titration. To achieve a homogeneous mixing in the cell, the stirrer speed was established at 300 rpm. To remove the effect of titrant diffusion across the syringe tip during the equilibration process, an initial 2 μL injection was removed from each data set before analysis. The data, specifically the heat normalized per mole of injectant, were processed with Origin 7 software from MicroCal.positive Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 19433; Gram-negative Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC 700603, and Enterobacter cloacae ATCC 700323.Cation-adjusted Mueller-Hinton broth (CA-MHB) for antimicrobial susceptibility testing by broth microdilution method was purchased from Beckton Dickinson (BD Difco™ BBL™). Following bacterial strains recommended for antibiotics testing were used in the study: Gram-®). Minimal Inhibitory Concentration (MIC) of antibiotics and xanthines was defined as their lowest concentration at which no visible bacterial growth was observed after 24 h stationary incubation at 37 °C. The following gradients of compounds concentration, obtained by serial 2-fold dilutions of medium, were applied: from 128 to 0.015625 µg/mL for antibiotics, and from 16 to 1 mg/mL for xanthines. From thus prepared solutions, 100 µL aliquots were transferred into 96-well plates. Next, wells were inoculated with 10 µL aliquots of bacterial suspension containing approximately 1 × 107 CFU/mL obtained from liquid cultures in CA-MHB medium  diluted in fresh medium. Checkerboard titration method was used to evaluate interactions of antibiotics and xanthines by applying two-dimensional combination of their concentration gradients. For agents with antibacterial activity, i.e., when MIC value was shown, the following gradient was applied: 2 × MIC, 1 × MIC, 0.5 × MIC, 0.25 × MIC, 0.125 × MIC, 0.06 × MIC, and 0.03 × MIC. Agents without antibacterial potential (xanthines mainly) were applied starting from the highest tested concentration, i.e., 16 mg/mL, and the following concentrations were tested: 16, 8, 4, 2, 1, 0.5 and 0.25 mg/mL. Results were analyzed with two following methods: calculation of Fractional Inhibitory Concentration Index (FICI) for each tested combination (according to Odds) [Antimicrobial potential of tested agents was determined by broth microdilutions method according to CLSI guidelines  in sterito Odds) , and isoto Odds) . FICI waMICA + B—the lowest concentration of compound A in the presence of the lowest concentration of compound B at which inhibitory effect is observed,MICA—MIC of compound A tested alone,MICB + A—the lowest concentration of compound B in the presence of the lowest concentration of compound A at which inhibitory effect is observed,MICB—MIC of compound B tested alone.For xanthines without antimicrobial effect on particular bacterial pathogens (MIC >16 mg/mL), their highest used concentration, i.e., 16 mg/mL, was considered as equal to or lower than 0.5 × MIC. FICI values were used to characterize following types of interaction: (i) synergistic for FICI ≤ 0.5, (ii) additive for FICI between 0.5 and 2.0, (iii) antagonistic for FICI ≥ 4.0 . All mic


A conventional RC column specimen and two RC composite columns designed with ECC were fabricated. To investigate the cyclic behavior of each specimen, a series of cyclic loading tests was performed under a reversed cyclic loading condition with a constant axial load. Test results showed that ECC columns exhibited higher cyclic behavior in terms of load carrying capacity, ductility, and energy dissipation capacity compared to the RC column. It was also found that when applying ECC to the column specimen with a length of 3.6 As the economic growth and development of construction technology has been rapidly expanding recently, there has been an increasing demand for huge structures, such as high-rise buildings, long span bridges, and huge underground structures. Although concrete is one of the most widely used construction materials in the construction industry due to its low cost and durability, it has drawbacks, such as low tensile strength, which is approximately 10% of its compressive strength, and inherent brittle failure under tension load. An engineered cementitious composite (ECC) has been developed to overcome the inherent disadvantages of normal concrete . ECC shoFischer and Li  investigTherefore, the purpose of this study is to experimentally investigate the effect of the replacement ratio of concrete with ECC at the plastic hinge region of columns on the cyclic behavior of RC composite columns.3 and a Blain fineness of 3570 cm2/g was used. The density and Blaine fineness of fly ash  were 2.15 g/cm3 and 3590 cm2/g, respectively. The density and Blaine fineness of blast furnace slag  used as a binder were 2.89 g/cm3 and 4230 cm2/g. PVA fiber  was used as a reinforcing fiber and its physical properties are listed in ECC and normal concrete were used as a matrix material in this study. Type I ordinary Portland cement  with a density of 3.15 g/cmThe mixture proportions of ECC and concrete used in this study are listed in 3.d6 to ECC5.d4) were manufactured. The replacement length (RL) of concrete with ECC for each specimen is listed in d from the column-base joint at the lateral displacement of 4.3 mm. Additional flexural cracks appeared near the column-base joint, as the cyclic load increased. The width of the initial flexural crack increased and flexural cracks occurred near the column-base joint when the lateral displacement was larger than 26.1 mm. As the lateral displacement level increased, several macro cracks occurred up to the top of the column by enlarging micro cracks. At the lateral displacement of 34.8 mm, a maximum lateral loading state of the RC specimen was observed and the crack width at the column-base joint increased. As the cyclic loading level increased, shear cracks occurred in the cover concrete of the column near the zone of the column-base joint. A rapid reduction of the load bearing capacity of the joint also occurred. Until the lateral displacement reached 52.2 mm, the spalling of the cover concrete and buckling of the longitudinal reinforcing bars were observed in the plastic hinge region within 0.84d (210 mm) from the column-base joint. The plastic hinge length was calculated using the formula suggested in the previous study [us study .3.d6 specimen, an initial micro flexural crack was observed near the bottom column at the lateral displacement level of 4.3 mm. As the lateral displacement increased, the number of micro cracks gradually increased and reached a maximum load at the lateral displacement of 40.6 mm after flexural cracking in the column-base joint. In comparison to the RC specimen, no macro cracks were observed at the column until the maximum load was reached. At the lateral displacement of 61.2 mm, the ECC3.d6 specimen finally failed, with multiple micro cracks near the height of 3.6d from the column-base joint. Unlike the RC specimen, spalling of the cover concrete and the buckling of longitudinal reinforcing bars near the plastic hinge zone was not observed. From the cracking pattern observation, it was found that the replacement of ECC effectively prevented the plastic hinge damage generated in the RC specimen under cyclic loads. The ECC5.d4 specimens showed similar fracture behavior to the ECC3.d6. As the ECC replacement length increased, the number of micro cracks increased and the crack spacing decreased. Both ECC3.d6 and ECC5.d4 specimens exhibited effective prevention of shear cracks, spalling of the cover concrete, and the buckling of the longitudinal reinforcing bars, compared to the RC specimen.For the ECC3.d6 and ECC5.d4 specimens were increased by 1.1 kN and 1.7 kN, respectively, compared to the RC specimen. This indicates that the increase of initial cracking load was insignificant with an increase in the RL. As the lateral displacement increased, the lateral force of ECC specimens increased. The longitudinal reinforcement of the column-base joint yielded at the range of load lever from 25.7 to 28.7 kN, which were higher than that of the RC specimen by 8.9% and 21.6%, respectively. As the RL increases, the lateral displacement measured at the top of the column also increased. The maximum load values of ECC3.d6 and ECC5.d4 specimens were increased by 9.3% and 21.4% higher than that of the RC specimen. The lateral displacement at maximum load state also increased by 23.0% to 31.8% compared to the RC specimen. This indicates increased ductility of the columns through the use of ECC. The ultimate displacement (RL of concrete with ECC. Therefore, it was found that the seismic performance of the column-base joint increased as the RL of concrete with ECC increased.On the other hand, the initial cracking load values of the ECC3.d6, and ECC5.d4 specimens were 2.88, 3.44 and 3.40, respectively. Compared to the RC specimen, the ECC series showed an improved ductility ratio by minimizing the spalling of the cover concrete and the buckling of the longitudinal reinforcement. Although the ultimate displacement of the ECC specimens increased with an increase in the RL, all ECC specimens exhibited a similar ductility ratio. This means that the effect of RL on the ductility ratio is not significant. The increase of the ductility ratio of ECC specimens is mainly due to tightly controlled bending and shear cracks in the plastic hinge zone through the strain hardening behavior and multiple micro cracks of ECC. This resulted in an increase in the displacement of ECC specimens at the yielding.The ductility of the specimens can be evaluated by calculating the ductility ratio, which is the ratio of ultimate displacement (3.d6 and ECC5.d4 were 41.7% and 101.3% higher than that of the RC specimen, respectively.The energy dissipation capacity of structures under cyclic loading conditions is an important factor for evaluating the seismic behavior of structural members. The energy dissipation of all specimens is shown in d or more. From the test results, it was found that the energy dissipation capacity and the strengthening effect were enhanced as RL increased. In the construction field, however, economic efficiency and required strengthening level should be considered when we select a suitable RL.d from the column-base joint, and buckling of the longitudinal reinforcing bars. In comparison to the RC specimen, ECC specimens exhibited effective prevention of shear cracks and spalling of the cover concrete, and the buckling of longitudinal reinforcement was not serious. Excellent tensile properties of the ECC, such as strain hardening behavior and multiple micro cracks, led to a minimization of spalling of the cover concrete by controlling flexural and shear cracks in the plastic hinge zone of the column.In the RC specimen, as the lateral displacement increased after the maximum load, the load decreased drastically. Local damage was also observed, such as spalling of the cover concrete near the plastic hinge on the length of 1.03.d6, and ECC5.d4 specimens were 2.88, 3.44 and 3.40, respectively. Although the ultimate displacement of the ECC specimens increased with an increase in the LR, all ECC specimens exhibited a similar ductility ratio. This means that the effect of LR on the ductility ratio is not significant. The increase of the ductility ratio of ECC specimens is mainly due to tightly controlled bending and shear cracks in the plastic hinge zone. This resulted in an increase of displacement of ECC specimens at the yielding.In the case of the ECC specimens, the ductility ratio of the RC, ECCd at the plastic hinge region of an ordinary RC column.The energy dissipation capacity was improved up to 101.3% higher than that of the RC specimen. Based the results of this experiment, it is recommended that the replacement length of concrete with ECC from the column-base joint is considered to be more than 3.6In this study, the effects of the replacement length of concrete with ECC were experimentally evaluated under reversed cyclic loading conditions. An RC specimen and two types of ECC specimens with different replacement lengths of concrete with ECC were fabricated and a series of cyclic load tests was carried out. From the test results, the following conclusions were drawn:


The emerging threat of antibiotic resistance is growing exponentially and antibiotic stewardship programs are cornerstone to fight against this global threat. The study aimed to explore the knowledge, perspectives and practices of physicians regarding various aspects of antibiotic stewardship program including antibiotic stewardship activities, rational use of antibiotics, antibiotic resistance, prescribing practices and factors associated with these practices.In this qualitative study, a total of 17 semi-structured, in-depth interviews with doctors of three tertiary care public sector hospitals in Bahawalpur and Rahim Yar Khan were conducted. The convenient sampling method was adopted to collect the data and the saturation point criterion was applied to determine the sample size. Thematic analysis approach was used to draw conclusions from the data.The analysis of data yielded five themes, 12 subthemes and 26 categories. The themes included, (i) perception about antibiotic use and antibiotic stewardship, (ii) antibiotic prescription practices, (iii) antibiotic resistance, (iv) limited strategies adopted by hospital administration to ensure quality and safe distribution of antibiotics, (v) implementation of antibiotic stewardship program: barriers, suggestion and future benefits. Doctors had misconceptions about the rational use of antibiotics. The perception regarding antibiotic stewardship programs was poor. Moreover, very few activities related to ASP existed. The participants gave many suggestions for successful implementation of ASP in order to reduce the burden of antibiotic resistance, including development of guidelines for the use of antibiotics, strict legislation regarding use of antibiotics, active participation of healthcare professionals and awareness program among general public about the use of antibiotics.This study concluded that poor knowledge of doctors regarding ASP, non-existence of antibiogram of hospital and lack of rules for the safe use of antibiotics were the main driving factors associated with irrational antibiotic prescription practices and development of AR.The online version contains supplementary material available at 10.1186/s12879-021-06043-5. Antibiotics are lifesaving drugs, and more than half of the hospitalized patients receive antibiotics , 2. AccoOver the past few years, unnecessarily overuse of antibiotics has led to the emergence of multidrug resistant (MDR) bacterial strains making them ineffective –7. AccorThe main factors increasing rates of MDR infections in Pakistan include the unwarranted use of broad spectrum antibiotics as well as insufficient infection prevention and control (IPC) and antibiotic stewardship polices , 20–22. In Pakistan, healthcare in the country is regulated by the Ministry of National Health Services Regulation and Coordination and the healthcare delivery system is comprised of primary, secondary and tertiary care . PrimaryThis study was conducted in two cities of the Punjab province of Pakistan i.e., Bahawalpur and Rahim Yar Khan. The Bahawal Victoria Hospital (BVH), Bahawalpur (1600 plus bedded facility) and the Civil Hospital, Bahawalpur (400 bedded facility) are two tertiary care hospitals located in the district of Bahawalpur , 35, andA qualitative study design  was adopThe interview schema was developed after comprehensive literature review to answer the research questions . The duration of interviews ranged from 20 to 30 min with an average duration of 25.5 min (SD = 3.83). The participants had minimum experience of two years and maximum experience of 10 years. Eleven participants were male and six were female. Characteristics of the participants are given in Table The thematic analysis of the data yielded five themes and 12 subthemes. The themes included: perception about antibiotic use and ASP, antibiotic prescription practices, AR, limited strategies adopted by hospital administration to ensure quality and safe distribution of antibiotics, and implementation of ASP: barriers, suggestion and future benefits.To understand the familiarity of doctors about the terms related to antibiotic use, they were asked about how they perceive the term rational use. Most of the study participants had inadequate knowledge about rational use of antibiotics (12 out of 17 respondents) as they had misconception that use of broad spectrum antibiotics is called empirical therapy and that it was rational use. All respondents (17 out of 17 respondents) agreed that irrational use of antibiotics was a problem in Pakistan. ASP was the term that most of the doctors were not familiar with. Doctors who were familiar with the term were those who had work experience at large private sector hospitals in Karachi and Lahore (3 out of 17 respondents) (Table Broad spectrum antibiotics were considered as better treatment option by the doctors (8 out of 17 respondents). Moreover, broad spectrum antibiotics were prescribed as prophylactic agents to patients for nosocomial infections. Patient’s poor financial status and limited resources in public sector hospitals were highlighted by nine doctors as main reasons why culture tests were not performed. Unreliable laboratory reports were highlighted (2 out of 17 respondents) as a reason for not sending specimens for cultures Table .Table 3Most of the respondents (15 out of 17) stated that situation of AR is alarming in Pakistan. The doctors highlighted frequent use of antibiotics for minor ailments (9 out of 17 respondents) and as one of the main reasons of AR. Six out of 17 respondents stated that poor compliance and improper dosing contribute to already existing problem of antibiotic resistance. Lack of doctors’ experience (2 out of 17) and quacks working as doctors in remote areas of Pakistan (8 out of 17 respondents) were also reported as contributing factors that aggravated the problem of resistance. Four out of 17 doctors described that antimicrobial resistance leads to increase in out-of-pocket expenses of patients Table .Table 4Table Most of the respondents agreed that there is no proper system to ensure the safe use of antibiotics (15 out of 17 respondents). They agreed that, although, policies and rules exist to ensure the quality and safe distribution of antibiotics but they are usually not followed. However, some study participants pointed out different strategies that were adopted to ensure safe use of antibiotics. Two out of 17 respondents stated that if a medicine was showing some adverse drug event, it was reported to hospital pharmacist. One respondent stated that pharmaceutical products containing antibiotics were sent to drug testing laboratory to check whether these meet quality standards. One respondent stated that one positive strategy to ensure rational use of antibiotics is to prescribe antibiotics from hospital essential list Table .Table 6Multiple reasons were highlighted by the respondents that were considered as barriers for successful implementation of antibiotic stewardship programme. Fifteen out of 17 respondents stated that no educational programme or awareness campaign existed to educate doctors about antibiotic stewardship. Lack of hospital antibiogram was considered as a major barrier which hindered the implementation of antibiotic stewardship program (10 out of 17 respondents). The respondents suggested that the government support and strict legislation regarding use and dispensing of antibiotics was important to ensure safe use of antibiotics (4 out of 17 respondents). More than half (9 out of 17 respondents) of the respondents stated that general public should be made aware to avoid self-medication of antibiotics, and they must also avoid visiting quacks (Table Antimicrobial resistance is an issue that has been increasing globally with an alarming rate making it a huge concern . Many inFindings of this study revealed that the doctors used to prescribe broad spectrum antibiotics as an empiric therapy. Our participants also agreed that habitual empiric prescribing practices lead to inappropriate use of antibiotics which subsequently lead to AR. These results were similar to studies conducted in Cambodia and Iran that described the similar kind of behavior , 52. EarThe present study also revealed that the doctors used to prescribe antibiotics in almost all hospitalized patients as a prophylactic measure to counter the nosocomial infections. Similarly, a study from India also revealed that doctors prescribed antibiotics in hospitalized patients to avoid superinfections . FindingA multicounty study conducted in European countries revealed that antibiotic guidelines help doctors in rational prescribing of antibiotics . HoweverThis study revealed that doctors had no idea about ASP, and it was relatively a new word for them. These findings were similar to an Indian study , while aThis study has few limitations as well. First, a convenient sampling method was used to collect data. In convenience samples, subjects who are more easily accessible to the researcher are more likely to be included and, therefore, not all qualified individuals in the target population have the same opportunity to participate and study results are not much reliable and necessarily generalizable to this population. Though, increasing the sample size in quantitative studies can improve the credibility of convenience samples, but this is difficult in qualitative studies because saturation point criterion is used as redundancy signal. However, considering aforementioned concerns, a mix of purposive and convenience sampling was adopted and we believe that the utilization of purposive sampling in the first phase surpassed any possible concerns associated with convenient sampling and saturation point criteria altogether. Second, our findings cannot be generalized to the whole of Pakistan as these depict the views of doctors working in two main cities of southern Punjab. However, the authors do not expect significant differences across the country, as the same healthcare system has been implemented and regulated throughout the country by the Ministry of National Health Services, Regulation and Coordination. Though this study provides an indication of what is happening in the whole province of Punjab, but there is a need for large scale multi-setting quantitative studies to underline more reliable and impactful country-level situation. Likewise, multi-perspective studies involving regulators, pharmacists, nurses and dispensers are encouraged to improve the antibiotic stewardship activities at each level of healthcare through evidence based recommendations.The strength of the present study is that it highlighted an important issue about which limited data is available from developing countries, for example, Pakistan. In terms of policy, our study findings warrant the need to put legislation to introduce the ASP in hospital settings in Pakistan. Moreover, National Action Plan of Pakistan on AR must be implemented to prohibit irrational use of antibiotics and to implement ASP. In practice terms, there is a dire need regarding training and involvement of doctors in ASP to minimize the surge in the antimicrobial resistance issue. Frequent seminars must be conducted on small and large scale to educate doctors about the benefits of implementing ASP, and warn them to prescribe antibiotics cautiously because of the fact that our study participants imperfectly understood the concept of empirical therapy and ASP. Yet education alone will not be enough, antibiotic prescribing guidelines and local antibiogram must be available in each hospital. In addition, collaborative efforts by all the healthcare professionals dealing with human as well as animal patients are needed to adopt “One Health Approach” with the aim to halt the spread of antibiotic resistance at each level. Future large scale interventional studies involving doctors, pharmacists, nurses, veterinary and agricultural experts and health policy makers are also required to assess the impact of ASP on health outcomes and to understand the potential barriers and facilitators for implementation of program.The study findings will not only aid countries with existing ASPs in forthcoming initiatives, but also those countries that are planning to implement ASPs can gain insight for effective introduction of programme. Specifically, the findings of this study highlight that countries with existing ASPs should focus on awareness and education of healthcare professionals about ASPs protocols, strict compliance with the antibiotic prescribing guidelines, active participation of healthcare professionals, and availability of local antibiogram in each healthcare facility. Whereas, the countries planning to implement ASPs can learn that special attention to the above mentioned aspects is mandatory for an effective introduction of activities. Moreover, the study findings will act as a strong base and impetus for both large scale multi-stakeholder qualitative studies and quantitative studies.The study concluded that the doctors working in the public sector hospitals of Pakistan had inadequate knowledge about ASP. There was no proper system in place to ensure the appropriate use of antibiotics. Similarly, local antibiogram data and antibiotic prescribing guidelines were not available in the hospital. There is dire need to implement the strategic objectives of national action plan of Pakistan on AMR in order to mitigate the adverse outcomes of AR in future.Additional file 1. Interview schema


Immunoglobulin G4 (IgG4)-related disorders are characterized by tissue hypertrophy due to IgG4-positive cell infiltration and increased serum IgG4 levels. IgG4-related hypophysitis (IgG4-RH) is characterized by pituitary hypertrophy, IgG4-positive cell infiltration, central diabetes insipidus, and increased serum IgG4 levels. IgG4-RH is diagnosed through diagnostic criteria. A few cases of IgG4-RH in children have been reported. We report a case of CDI with increased serum IgG4 levels that failed to meet the diagnostic criteria of IgG4-RH. The patient developed polyuria and polydipsia at age 11 and was diagnosed as having idiopathic CDI at age 12. The patient was not treated with steroids and is well controlled with antidiuretic hormones. It has been reported that pediatric IgG4-RH differs from that of adults in several respects. We believe that the pediatric IgG4-RH may not fit the diagnostic criteria of adults. There may be also other cases of increased serum IgG4 levels in pediatric patients with idiopathic CDI. We do not know if they are subtypes of IgG4-RH or if serum IgG4 levels are by chance raised in CDI, however, we report them here because IgG4-RH in children may be different from that in adults. Immunoglobulin G4 (IgG4)-related disorders are characterized by enlargement and thickening of systemic organs , high serum IgG4 levels, marked infiltration of IgG4-positive plasma cells, and fibrosis on histopathology , 2. HypoAlthough IgG4-RH may be infrequent, the symptoms show that central diabetes insipidus (CDI) and IgG4-RH generally appear in middle-aged and elderly males and young females , with liCDI is characterized by an insufficient secretion for AVP production from the hypothalamus-neurohypophyseal system after adequate stimulation . There aWe do not know if they are subtypes of IgG4-RH or if IgG4 was accidentally raised in CDI, however, we report them here because IgG4-RH in children may be different from that in adults.IgG4-RH can be diagnosed using established diagnostic criteria formulated by Takagi et al.  Table 11 and by The patient was a 12-year-old Japanese male born at 37 weeks of gestation from a non-consanguineous marriage, with a birth weight of 2776 g and birth height of 47.3 cm. No abnormalities had been noted during his perinatal period. No atopic dermatitis, or immune/collagen diseases, as well as immune/collagen diseases in the family history, were noted. At 11 years of age, the patient developed polydipsia and polyuria, with increased water intake and nocturia consequently being noted, however, the patient had no fever, vomiting, constipation, or growth failure. Subsequently, the patient underwent a medical examination by a physician, who suspected diabetes insipidus. As such, the patient was referred to our department for medical treatment at 12 years old.No concomitant symptoms, including headache or visual field disturbances associated with pituitary lesions, were observed. Blood tests revealed the following results: sodium, 143 mmol/L; plasma osmolality, 282 mOsm/kg; arginine vasopressin (AVP), 0.7 pg/mL; urine osmolality, 154 mOsm/kg; urine specific gravity, 1.004; and blood glucose, 91 mg/dL. The hypertonic saline infusion test showed elevated sodium (151 mmol/L) and plasma osmolality (299 mOsm/kg) with no change in AVP(0.9 pg/mL), urine osmolality (154 mOsm/kg), and urine specific gravity (1.004). Moreover, the pitressin tolerance test showed the following results: plasma osmolality, 299 mOsm/kg; urine osmolality, 591 mOsm/kg; and urine specific gravity, 1.014. Based on these results, a diagnosis of central diabetes insipidus (CDI) was established.3). All anterior pituitary hormone function tests are summarized in Table 3).Contrast-enhanced magnetic resonance imaging (MRI) of the head did not reveal a mass-like shadow extending from the pituitary stalk to the pituitary gland Figures -1B. The We herein report a case of a 12-year-old male patient with CDI and increased serum IgG4 levels. Symptoms indicating anterior pituitary dysfunctions  or organ inflammation had not been observed. Although Leporati’s diagnostic criteria for IgG4-RH have been utilized worldwide . ConsideOne study showed that CDI was the most frequent endocrinological dysfunction in adults with IgG4-RH (70.2%) . HoweverGenerally, good response rates have been noted with glucocorticoid treatment for IgG4-RH (97.2% in adults) . AlternaOur patients have had no exacerbation of symptoms or complications despite not undergoing steroid therapy. In near future, it is necessary to study more cases of pediatrics CDI to determine the number of cases as well as the symptoms of increased serum IgG4 levels. The patient’s weight had decreased since the start of the AVP treatment for CDI. We consider the possibility that AVP treatment reduced stress by improving polydipsia and polyuria, especially at night, and ensuring good sleep quality.We report a case of pediatric CDI only with increased serum IgG4 levels that did not fulfill the diagnostic criteria for IgG4-RH. Although IgG4-RH in children is very rare, there may be cases of increased serum IgG4 levels in children with CDI only. Although the diagnostic criteria for IgG4-RH were not fulfilled in this case, careful follow-up to check for the appearance of IgG4-RD may be necessary in the future.


We observed that compared to control animals, rats exposed to LBN conditions during postnatal days 2–9 showed increased breakpoints for heroin self-administration under a progressive ratio schedule of reinforcement, impaired extinction of heroin-seeking behavior, and increased reinstatement of heroin-seeking behavior induced by heroin-associated cues. No effect of LBN rearing conditions were observed on the acquisition and maintenance of heroin self-administration, and no sex differences in heroin intake were observed. LBN and control reared animals showed no differences in the excitability of AIC-NAc pyramidal neurons, but animals treated with repeated heroin showed decreased excitability of these neurons through a significant increase in rheobase and reduction in action potentials induced by depolarizing currents. Together, these results suggest that ELS exposure produces exacerbations of heroin seeking behavior without parallel effects on AIC-NAc excitability, although heroin itself reduces the excitability of these neurons.A number of retrospective studies have demonstrated adverse childhood experiences are associated with increased vulnerability to substance use disorders, including opioid use disorders (OUDs). These adverse childhood experiences, also referred to as early life stress (ELS), can be modeled in laboratory animals by various paradigms including limited bedding and nesting (LBN) procedures. Studies using rodent models of ELS have been shown to recapitulate various aspects of OUDs, including relapse propensity and perseverance of drug-seeking behavior. In the current study, we utilized the LBN paradigm to explore potential effects on heroin self-administration, extinction, and relapse-like behaviors in male and female rats. We also utilized  Opiate abuse and addiction pose a substantial financial economic burden, exceeding over one trillion dollars in the United States in 2017 . The epiIn humans, ELS includes a myriad of experiences including physical, emotional or sexual abuse, neglect, and instability of maternal care. Such events have been shown to increase risk of developing impairments in coping skills, affective processing, and impulse control, all of which are characteristics of individuals with OUDs . Such maThe insular cortex shares reciprocal connections with various brain regions including limbic regions associated with reward, such as the nucleus accumbens (NAc). Functional neuroanatomical studies have demonstrated that the anterior insular cortex (AIC) mediates a number of behaviors including cognitive, emotional, integrative and social functions, including attention, decision-making, and incentive and risk assessment. In rodents, lesions of the AIC or pharmacological modulations of this region alter cocaine intake and cocaine-seeking behaviors , conditiIn the current study we aimed to explore how ELS, as modeled through the LBN paradigm, alters heroin seeking through intravenous self-administration (IVSA), extinction of heroin-seeking, as well as reinstatement elicited by exposure to heroin-paired cues. Further, we also explored the effects of LBN on the single-cell physiology and excitability of AIC neurons projecting to the NAc. Additionally, we examined the effects of experimenter delivered heroin to LBN reared and control animals to determine if heroin further altered single cell physiology and excitability of these neurons, as such alterations may give rise to behavioral alterations such as heroin seeking and relapse.ad libitum for the duration of the study. All experiments conducted were approved by the Institutional Animal Care and Use Committee at Arizona State University.A total of 39 Long-Evans rats were used in the current study. Four pregnant dams were ordered at gestation day 10 from Charles River Labs . All dams were kept in a temperature-controlled vivarium (22–24°C) at Arizona State University on a standard reverse light-dark cycle (12 hr light/12 hr dark). Rats were single housed in conventional polycarbonate cages until pups were weaned. All animals were given standard rodent chow and water via anogenital distance. To minimize potential genetic influences, pups from all litters were randomly assigned to one of two conditions (LBN or control), and sex was kept at a 1:1 ratio. To minimize potential litter influences, dams assigned to the LBN group were placed with their assigned pups in a standard cage containing a wire mesh floor designed to maintained animals ∼2.5 cm above the cage floor, which contained a thin layer of standard bedding. However, due to some pups becoming entrapped in the mesh during the 1st day of LBN procedures (PND 2), wire meshes were removed from the cages and dams assigned to the LBN condition were provided only with ∼100 ml of standard bedding and a single folded paper towel to use as nesting material  were anesthetized with isoflurane  vaporized in oxygen at a flow rate of 2 L/min and implanted with polyurethane catheters  2–2.5 cm into the right jugular vein and were tunneled subcutaneously to the posterior side of the animal and connected to an indwelling back port (Instech). Dental cement was used to adhere the catheter to the indwelling port, and the port was sutured ∼2 cm caudal from the shoulder blades using 4–0 vicryl braided sutures. Catheters were flushed daily with heparin (70 U/ml) containing timentin (66.6 mg/ml) during recovery to maintain patency and minimize postoperative infections. Meloxicam  was administered immediately following surgery and once daily for the first 3 days of recovery to minimize discomfort. Throughout the remainder of the self-administration phase of the experiment, heparin (10 U/ml) was administered daily. Animals used in Experiment 2  were anesthetized as described above underwent stereotaxic injections of the retrograde serotype AAV expressing EGFP  into the nucleus accumbens core (NAc) at a volume of 300—400 nl and at the following coordinates in mm from bregma and skull surface: A/P: + 2.5; M/L ± 1.6; DV: −6.1. The AAV was delivered over a 5 min period and the injector was left in place for an additional 10 min to allow for diffusion. The wound was then sutured closed and triple antibiotic ointment was applied to the suture site. Meloxicam (1 mg/kg s.c.) was administered immediately following surgery and for 6 consecutive days during recovery.All animals used in Experiment 1 (via nosepoke on an FR1 reinforcement schedule in 3 hr daily sessions. A stimulus light and audible cues (2900 Hz tone and the sound of the syringe pump motor) were presented during each heroin infusion. After 15 days of self-administration, rats were subjected to progressive ratio (PR) testing where an exponential increase in the number of active nosepokes  was required to obtain each successive infusion. PR sessions were conducted in 3 hr daily sessions across for three consecutive days, and resulting data were averaged across days for analysis. Animals were then allowed to re-establish baseline IVSA patterns on an FR1 schedule for 2 days. Next, animals underwent extinction procedures where active nosepokes no longer produced heroin infusions or cue presentations. After reaching extinction criteria  animals were placed in their home cages and left undisturbed for 2 days prior to cue-induced reinstatement of heroin-seeking. For reinstatement sessions, previously paired heroin-associated cues  were presented upon active nosepokes. On the day following cue reinstatement testing procedures, active nosepoke responding was again extinguished using the same procedures and criteria above. A timeline of these procedures is outlined in All self-administration procedures were conducted in Med Associates operant conditioning chambers as described elsewhere . Animals2, rapidly decapitated and brains were extracted. Brains were placed into ice-cold carbogen (95% O2/CO2) saturated cutting solution containing the following: 120 mmol/l NaCl, 25 mmol/l NaHCO3, 10 mmol/l dextrose, 3.3 mmol/l KCl, 1.23 mmol/l NaH2PO4, 1.8 mmol/l CaCl2, and 2.4 mmol/l MgCl2, osmolarity adjusted to 295 ± 5 mOsm and pH adjusted to 7.40 ± 0.03. Coronal slices (300 μm) containing the insular cortex were then prepared in ice-cold cutting buffer using a vibrating tissue slicer . Slices were then transferred to a holding chamber filled with recording artificial cerebrospinal fluid (aCSF) containing in the following: 120 mmol/l NaCl, 25 mmol/l NaHCO3, 3.3 mmol/l KCl, 1.23 mmol/l NaH2PO4, 0.9 mmol/l CaCl2, 2.0 mmol/l MgCl2, and 10 mmol/l dextrose, osmolarity adjusted to 295 ± 5 mOsm and pH adjusted to 7.40 ± 0.03. In the holding chamber, the recording solution was continuously saturated with carbogen and incubated at 34°C for 45 min, then allowed to cool at room temperature before being transferred to the recording chamber. Once transferred to the recording chamber, slices were perfused continuously with carbogen saturated recording solution. Pyramidal cells within the insular cortex expressing EGFP were visualized using an Olympus BX51WI microscope with infrared DIC microscopy and equipped with a blue collimated LED (ThorLabs). Whole-cell recordings were made from the soma of pyramidal neurons expressing EGFP using recording pipettes (7–15 mΩ) made from thin-walled capillary tubes containing an intracellular solution containing the following: 135 mmol/l K-gluconate, 12 mmol/l NaCl, 1 mmol/l K-EGTA, 10 mmol/l HEPES, 2 mmol/l Mg-ATP, and 0.38 mmol/l tris-GTP. Osmolarity was adjusted to 285 ± 5 mOsm and pH adjusted to 7.30 ± 0.01. Upon establishing a giga-seal, the cell membrane was ruptured and cells were allowed to equilibrate for 5 min until a steady state resting membrane potential was reached. In voltage clamp mode, a 10 mV square pulse was delivered via the recording pipette and 100 capacitive transients were recorded and averaged to determine membrane capacitance/resistance and series resistance. Recordings were then switched to current clamp mode, where rheobase and input-output (I-O) curves were assessed to determine the amount of injection current required to elicit action potential spiking. A total of 15 depolarizing current pulses  were used. Action potential (AP) spike threshold, amplitude, rise time, and decay time were also measured, as were after-hyperpolarization (AHP) amplitude and duration. Each of these protocols have been described in detail in our prior studies (n = 17–18 cells/group).Prior to whole-cell electrophysiology, animals used for Experiment 2 were randomly assigned to receive once daily intraperitoneal (i.p.) injections of either saline or heroin (2 mg/kg) for five consecutive days. This dosage of heroin was selected given its ability to produce conditioned place preference (CPP) in rodents . One hou studies , 2021. Spost hoc t-tests were conducted where appropriate and p-values <0.05 were considered statistically significant. The number of post hoc comparisons for all two-way ANOVAs was six. Additionally, unpaired t-tests were used to assess changes in rheobase, action potential amplitude, action potential rise time and action potential decay time between treatment groups . Lastly, a two-way ANOVA with treatment  and input current amplitude as factors was used to assess changes in excitability. Bonferroni’s post hoc comparisons were utilized to examine changes in the number of action potentials elicited at each current input. Statistical tests were performed in GraphPad Prism 8.0, and p < 0.05 was considered statistically significant. Values are represented as mean ± standard error of the mean (SEM).Self-administration data were analyzed utilizing a two-way ANOVA with session and rearing condition (LBN and control) as considered factors. Extinction and re-extinction data were analyzed using a survival analysis, where LBN and control reared animals were compared. Cue-induced reinstatement data were analyzed utilizing a two-way ANOVA with session (extinction and cue-induced reinstatement) and rearing condition (LBN and control) as considered factors. Electrophysiological data were analyzed using a two-way ANOVA, where group (LBN and control) and treatment  were considered factors. Bonferroni-corrected F16,336 = 5.4; p < 0.0001) but not rearing condition  and no interaction was observed . A Bonferroni’s post hoc analysis revealed no differences between rearing condition at any session , rearing condition  or interaction  in the total number of infusions earned across self-administration sessions 1–15. An unpaired t-test revealed that LBN and control animals did not differ in the total number of infusions earned across self-administration sessions 1–15 , rearing condition  as well as a significant interaction   . FollowiF1,61 = 4.3; p < 0.05) but not treatment  or interaction  between drug and treatment on cellular capacitance. A post hoc Bonferroni comparison revealed no significant differences between groups , treatment  as well as a significant interaction between these two factors . A Bonferroni’s post hoc comparison revealed a significant difference in the number of spikes elicited between saline and heroin treated animals at current inputs at and above 100pA. Heroin significantly decreased the number of action potentials elicited relative to saline, with depolarizing currents of 100 pA , 115 pA , 130 pA , 145 pA , 160 pA , and 175 pA . These results are shown in First, we examined heroin-induced changes in rheobase in AIC-NAc projection neurons. An unpaired In the present study, we explored the effects of LBN rearing conditions on heroin self-administration, progressive ratio responding, extinction and cue-induced reinstatement. Additionally, we examined the effects of repeated heroin or saline exposure on AIC-NAc projecting pyramidal cell electrophysiology and excitability in LBN and control reared animals. Here we report LBN has no significant effect, relative to control rearing, on the acquisition of heroin self-administration in either male or female animals. Due to the lack of sex differences in acquisition, data from female and males were collapsed within each group for subsequent analyses. When placed into a progressive ratio schedule of reinforcement, LBN reared animals earned significantly more infusions than control reared animals. LBN reared animals also showed impaired extinction learning relative to controls both before and after reinstatement testing, and LBN reared animals also showed potentiated cue-induced reinstatement of heroin-seeking. These observations are highly consistent with a recently published study that demonstrated LBN rearing conditions did not alter acquisition of heroin self-administration, but did impair extinction learning and potentiate cue-induced reinstatement . Taken tFurthermore, we demonstrate that LBN has no effect on baseline electrophysiological properties or excitability of AIC-NAc projecting pyramidal cells, which have recently been implicated in opioid reward and relapse . HoweverHeroin users have been found to experience higher levels of early life stress including childhood trauma such as physical punishment, physical neglect and penetrative sexual abuse . ExposurPrevious studies have shown that insular structure and function are adversely affected by both opiates and ELS. In fact, a meta-analysis of 12 neuroimaging studies of opiate-dependent individuals has recently shown evidence for gray matter atrophy within the insular cortex, accompanied by abnormal functional connectivity, resting state activity as well as white matter composition . SimilarIn the current study, we did not examine electrophysiological changes in AIC-NAc pyramidal neurons following heroin self-administration, extinction or reinstatement. Given that experimenter delivered and IVSA can have different effects on cellular excitability, it is possible that AIC-NAc pyramidal neurons display differences following each of these phases of IVSA as compared to experimenter delivered used in the current study. While there are limited studies addressing the differences in AIC-NAc pathway following contingent versus non-contingent heroin delivery, studies assessing effects of heroin on other pathways have reported some physiological differences. For example, non-contingent heroin delivery was found to reduce GABAergic neuronal firing within the ventral tegmental area (VTA), whereas heroin self-administration (contingent) initially results in a burst of GABAergic activity during nose-poke/approach activity, which was followed by a dramatic reduction in firing, below pre-approach levels . As wellIn the current study, we did not observe differences between AIC-NAc projection neurons recorded from heroin-treated LBN and control reared animals. This finding suggests that heroin significantly reduces the excitability of this pathway, regardless of LBN exposure. However, these lack of effects are possibly due to the short duration  of heroin exposure. Given that LBN vs. control animals showed no differences in heroin IVSA in Experiment 1, it is not surprising that experimenter delivered heroin did not produce any differences in AIC-NAc excitability between ELS and control reared animals. It is likely that longer exposure (15 + days as used in the IVSA paradigm) to heroin as well as withdrawal would reveal differences in AIC-NAc projection neuron excitability between ELS and control reared animals. To fully explore this hypothesis, future studies exploring the effects long-term heroin exposure as well as heroin withdrawal on this pathway are necessary. Additionally, these studies would lend insight into potential neuronal underpinnings that lead to hindered extinction to heroin-paired cues as well asvia functional connectivity analyses in heroin-dependent patients that degree centrality values for the insula and NAc were negatively correlated with relapse frequency (In the current study, we demonstrated that LBN during early postnatal development increases the vulnerability toward heroin seeking behavior later in life, as manifested by impaired extinction learning and increased reinstatement magnitude in response to heroin-paired cues. To our knowledge, we have also demonstrated for the first time that LBN increases demand for heroin as assessed by progressive ratio procedures. Furthermore, we demonstrate that repeated non-contingent exposure to heroin has inhibitory effects on the excitability AIC-NAc projection neurons, which could significantly alter connectivity between these two regions and promote inappropriate plasticity within the accumbens. In agreement with this, it was recently demonstrated requency . 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 Institutional Animal Care and Use Committee, Arizona State University.JL-J performed the electrophysiology experiments and analyses and wrote the original draft of the manuscript. PO and EN performed surgical procedures, conducted the behavioral experiments, and edited the manuscript. MFO conceptualized the study, performed surgical procedures, conducted analyses, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.