Introduction: The accelerating threat of multidrug-resistant bacteria (MRB) forces health care providers to use antibiotics more rationally. Antibiotic stewardship programs (ASP) are a proven and safe way to achieve that goal. They have been comprehensively studied in adults but data from secondary care pediatric hospitals are lacking.Material and Methods: In our study an ASP with standard operating procedures (SOPs), audits, a weekly ward round with experts in pediatric infectious diseases and an antibiotic pocket-card for selected infectious diseases was established in July 2017 in a Munich municipal secondary care children's hospital. All antibiotic prescriptions on general pediatric wards were reviewed each in the first quarter of 2017 and 2018. The primary outcome was adherence to treatment guidelines. Secondary outcomes were substance consumption, duration of therapy and death.Results: After the ASP was implemented guideline adherence increased significantly from 33 to 63%. The consumption of cephalosporins decreased significantly (−60%), whereas aminopenicillin use increased accordingly (+120%). Neither in the pre- nor in the post-intervention group deaths occurred.Discussion: Data on ASP in pediatric secondary care hospitals are scarce. Most previous studies have been performed at tertiary care/university children's hospitals. We demonstrate a significant improvement in guideline adherence regarding antibiotic treatments after the implementation of an ASP. Cephalosporin consumption decreased which might be relevant for the selection of MRB . Results are limited by the single-center design and the short observation period. The study encourages the implementation of ASPs in secondary care children's hospitals. Staphylococcus aureus (MRSA)   are a leading global health care threat , 2. The s (MRSA) , 4. A wici (VRE) , 6. Furtci (VRE) , 8.defined daily doses (DDD). The DDDs are standardized and the extent of antibiotic usage can be determined by requesting the amount of orders in the clinical pharmacy. This amount can then be related to the patient days (PD). One PD is 1 day of inpatient treatment of one patient in the hospital. Together this represents an easy way to monitor the amount of antibiotic usage. In children and infants the drug dosage is routinely adjusted to the body weight. To measure the consumption of antibiotics with DDD is therefore not feasible. The best solution is to measure days of therapy (DOT). One DOT means that a patient is treated for 1 day with one specific drug. Since sometimes more than one drug is used simultaneously DOTs can be overlapping. This is the reason why the total duration of therapy should also be evaluated using the item length of therapy (LOT)  .In a Cochrane systematic review in adults and children one of the outcome variables was the adherence to treatment guidelines and how ASPs affect it. The authors found 29 randomized controlled trials (RCT) with a total of 23,394 patients. They demonstrated an increase of guideline adherence from 43 to 58% after ASPs had been launched. The mean duration of antibiotic treatment decreased from eleven to 9 days . The interventions were deemed safe by the authors as mortality did not change upon ASP implementation –12. SmitKinderklinik München Harlaching). The hospital consists of three general pediatric wards and a neonatal intensive care unit (NICU). All antibiotic prescriptions for inpatients were collected for the first quarter of 2017 and 2018. We excluded the NICU and antibiotic prophylaxis  from deductive statistics. Data were collected from patient charts and discharge reports. In cases in which a further antibiotic treatment after discharge had been recommended we assumed it accomplished as recommended in the reports. This led to consideration of the entire antibiotic treatment of each patient.We investigated antibiotic prescriptions in a municipal secondary care pediatric hospital in Munich, Germany (standard operating procedures (SOPs) for selected infectious diseases and a pocket-card, which summarizes the key information of the SOPs. SOPs were established for acute otitis media (AOM), pneumonia, meningitis, tonsillitis, and UTI.In July 2017 anASP was implemented. It consisted of a prospective audit with feedback by experts in pediatric infectious diseases, in-house treatment guidelines as Deutsche Gesellschaft für Pädiatrische Infektiologie, DGPI e.V.)  . In the Clostridium difficile (CDC), infection with MRB, length of stay, detection of Respiratory Syncytial Virus (RSV) and death.Secondary outcomes were the usage of specific classes of antibiotic drugs. They were measured in DOT. Other secondary outcomes were LOT, amount of different prescribed antibiotics, overall prescription rate, colitis caused by Data were analyzed with descriptive statistical methods . Results were statistically tested using IBM SPSS Statistics®. We performed the Chi Square Test for the primary outcome and Mann-Whitney tests for secondary outcomes with non-parametric data. The level of significance was adjusted using Bonferroni correction for multiple testing.p-value was 0.000016 in the Chi Square test.The primary outcome “adherence to treatment guidelines” increased significantly with the implementation of our ASP. The considered diagnoses for the primary outcome were AOM, pneumonia, meningitis, tonsillitis and UTI. In the pre-intervention group 100 cases with antibiotic prescriptions within the defined diseases were determined of which 33 were treated according to treatment guidelines. After the intervention 101 cases were identified in the defined period and 64 of them were treated according to the newly established SOPs see . The p-vGuideline-conform treatment differed substantially between the most frequent diagnoses see . EspeciaIn total 329 patients were treated in the two observation periods and met the inclusion criteria. There were eleven cases with insufficient documentation that were not included in the analyses. Patients had a mean age of 3.2 years with a standard deviation of 4.3 years. Median age was 1.4 years with a broad variance comprising a minimum of 0 and a maximum of 17.9 years of age. Of the admitted patients, 40 of 329 had been treated with antibiotics prior to admission to the hospital. In the first quarter of 2017, 852 patients were admitted to the hospital. In this period, 164 of 852 patients were treated with antibiotics (19.2%). After the intervention, 165 of 882 patients received antibiotics (18.7%), indicating no substantial change in the rate of antibiotic treatments.The use of cephalosporins decreased significantly after the intervention. Especially the use of second generation cephalosporins was reduced. Accordingly, the consumption of aminopenicillins with and without beta-lactamase inhibitors (BLI) increased significantly see . The amoThe diagnoses leading to antibiotic treatment are given in Here, we demonstrate effectiveness of an antibiotic stewardship program in a secondary care municipal pediatric hospital. Treatment adhering to guidelines markedly increased after our intervention and cephalosporin consumption could be replaced by aminopenicillins in many cases. Guideline adherence is one of several important items in ASPs. It was chosen as the primary outcome parameter for this study as it is unambiguously measurable and short term effects for this item were expected.There are some data regarding sustainability of ASPs . In studThe majority of patients in our study received antibiotic treatment due to pneumonia. In another German study investigating the effect of a pediatric ASP guideline adherence for the treatment of pneumonia was a separate outcome variable. The authors demonstrated a decrease in the use of second-generation cephalosporins from 50 to 6% . In lineThe use of third-generation cephalosporins did not change significantly. This class of antibiotics was mainly prescribed in patients with UTIs and was contained in our SOPs for this indication. Overall, the consumption of cephalosporins decreased significantly while aminopenicillin use increased. To draw clear conclusions on how replacing cephalosporins by aminopenicillins affects the prevalence of MRB has to be investigated by larger studies. The two cases of MRB-related UTIs are not sufficient to draw any conclusions. In one previous study no direct impact on the prevalence of MRB had been observed .How physicians adapted the initial treatment was not evaluated in our study, nor did we evaluate the diagnostic process, which is an important part for the optimal treatment in infectious diseases. There is growing interest in the field for optimal diagnostic processes which is now often labeled as “diagnostic stewardship” , and we Cost-effectiveness for pediatric ASPs was analyzed in one German and one US study at university children's hospitals with remarkable cost savings , 13. It Our study is limited due to its single-center design, the relatively small patient number, and the short observation period of one quarter each in 2017 and 2018. Larger multi-center studies are warranted to confirm and possibly expand our results. Innovations in the daily workflow like electronic prescription and documentation will make it easier to evaluate ASPs in the future. Studies already showed an improvement of the prescription process in a pediatric hospital with the launch of an IT-based prescription (The datasets generated for this study are available on request to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.The work comprises part of the doctoral thesis by JM.JM designed the study, analyzed data, and wrote the manuscript. NR designed and supervised the study and wrote the manuscript. PO and MB helped with data collection and establishment and implementation of SOPs. UB and SB assisted in study design and establishment of SOPs and reviewed the manuscript.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.


Mice lacking c-Met either in Kupffer cells, α-SMA+ and CK19+ cells, or bone marrow-derived immune cells displayed earlier and faster progressing steatohepatitis during dietary treatments. Severe fatty liver degeneration and histomorphological changes were accompanied by an increased infiltration of immune cells and a significant upregulation of inflammatory cytokine expression reflecting an earlier initiation of steatohepatitis development. In addition, animals with a cell-type-specific deletion of c-Met exhibited a strong generation of reactive oxygen species (ROS) by dihydroethidium (hydroethidine) (DHE) staining showing a significant increase in the oxidative stress response especially in LysCre/c-Metmut and MxCre/c-Metmut animals. All these changes finally lead to earlier and stronger fibrosis progression with strong accumulation of collagen within liver tissue of mice deficient for c-Met in different liver cell types. The HGF/c-Met signaling pathway prevents from steatosis development and has a protective function in the progression to steatohepatitis and fibrosis. It conveys an antifibrotic role independent on which cell type c-Met is missing . These results highlight a global protective capacity of c-Met in NASH development and progression.Nonalcoholic steatohepatitis (NASH) is the most common chronic, progressive liver disease in Western countries. The significance of cellular interactions of the HGF/c-Met axis in different liver cell subtypes and its relation to the oxidative stress response remains unclear so far. Hence, the present study is aimed at investigating the role of c-Met and the interaction with the oxidative stress response during NASH development in mice and humans. Conditional c-Met knockout (KO) lines (LysCre for Kupffer cells/macrophages, GFAPCre for  Nonalcoholic fatty liver disease (NAFLD) is a chronic and progressive liver disease which is evolving to be one of the leading causes for liver transplantation –3. As thMet, the cellular receptor tyrosine kinase for the hepatocyte growth factor (HGF), plays a pivotal role in mediating processes like stem cell growth, wound healing, motility, and morphogenesis . PreviouThe importance of c-Met gets more obvious when considering that mice with a deletion of this receptor exhibit serious developmental defects of several muscle groups , placentc-Met is predominantly expressed on cells with endothelial and epithelial origins and has initially been described as a protooncogene . In the During chronic liver injury, especially in NASH, c-Met has been shown to play a benefitial, hepatoprotective role by suppressing chronic inflammation and progression to fibrosis . The genα-SMA+/CK19+ cells, or bone marrow-derived immune cells), accelerated the disease outcome in diet-induced steatohepatitis and fibrosis.Based on the current knowledge, the aim of this study was to elucidate the particular role of c-Met on the different liver cell types during the development and progression of diet-induced steatohepatitis in two distinct murine feeding models. Mice with a deficiency of c-Met in either Kupffer cells/myeloid cells, cells under the glial fibrillary acid protein promotor, or bone marrow-derived immune cells were treated with high-fat diet (HFD) or methionine-choline-deficient diet (MCD). This study is the first to publish that the deletion of c-Met, insignificant on which liver cell types , Recklinghausen, Germany) and approved by the LANUV Committee (permit number: TV11018G). All experiments were performed in accordance with the German guidelines for animal housing and husbandry.52 liver specimens were obtained according to the local ethics committee rules from liver explantation or liver resection recruited at the RWTH Aachen University Hospital . When intrahepatic lesions were present, the tissue for this study was collected from the most distant section of the specimen. Overall, we analyzed 12 samples from patients with HBV, 17 HCV, 6 PBC, 8 PSC, 5 alcoholic cirrhosis, and 4 NASH patient samples.fl/fl and LysCre c-Metmut, conditional c-Metfl/fl and GFAPCre c-Metmut, and conditional c-Metfl/fl and MxCre c-Metmut mice all in C57BL/6J background were used as experimental animals. At least five animals per group and time point were treated in parallel. Respective c-Metfl/fl animals were always littermates to the corresponding c-Metmut mice. All experiments were repeated at least twice. Experimental animals were housed in the animal facility of the RWTH Aachen University Hospital with 12-hour light/dark cycles and water and food ad libitum available. Treatments were in accordance with the criteria of the German administrative panel on laboratory animal care.Male C57BL/6J wild-type mice, conditional c-MetAll dietary treatments were performed with 8–12-week-old male mice weighing at least 25 g. Mice were fed either with chow diet, methionine-choline-deficient diet (MCD) , or high-fat diet (HFD) , 20 kcal% fructose, and 2% cholesterol) . Wild-type and knockout mice showed a food intake of about 6 g per day without any differences between chow- and diet-treated animals.Further material and methods' data are provided in Supplementary Materials .α-SMA+/CK19+ cells, and bone marrow-derived immune cells in all three tissue-specific genetic settings. These controls show the presence of the c-Met WT allele in the varying liver cell types not targeted by the specific Cre promotor , we first conducted genomic PCR genotyping, detecting the recombinant and nonrecombinant c-Met allele. Analysis was performed for hepatocytes, Kupffer cells/macrophages, fl/fl and LysCre/c-Metmut littermates were fed either chow, methionine-choline-deficient diet (MCD) (4 weeks), or high-fat diet (HFD) (24 weeks) ad libitum. A significant increase in serum transaminase levels in LysCre/c-Metmut animals as well as in the MCD and HFD models points to a severe disease progression of nonalcoholic steatohepatitis (NASH) , and HFD (24 weeks) feeding. After treatment with both steatohepatitis-induced diets, LysCre/c-Metmut mice displayed a significantly higher accumulation of hepatic triglycerides compared to c-Metfl/fl controls ) staining  and 3B. Our results thus indicate that the proinflammatory immune response provoked by oxidative stress may be mechanistically at least in parts responsible for the more invasive phenotype in mice with a c-Met deficiency in Kupffer cells.α-SMA which revealed a significant increase in LysCre/c-Metmut mice after MCD and HFD feeding compared to equally treated c-Metfl/fl animals , and CK19  which gets evident by significantly increased serum transaminase levels in GFAPCre/c-Metmut animals compared to equally treated c-Metfl/fl controls  and HFD (24 weeks) feeding  . A multiry tract ) in c-Me9+ cells  and 6. Fcontrols . Histolo Figures . To assey models . In linereatment .The data suggest that a deficiency of c-Met in different liver cell types expressing the GFAP promoter leads to a worsened disease outcome in two independent mouse models of steatohepatitis.mut) resulted in severe maintenance of survival compared to controls (c-Metfl/fl)   and alanine aminotransferase (ALT) levels  MxCre promoter. After bone marrow transplantation, the efficiency of the c-Met deletion was investigated by a knockout PCR approach. The 372 bp PCR product shows the effective deletion of c-Met either in the donor or in the associated recipient . Surprisetfl/fl) . In supp Figures . Represe) levels  and high) levels  in MxCrec-Metmut . To testcontrols .fl/fl and MxCre/c-Metmut mice after chow, MCD, or HFD feeding. Comparable to what was found in c-Metfl/fl and LysCre/c-Metmut animals, mice deficient of c-Met in hematopoietic cells displayed a significantly greater amount of TUNEL- and DHE-positive cells whereas no difference in the expression of the antioxidative regulator Nrf2 could be detected after MCD and HFD treatment . To investigate whether this is a general phenomenon when c-Met signaling is blocked, we also performed TUNEL and DHE staining in c-Metreatment , and 8C.β, Collagen1a1, and α-SMA was measured via real-time PCR. All investigated markers showed increases in MxCre/c-Metmut animals compared to c-Metfl/fl controls after dietary treatments whereas the increase was clearly more pronounced in MCD fed mice compared to HFD fed mice. HFD-treated animals displayed only a minor but visible upregulation  of patient samples. The correlation clearly demonstrates a decrease in c-Met expression with less severe disease development  c-Met is essentially involved in this process.We here hypothesized that c-Met prevents from NASH-related fibrosis and wanted to clarify in detail on which cell type  led to severe steatosis, steatohepatitis, and fibrosis progression in two different mouse models of diet-induced steatohepatitis in all three used Cre lines  leads to an increase in the oxidative stress response and apoptotic cell death under dietary treatment.As hepatic manifestation of the metabolic syndrome, the development of NASH is associated with an impaired insulin metabolism. Varkaris et al. could show in 2013 that the insulin-like growth factor-1 (IGF-1) signaling through the IGF-1 receptor is able to delay the induction of c-Met activation, thereby showing a direct interaction between the HGF/c-Met signaling axis with metabolic changes as they can occur in steatohepatitis . Thus, s Figures . Further Figures . These rIt is well described that the infiltration of proinflammatory monocytes contributes to NASH progression . The rolmut animals display earlier fat droplet accumulation, a stronger onset of insulin resistance, and an increase in the proinflammatory immune response finally leading to massive extracellular matrix deposition  that express the GFAPCre promotor play a major role in the development of liver fibrosis . Under imponents . Previou+ T cells upon proinflammatory cytokine exposure during tissue injury [The infiltration of immune cells is one of the characteristic differences between simple steatosis and the more severe steatohepatitis. Recent investigations describe an immunoregulatory function of c-Met on hematopoietic progenitor cells , 52. Fure injury , 54. In e injury . DefectiTaken together, our findings surprisingly show that we could find no difference in the degree and outcome of diet-induced steatohepatitis initiation and progression independent on which cell type c-Met is lacking. This indicates a global role of c-Met in a cross talk between intrahepatic cells involved in steatohepatitis development and a protective effect of c-Met in fibrosis generation. Mechanistically, the knockout of c-Met leads to an imbalance of different pro- and anti-inflammatory pathways with an increase in apoptotic cell death together with a strong activation of ROS production which may in part explain the increase in liver injury leading to severe fibrosis progression.On the contrary, there is a growing quantity of clinical trials, e.g., ficlatuzumab, which is a humanized HGF inhibitory monoclonal antibody preventing HGF/c-Met signaling by blocking the ligand-mediated activation which is actually in a phase 1b clinical trial for the treatment of non-small-cell lung cancer . Further


Ongoing, moderate noise exposure does not instantly damage the auditory system but may cause lasting deficits, such as elevated thresholds and accelerated ageing of the auditory system.The neuromodulatory peptide urocortin‐3 (UCN3) is involved in the body's recovery from a stress response, and is also expressed in the cochlea and the auditory brainstem.Lack of UCN3 facilitates age‐induced hearing loss and causes permanently elevated auditory thresholds following a single 2 h noise exposure at moderate intensities.Outer hair cell function in mice lacking UCN3 is unaffected, so that the observed auditory deficits are most likely due to inner hair cell function or central mechanisms.Highly specific, rather than ubiquitous, expression of UCN3 in the brain renders it a promising candidate for designing drugs to ameliorate stress‐related auditory deficits, including recovery from acoustic trauma.Environmental acoustic noise is omnipresent in our modern society, with sound levels that are considered non‐damaging still causing long‐lasting or permanent changes in the auditory system. The small neuromodulatory peptide urocortin‐3 (UCN3) is the endogenous ligand for corticotropin‐releasing factor receptor type 2 and together they are known to play an important role in stress recovery. UCN3 expression has been observed in the auditory brainstem, but its role remains unclear. Here we describe the detailed distribution of UCN3 expression in the murine auditory brainstem and provide evidence that UCN3 is expressed in the synaptic region of inner hair cells in the cochlea. We also show that mice with deficient UCN3 signalling experience premature ageing of the auditory system starting at an age of 4.7 months with significantly elevated thresholds of auditory brainstem responses (ABRs) compared to age‐matched wild‐type mice. Following a single, 2 h exposure to moderate (84 or 94 dB SPL) noise, UCN3‐deficient mice exhibited significantly larger shifts in ABR thresholds combined with maladaptive recovery. In wild‐type mice, the same noise exposure did not cause lasting changes to auditory thresholds. The presence of UCN3‐expressing neurons throughout the auditory brainstem and the predisposition to hearing loss caused by preventing its normal expression suggests UCN3 as an important neuromodulatory peptide in the auditory system's response to loud sounds. Ongoing, moderate noise exposure does not instantly damage the auditory system but may cause lasting deficits, such as elevated thresholds and accelerated ageing of the auditory system.The neuromodulatory peptide urocortin‐3 (UCN3) is involved in the body's recovery from a stress response, and is also expressed in the cochlea and the auditory brainstem.Lack of UCN3 facilitates age‐induced hearing loss and causes permanently elevated auditory thresholds following a single 2 h noise exposure at moderate intensities.Outer hair cell function in mice lacking UCN3 is unaffected, so that the observed auditory deficits are most likely due to inner hair cell function or central mechanisms.Highly specific, rather than ubiquitous, expression of UCN3 in the brain renders it a promising candidate for designing drugs to ameliorate stress‐related auditory deficits, including recovery from acoustic trauma. Protection from acoustic trauma is an important feature of the auditory system that helps to preserve our hearing over decades of life. Recent studies have shown that even moderate noise levels that were previously considered to be non‐damaging can effectively speed up the ageing of the auditory system and cause permanent changes in the physiology of auditory circuits  controls the level of the outer hair cells’ contribution to cochlear amplification, and the lateral olivocochlear system (LOC) modulates the excitability of the auditory nerve  and measurements of distortion products of otoacoustic emissions (DPOAEs), before and following acoustic exposure to moderate noise in wild‐type mice and in mice lacking UCN3 signalling.In the present study, we investigate whether UCN3 expression outside the hypothalamic–pituitary–adrenal axis helps to protect the cochlea during exposure to moderate noise. We use et al. et al. CAG::loxP‐STOP‐loxP‐tdTomato mice  as previously described , approved by the regional authority (Regierung von Oberbayern) under file no. 55.2‐1‐54‐2532‐38‐13) and conform to the principles and regulations as described in the editorial by Grundy . Experim−1 body weight; i.p.) and were perfusion‐fixed with 4% paraformaldehyde (PFA) intracardially. Following overnight postfixation in 4% PFA, brainstems were sectioned coronally at 50 µm using a vibrating blade microtome . After rinsing in phosphate‐buffered saline (PBS), sections were transferred to a blocking solution containing 1% bovine serum albumin, 0.5% Triton X‐100 and 0.1% saponin in PBS. The floating sections were then incubated for 48 h at 4°C in blocking solution containing primary antibodies against calbindin . Sections were washed three times in PBS for 15 min and incubated with secondary antibodies  overnight at 4°C. Sections were washed in PBS, mounted on slides and coverslipped with Vectashield mounting medium .Mice received an overdose of pentobarbital (400 mg kgAfter perfusion‐fixation (see above), temporal bones were removed and post‐fixed in 4% PFA overnight. Following post‐fixation, the temporal bones were decalcified in 4% EDTA (pH adjusted to 7.4 with NaOH) for several days. Cochlear tissue including the organ of Corti was isolated and processed using immunohistochemical methods (see above). After immunohistochemical staining, tissue was treated with Phalloidin‐Atto 488  and 4′,6‐diamidino‐2‐phenylindole  for 20 min, followed by two 10 min washes in PBS before coverslipping.After perfusion‐fixation (see above) and decalcification, cochleae were bisected through the modiolus and embedded in Tissue Tek. Cryostat sections were cut at 12 µm and mounted on Superfrost Plus (Thermo Fisher Scientific) slides. Immunocytochemistry using antibodies for phalloidin and DAPI was performed directly on the slide at the concentrations described above.i.p. injection of MMF . During the experiments, mice were placed on a temperature‐controlled heating pad  in a soundproof chamber . Disposable needle electrodes  were placed subdermally at the vertex of the mouse's head (reference), ventral to the pinna (active (−)) and near the base of the tail (ground). Electrodes were attached to a low impedance headstage  and connected to the auditory processor  with an optical input cable through a preamplifier . SPIKE software  was used to calibrate the speakers in situ , generate stimuli and record ABR waveforms. Stimuli consisted of broadband clicks (100 µs duration) or pure tones  presented 1000× at varying intensities . For individual frequencies, responses to the 1000 stimulus repetitions were averaged and thresholds were determined as the sound intensity at which at least two peaks could be distinguished in the average ABR waveform. If no threshold was detected at 85 dB SPL, a value of 90 dB SPL was given as threshold. When possible, the genotype of the animal was blinded to the experimenter ; otherwise, the thresholds were assessed offline by a second person who then was blinded to the genotype. At the end of the experiment, animals were either revived using an i.p. injection of the MMF antidote AFN  or they received an overdose of pentobarbital  following the last measurement at the 14 day post‐exposure time point.Auditory brainstem responses were recorded in mice anaesthetized with an −1 body weight; i.p.) immediately after ABR measurement; therefore, repeated measures were not obtained for this part of the study. The average age was not significantly different between genotypes . ABR thresholds of WT and UCN3 KO mice were plotted against the animals’ age and the data were fitted by an exponential rise to maximum function: f = a(1 − exp(−bx)). These fits suggested differences between the two datasets occurring with increasing age. A change detection algorithm  and UCN3 KO mice aged between 1 month and 2 years. For the ageing study, each animal received an overdose of pentobarbital : WT: 3.64 (3.48–3.97) months, n = 15; UCN3 KO: 4.33 (3.41–4.56) months, n = 15; Mann–Whitney rank sum test: P = 0.575).Bandpass filtered noise between 8 and 16 kHz was generated using RPvdsEx (Tucker Davis Technologies) and was presented bilaterally via earphones  for 2 h at a level of 94 or 84 dB SPL. During the noise exposure, animals were under MMF anaesthesia (see above) and placed on a temperature‐controlled heating pad  in a soundproof chamber . ABRs were recorded before noise exposure (naïve), immediately after (= day 0), 1 day later, 7 days later, and 14 days later . Recovery of auditory thresholds was tested against a baseline of threshold variability acquired from repeated ABR measures of six animals at multiple time points (similar to those of the noise‐exposed mice) without acoustic exposure. These data gave us a baseline for threshold change significance testing and showed that our ABR protocol did not induce threshold shifts over the same time course as our noise exposure experiments. The average change in threshold for these control animals without noise exposure was −0.4 ± 1.0 dB SPL. This baseline variability is represented as horizontal grey shaded areas in Figs DPOAEs were recorded in C57BL/6J wild‐type mice and UCN3 KO mice. Mice  were anaesthetized with MMF (see above) and physiological temperature was maintained by a heating pad during the experiments. All DPOAEs were recorded in a soundproof chamber. Sound signals to elicit DPOAEs were generated and recorded with an external sound card, RME Fireface UC 24‐bit  at a sampling rate of 192 kHz and controlled by custom‐written MATLAB scripts . To use low‐latency multi‐channel ASIO, the SoundMexPro application  was employed for interfacing in the MATLAB environment. A custom‐made acoustic coupler (based on the Eaton‐Peabody‐Laboratory (EPL) Acoustic System) consisting of a microphone  and two micro dynamic speakers  was inserted into the ear canal of anaesthetized animals under visual control such that the tip of the coupler was at a distance of about 1 mm from the tympanic membrane.in situ to guarantee a flat frequency and phase response at the tip of the acoustic coupler. For that, the ear canal was stimulated with band‐pass filtered white noise (0.2–40 kHz) and a compensational impulse response was calculated for each loudspeaker. This compensational impulse response was used to correct all acoustic stimuli delivered by the loudspeakers.Before conducting the experiments, the amplitude and phase response of the probe microphone was corrected by comparing it to the flat amplitude and phase response of a reference microphone . Before each trial, the two loudspeakers generating DPOAE primary tones were calibrated f1 and f2 (f2/f1 = 1.23), with f1 level set to 65 dB SPL and f2 level set to 55 dB SPL, were presented 32 times with a stimulus duration of 500 ms. The frequency ratio of the primaries (f2/f1) and the intensity level were held constant while eliciting DPOAEs at several different frequencies with f2 between 4 and 40 kHz increasing in increments of 4 kHz steps. Thereby, so called DP‐grams, DPOAE magnitude as a function of and f2, were generated. The cubic DP (2f1 −  f2) was extracted from the ear canal sound pressure after time‐domain averaging. By averaging the magnitudes of six spectral lines below and six above the respective cubic DP, the noise floor was calculated. DPOAE recordings with DPOAE levels less than 6 dB above noise floor were not included in the analysis and rejected. At the end of the experiment, animals were either revived using an i.p. injection of the antidote AFN  or they received an overdose of pentobarbital  following the last measurement at the 14 day post‐exposure time point.To evoke DPOAEs, two primary tones n = 26 animals/3 litters) under MMF anaesthesia (see above) using a Neurostar stereotaxic injection system . The superior olivary complex (SOC) was targeted at the interaural midline level  and 0.5 µl of the viral vector solution was injected slowly over the time course of 3 min. This virus is taken up by all neurons, but only those neurons expressing the enzyme cre‐recombinase (in our experiment only those that transcribe UCN3) will produce diphtheria toxin and subsequently degenerate. At the end of the injection, mice were injected with the analgesic Metacam . The animals were then sutured and the AFN antidote (see above) was given to terminate anaesthesia. After the operation, each animal was placed in a single cage, which was irradiated by an infrared heat lamp, and continuously observed until the animal has recovered to the point where it has full motor coordination and begins normal feeding and grooming behaviour. At that point the animal was united with its original group. So far, we have had good experiences with this form of return to the group: even in males, there were no complications with re‐integration. During post‐operative recovery the animals received an injection of Metacam every 24 h for 3 days. The health status of the animals was evaluated daily for at least the next 5 days using a score sheet including body weight, posture, reaction to handling, physical appearance as well as motor and social behaviour. All animals used in this study had a total score of zero on day 5, rendering them comparable to untreated mice in the colony; 28 to 49 days were allowed for expression of diphtheria toxin, before the ABR and noise exposure protocols were then performed as described in previous paragraphs. After the 2 week time point, animals received an overdose of pentobarbital , were perfused intracardially, and the brains were examined following sectioning to assess the cre+ or cre− genotype. Note, that up until this point the experimental group (cre+: showing reduced UCN3 expression in the auditory brainstem) and the control group  were completely blinded to the experimenter. Animals of each genotype did not differ in age at the time of noise exposure . Tissue sections were counterstained with microtubule‐associated protein 2  CH22103, 1:1000; secondary: donkey anti‐chicken aminomethylcoumarin acetate (AMCA), 703‐156‐155 Dianova, Hamburg, Germany, 1:100) following the protocol above.UCN3‐tdTomato mice were injected with rAAV8/EF1α‐mCherry‐Flex‐dtA (AAV‐DTA) on postnatal day 22–23 (t test for comparing two groups and ANOVA for comparing three or more groups). When the normality assumption was violated, non‐parametric tests  were used. A paired t test or Mann–Whitney rank sum test was used when two data sets were compared in before–after conditions. Differences were considered statistically significant at P ≤ 0.05 and presented in the figures as n.s. for non‐significant differences and as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 for significant differences. In the test all actual P‐values are given for values >0.001.In the text, data are presented as median (interquartile range) or as mean ± SEM. In the figures, data are presented as medians (lines in boxes); 25/75 quartiles (boxes); and 10/90 percentiles (whiskers) in addition to individual data points. Statistical analyses of the data were performed with SigmaStat/SigmaPlot . Normality was tested by the Shapiro–Wilk test. Comparisons between different datasets were made depending on the distribution of the data using parametric tests for normally distributed data . Additionally, cochlear whole mount sections of UCN3‐tdTomato reporter mice were prepared and counterstained with phalloidin and DAPI to visualize the surrounding pillar cells and nuclei of the auditory hair cells, respectively, alongside UCN3 expression as indicated by the tdTomato fluorescence signal . The image shows three rows of outer hair cells (OHCs) and one row of IHCs. Again, UCN3 positive puncta were observed at the base of the IHCs (white arrows), resembling the pattern of the afferent ribbon synapses . From the UCN3‐expressing neurons in the VCN, labelled fibres project towards the DCN, which suggests at least some of these cells are multipolar cells . The presence of UCN3 positive calyx of Held synapses in the lateral MNTB  implies that a subpopulation of the UCN3‐expressing neurons in the VCN are globular bushy cells, which are known to give rise to calyx of Held synapses . The dorsal periolivary nucleus (DPO) contains a rich network of UCN3‐expressing fibres and neuronal somata . Interestingly both of these para‐ (in guinea pig) and periolivary  nuclei are also host to neurons of the efferent feedback system to the cochlea  and neuropil staining in the dorsal cochlear nucleus  and UCN3 KO (red) populations. Presentations of different sound intensities were used to determine auditory thresholds . ABR thresholds were plotted against each animal's age in order to estimate if and at what age hearing thresholds of UCN3 KO mice deviate significantly from the WT mice. The progressive elevation of ABR thresholds with increasing age could be predicted by fitting exponential rise to maximum functions to each dataset . These estimates suggested a faster elevation of ABR thresholds with increasing age in UCN3 KO mice. The difference in ABR thresholds between WT mice and UCN3 KO mice became significant at 4.73 months , which therefore became the age of division between young and old mice. Audiograms comparing young and old animals of each genotype are depicted in Fig. D. Young animals of UCN3 KO and WT mice have similar and overlapping thresholds, but old UCN3 KO animals tended to have higher ABR thresholds than aged WT mice at all frequencies. This genotype‐specific elevation of ABR thresholds was quantified across the audiogram and revealed no statistically significant difference for young mice . Old UCN3 KO mice had significantly higher ABR thresholds (UCN3 KO: 83.75 (61.41–88.44) dB SPL) compared to age‐matched WT mice (79.72 (53.05–84.44) dB SPL; Wilcoxon signed rank test: P = 0.004; Fig. F). To remove the confounding effect of differences in age‐induced hearing loss from our subsequent experiments, we used only young animals <4.73 months to study the impact of UCN3 on noise‐induced hearing loss.Different mouse strains show distinct susceptibility to age‐induced hearing loss with the C57BL/6 strain being especially prone  and two subsequent time points following exposure. Decreases in wave I amplitude were observed for both genotypes immediately after exposure. However, WT wave I amplitudes recovered to near control amplitudes while UCN3 KO amplitudes remained reduced at the day 1 time point . Waves II–IV seem to show longer lasting effects in both genotypes, which will have to be addressed in a separate study by corresponding cellular physiology in the different processing stages of the ascending auditory pathway. Audiograms for the group of animals exposed to 94 dB SPL noise are shown in Fig. D and E. Threshold values in the naïve condition were subtracted from threshold values at each post‐exposure time point to obtain a threshold shift value. Threshold shift values were averaged across the audiogram and compared between genotypes as well as against a baseline acquired from a group of animals tested at several time points without exposure . Following noise exposure, threshold shifts in UCN3 KO mice were larger when compared to WT mice especially for recovery times of more than 1 day (WTday0: 10 (7.8–22.8) dB; UCN3 KOday0: 25 (14.5–35.62) dB; P = 0.104; WTday1: 5 (0–12.0) dB; UCN3 KOday1: 15 (10–33.8) dB; P ≤ 0.001; WTday7: 0 (0–5) dB; UCN3 KOday7: 8.5 (5–15) dB; P ≤ 0.001; WTday14: 0 (–2.1 to 2.5) dB; UCN3 KOday14: 7.5 (5–10.6) dB; P = 0.017; Kruskal–Wallis one‐way ANOVA on ranks: P ≤ 0.001, all pairwise multiple comparison procedures (Dunn's method)). Moreover, in UCN3 KO mice, changes in threshold remained significantly different from baseline even 2 weeks after exposure  at 2 weeks: P = 0.014). Conversely, ABR thresholds for WT animals were significantly elevated immediately after exposure but returned to baseline after 1 week of recovery  immediately after exposure: P ≤ 0.001 and at 1 week: P = 0.325). These data suggest that UCN3 KO mice are more susceptible to long lasting threshold shifts after a single 94 dB SPL noise exposure when compared to their WT counterparts. We next tested whether an even lower intensity, ‘safe’ noise level would elicit noise‐induced hearing loss in UCN3 KO animals.To investigate the role of the stress peptide UCN3 in the auditory system's response to loud sounds, we adapted a noise exposure protocol that is reported to induce mild acoustic trauma without damaging the peripheral or central auditory system : WTday0: 2.5 (0–5) dB, P = 0.076; WTday1: 0 (0–5) dB, P = 0.145; WTday7: 0 (0–5) dB, P = 0.245; WTday14: 0 (−3.8 to 0) dB, P = 1; Fig. G, I). The UCN3 KO mice, however, showed significant changes in threshold especially at higher frequencies. Even averaging the dB shift across the whole audiogram showed persistent changes in threshold for at least two weeks after exposure : UCN3 KOday0: 10 (7.5–17.5) dB, P ≤ 0.001; UCN3 KOday1: 6 (2.5–10) dB, P ≤ 0.001; UCN3 KOday7: 6 (0–10) dB, P ≤ 0.001; UCN3 KOday14: 5.8 (2.5–10) dB, P = 0.003; Fig. H, I). Additionally, these shifts were greater than those of the WT mice at each time point : day 0: P ≤ 0.001; day 1: P ≤ 0.001; day 7: P = 0.027; day 14: P = 0.009; Fig. I). These data indicate that noise levels which are considered safe for WT animals cause significant and long‐lasting threshold shifts in mice that lack UCN3 signalling.A single 2 h noise exposure at 84 dB SPL is suggested to result in little or no temporary threshold shifts in normal‐hearing individuals (Gourevitch ay14: 0 −.8 to 0 day14: 0 −.8 to 0 day14: 0 −.8 to 0 dA and B). After noise exposure, DPOAE magnitudes were only slightly decreased in both genotypes and recovered along similar timelines . Changes of DPOAE magnitudes of post‐exposure levels compared to pre‐exposure levels (control condition) were calculated and averaged across all frequencies for each animal and plotted in Fig. C. Similar to ABRs, frequencies above 8 kHz, which was the lower limit of the noise exposure bandwidth animals were exposed to, were most affected. To conclude, changes in DPOAE magnitude were not significantly different between WT and UCN3 KO animals at any time point . These data therefore suggest that OHC function is not significantly impaired in UCN3 KO animals compared to WT controls.Given our results regarding increased susceptibility to and prolonged recovery from noise exposure, we wanted to test whether OHC function was similarly affected. Our histochemical analysis suggested that UCN3 immunoreactivity was most pronounced in areas surrounding IHCs; however, we additionally assessed OHC function using DPOAEs as a metric following our noise exposure protocol. We used the 94 dB SPL exposure and the same time points as in the ABR assessment before. DPOAEs were evoked in WT and UCN3 KO mice across the audiogram  enabled targeted ablation of UCN3 positive brainstem neurons without affecting UCN3‐expressing neurons in other brain areas. Viral injections were performed on entire litters born to UCN3‐cre × Ai9‐flox (tdTomato) breeders. The offspring of these breeding pairs included pups of which some expressed cre‐recombinase in UCN3 neurons (cre+) and others did not (cre−). Histological examination was used to confirm genotypes and was not carried out until after all noise exposure and ABR recordings were completed and analysed so that the experimenter was blind to the effect of the virus injection. A month following viral infection, the ABR and noise exposure (94 dB SPL) protocols were performed and hearing thresholds were assessed at day 0, 1, 7 and 14 following exposure. The resulting audiograms are plotted in Fig. B and C for cre− and cre+ mice, respectively. Shifts in auditory thresholds following noise exposure are shown in Fig. D for the cre− animals and the cre+ animals. Both sets of animals experienced similar threshold shifts immediately following noise exposure (cre−day0: 13.5 (3.9–26.4) dB; cre+day0: 17.5 (7.2–28.9) dB; t test: P = 1.000; Fig. D), but auditory thresholds in the cre− animals recovered faster than in the cre+ animals and were no longer significantly different from the baseline at the 7 and 14 day time points : cre−day0: 13.5 (3.9–26.4) dB, P ≤ 0.001; cre−day1: 7.1 (0.9–19.9) dB, P = 0.004; cre−day7: 5.6 (−0.4 to 11.6) dB, P = 0.116; cre−day14: 4.2 (1.5–7.5) dB, P = 0.523; Fig. E). In contrast, auditory thresholds in cre+ animals recovered only slowly and remained different from baseline even after 2 weeks : cre+day0: 17.5 (7.2–28.9) dB, P ≤ 0.001; cre+day1: 16.9 (7.2–29.6) dB, P ≤ 0.001; cre+day7: 12.3 (3.3–18.4) dB, P ≤ 0.001; cre+day14: 12.3 (4.9–18.0) dB, P ≤ 0.001). This maladaptive recovery from mild acoustic trauma in cre+ mice was reminiscent of the UCN3 KO phenotype  and suggests a protective role for local UCN3‐expressing neurons in the auditory brainstem.Besides UCN3 expression in the auditory brainstem, UCN3 positive neurons are present in other brain regions involved in regulation of the body's stress axis  and DPO (in other rodents) can be retrogradely labelled via cochlear injections  or specifically in the brainstem (cre+ mice in this study) can lead to deficits in recovery from acoustic noise‐induced stress, similar to the function of UCN3 in the HPA axis , German Ministry of Science and Education (Grant No 01EO1401).This research was supported by the German Research Council (KO2207/3‐1, SFB870/2‐A10 and GS‐82 Graduate School of Systemic Neurosciences GSN


Oxidative stress plays a key role in the pathogenesis of PD. Mutations in LRRK2 have been shown to increase susceptibility to oxidative stress. To explore mechanisms underlying susceptibility to oxidative stress in LRRK2 mutants, we generated stable Caenorhabditis elegans (C. elegans) strains in which human LRRK2 proteins including wild type LRRK2 (WT), G2019S LRRK2 (G2019S), and G2019S-D1994A kinase-dead LRRK2 (KD) were expressed in all neurons. Human 14-3-3 β was injected into LRRK2 transgenic worms to allow co-expression of 14-3-3 β and LRRK2 proteins. We found that G2019S transgenic worms had increased sensitivity to stress (heat and juglone treatment) and impaired stress-induced nuclear translocation of DAF-16. In addition, G2019S inhibited ftt2 (a 14-3-3 gene homolog in C. elegans) knockdown-associated nuclear translocation of DAF-16. Comparably, overexpression of human 14-3-3 β could attenuate G2019S-associated toxicity in response to stress and rescued G2019S-mediated inhibition of sod-3 and dod-3 expression. Taken together, our study provides evidence suggesting that 14-3-3-associated inhibition of DAF-16 nuclear translocation could be a mechanism for G2019S LRRK2-induced oxidative stress and cellular toxicity. Our findings may give a hint that the potential of 14-3-3 proteins as neuroprotective targets in PD patients carrying LRRK2 mutations.Mutations in  Parkinson’s disease  is a neurodegenerative disorder caused by genetic and environmental factors. A substitution of serine for glycine at position 2019 (G2019S) in the kinase domain of leucine-rich repeat kinase 2  represents the most prevalent genetic mutation in PD . FurtherC. elegans , is an important transcriptional regulator of genes that rapidly respond to and neutralize the effects of oxidative stress, such as superoxide dismutase . Translo elegans , and thi elegans .C. elegans  LRRK2, and injected 14-3-3 β into these LRRK2 transgenic worms. We discovered that pan-neuronal expression of G2019S LRRK2 caused defects in stress resistance and impaired stress-induced or 14-3-3 protein-associated DAF-16 nuclear translocation in C. elegans. We also investigated G2019S LRRK2-induced defects in sod-3 and dod-3 mRNA expression by modulating DAF-16 localization. Our data suggest that human 14-3-3 β [MIM: 601289] protein could rescue G2019S LRRK2-associated toxicity in response to stress.We hypothesized that G2019S LRRK2 reduces stress resistance by inhibiting DAF-16 nuclear translocation, which may be mediated by C. elegans strains expressing human wild type LRRK2, G2019S LRRK2 and G2019S D1994A KD LRRK2 in all neurons to investigate the responses of different LRRK2 strains to two different kinds of stress conditions: thermal and oxidative ; whereas, KD could rescue G2019S-mediated loss of heat resistance . Meanwhile, similar results were achieved in oxidative stress experiments. RFP control worms exhibited 15.9% ± 3.0% survival after exposure to juglone for 7 h, while survival rates of WT, G2019S and KD transgenic worms were 10.4% ± 2.6%, 6.4% ± 2.0%, and 18.3% ± 3.2%, respectively. Compared with the control strain, WT showed similar results, but G2019S significantly decreased the survival rate ; whereas, KD could rescue G2019S-mediated loss of juglone resistance .We generated C. elegans. Translocation of DAF-16 from the cytoplasm into the nucleus is necessary for its transcriptional activity (C. elegans strain expressing DAF-16::GFP (TJ356) was used to investigate the role of DAF-16 in G2019S-mediated loss of stress resistance. This strain has been shown to respond to environmental stress by displaying DAF-16 nuclear translocation. Three different status of DAF-16 were show in Figure p < 0.01 for heat treatment; Figure p < 0.01 for juglone treatment). However, KD could rescue G2019S-mediated inhibition of DAF-16 nuclear translocation in response to heat stress  or juglone .DAF-16, a FoxO transcription factor, is a key player in stress resistance in activity . A C. elUnder stress, percentages of worms exhibiting nuclear translocation of DAF-16 were similar among WT-TJ356, KD-TJ356 and TJ356 strains . Thus, our data suggest that G2019S blocks DAF-16 nuclear translocation under stress conditions.sod-3 and dod-3. In response to heat stress, we found reduced expression levels of sod-3 and dod-3 in the G2019S transgenic C. elegans strain compared with controls; notably, both genes are under the regulation of DAF-16. As shown in Figures sod-3  and dod-3 , while other LRRK2 transgenic strains showed no significant difference from the control strain .DAF-16 controls redox metabolism by regulating the expression of anti-oxidative stress molecules and lifespan-associated genes, such as sod-3 and dod-3 of all strains in response to juglone. Compared with the control strain, the G2019S transgenic strain significantly reduced expression levels of sod-3 and dod-3. As shown in Figures sod-3  and dod-3 , while other LRRK2 transgenic strains showed no significant difference from the control strain . Thus, our data suggest that G2019S modulates the regulation of stress-resistance genes.Similarly, we examined the mRNA expression levels of C. elegans 14-3-3 protein ortholog FTT-2 acts as a key regulator of DAF-16 by binding to DAF-16 and regulating its sub-cellular localization via sequestration in the cytoplasm , whereas DAF-16::GFP was predominantly localized in the nuclei of the TJ356 strain receiving ftt-2 RNAi . Compared with the TJ356 control strain, G2019S-TJ356 exhibited a significantly decreased number of nuclear DAF16::GFP ; whereas, KD could rescue G2019S-mediated inhibition of DAF-16 nuclear translocation . Thus, our data suggest that G2019S inhibits ftt-2 -induced DAF-16 nuclear localization.The ytoplasm . Given t Figures  and TJ35 Figures  fed RNAii Figure . FurtherC. elegans strains co-expressing human 14-3-3 β and LRRK2 by injecting a plasmid containing 14-3-3 β fused with a green fluorescent protein (GFP) marker into WT, G2019S and KD transgenic worms. Subsequently, WT-14-3-3 β, G2019S-14-3-3 β and KD-14-3-3 β were selected using the GFP marker , while the survival rate of WT (or KD) was similar to WT-14-3-3 β (or KD-14-3-3 β) , while the survival rate of WT (or KD) was similar to WT-14-3-3 β (or KD-14-3-3 β) ; whereas, the survival rate of WT (or KD) was similar to WT-14-3-3 β (or KD-14-3-3 β) ; whereas, the survival rate of WT (or KD) was similar to WT-14-3-3 β (or KD-14-3-3 β) . Collectively, the present data suggest that increased stress susceptibility of G2019S LRRK2 is associated with inhibition of DAF-16 nuclear translocation in a 14-3-3-associated manner. Given the critical role of DAF-16 in stress resistance, our findings imply that DAF-16 may play an important role in 14-3-3-mediated attenuation of G2019S LRRK2 toxicity.In the present study, we examined the role of 14-3-3 and DAF-16 proteins in LRRK2 pathophysiology in C. elegans expressing human WT LRRK2 and PD-linked mutant G2019S LRRK2 or G2019S kinase dead LRRK2 in all neurons. Consistent with previous studies, sensitivity to stress was significantly increased in G2019S worms.We generated transgenic C. elegans, plays an important role in stress resistance. DAF-16 is activated by nuclear translocation in response to environmental stress. Once DAF-16 is activated, it activates expression of downstream genes such as sod-3 and dod-3 to increase stress resistance. Consistently, under heat and juglone stress, nuclear translocation of DAF-16 was evident in most C. elegans of both control and WT strains; whereas, DAF-16 was only detected in a relatively small population of G2019S C. elegans. Furthermore, G2019S-induced impairment of DAF-16 nuclear translocation resulted in loss of stress resistance, suggesting that blockage of DAF-16 nuclear translocation may be one mechanism by which G2019S elicits toxicity.DAF-16, the only FoxO transcription factor in Previously, LRRK2 has been shown to directly phosphorylate FoxO, an analog of DAF-16, initiating an apoptotic pathway . Phosphosod-3 and dod-3 in G2019S worms. Given that sod-3 and dod-3 are direct downstream genes of DAF-16, our results suggest that 14-3-3 may rescue G2019S-mediated loss of stress resistance via modulation of DAF-16 activity.14-3-3 proteins are a key hub for dysregulated proteins in transcriptional analysis of PD patients . All 14-Phosphorylation of DAF-16 is a key event in DAF-16 signaling pathways. Phosphorylation can promote or inhibit DAF-16 nuclear translocation depending on the function of kinases. For example, phosphorylation by Akt restricts DAF-16 to the cytoplasm, while JNK promotes nuclear translocation .We found that G2019S LRRK2 could block stress/14-3-3 knockdown-associated nuclear translocation. This event is kinase-dependent, as the kinase dead strain of G2019S completely rescued this event. Given that LRRK2 can directly interact with 14-3-3 and DAF-16, both interactions may have a role in nuclear translocation of DAF-16. Interestingly, pathogenic mutations such as R1441C, R1441G, R1441H, Y1699C, and I2020T, but not G2019S, disrupt the interaction with 14-3-3 . FurtherLRRK2 has been reported to phosphorylate FoxO1 at S319 . We alsoC. elegans strains were obtained from the Caenorhabditis Genetics Center or elsewhere, as indicated, including Bristol N2, TJ356, and BZ555 strains. All strains used were maintained and handled as previously described  with the pu51p vector. WT LRRK2 and mutant (G2019S and G2019S KD) LRRK2 cDNA fragments were obtained by cutting between NheI and BamHI sites of modified pcDNA3.1 and inserting fragments into pu51p between NheI and KpnI sites of pu51p. Additionally, we constructed transgenic a pu51p-14-3-3 β-GFP plasmid expressing 14-3-3 β protein fused with eGFP. First, the enhanced GFP sequence was inserted into XhoI and SacI sites of pu51p. Second, the PCR product of 14-3-3 β was inserted into NheI and XhoI sites immediately upstream of GFP in the modified pu51p to create pu51p-14-3-3-GFP. All RNAi vectors were purchased from Open Biosystems .Human cDNA encoding full-length FLAG-tagged human WT LRRK2 and mutants (LRRK2 G2019S and G2019S KD) generated by site-directed mutagenesis were previously constructed in pcDNA3.1 vectors, as described . The C. 14-3-3 β transgenic nematodes were created by injecting 100 ng/μL of pu51p-14-3-3β-GFP plasmid. This cocktail of DNA was injected into the gonad of young adult hermaphrodites of the N2 strain using a previously described method (2) and grown further to generate F1 progenies. Two hundred F1 nematodes were individually grown in a single plate and selected for 75% transmittance. Four nematodes were picked from every F2 population with 75% transmittance, and then individually grown in single plate and selected for 100% transmittance. With a yield of 1–2% stable integrants, transgenic nematodes were out-crossed with Bristol N2 nematodes at least five times and PCR analyzed for the presence of transgenic cDNA. LRRK2/DAF-16::GFP strains (carrying GFP) were created by conventional crossing of WT LRRK2 or mutant LRRK2 strains with TJ356 (DAF-16::GFP), and verified by checking both RFP and GFP markers. Representative integrated strains from each strain were used in repeated experiments throughout this study, and their detailed genotypes and strain designations are listed in Table LRRK2 transgenic nematodes were created by co-injecting a cocktail of DNA containing 50 ng/μL of pu51p-WT LRRK2, pu51p-G2019S LRRK2, or pu51p-G2019S KD LRRK2 plasmid along with the co-marker pu51p-RFP (100 ng/μL). Human d method . InjecteNematodes were synchronized either by a bleaching method or letting nematodes lay eggs for 2–3 h.Assays were performed using two dishes of 25 adult nematodes for each condition and strain. Nematodes were exposed to NGM agar containing 400 μM juglone or heated to 35°C. Treatment with juglone was begun on adult day 3, while treatment at 35°C was begun on adult day 2. Nematode viability was checked every hour with a soft touch at the tip of pharynx of the nematode. Nematodes that crawled up the side of the plate were excluded from all analyses.ftt2 open reading frame (ORF) (Open Biosystems) or empty vector. On adult day 2 of the next generation, nematodes were used in several experiments.RNAi knockdown assay was performed according to n ≥ 120 or greater).For quantification of DAF-16::GFP localization, synchronized eggs from TJ356 animals  or other strains (as indicated) were seeded onto either control or appropriate RNAi plates. For stress response experiments, adult day 1 worms were transferred to new plates and subjected to heat shock (35°C) or plates containing 400 μM juglone. GFP localization was then analyzed using a Nikon AZ100 fluorescent microscope at 5× magnification. An animal was scored as having nuclear GFP if more than one head hypodermic nuclei contained DAF-16::GFP. For single time point experiments, worms were blindly scored for the presence or absence of GFP accumulation within the nuclei of indicated cells (g for 1 min. The worm pellet was homogenized in ice-cold lysed buffer (1% Triton X-100 in PBS) supplemented with a cocktail of protease inhibitors (Roche), and then sonicated five times for 5 s each. The mixture was then incubated on ice for 30 min. Homogenates were clarified after centrifugation at 14,000 × g for 20 min. The supernatant was collected for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using anti-14-3-3  for FTT-2 detection. An anti-actin antibody  was used for protein control.Protein extracts were prepared from mixed-stage worms grown to near confluence on five 60-mm NGM plates. Worms were washed with M9 buffer and collected by brief centrifugation at 3,000 × C. elegans was extracted using an E.Z.N.A. total RNA extraction kit (OMEGA Biotek). Intact RNA was checked by running a 1.0% agarose/formaldehyde gel and quantified spectrometrically (Beckman Coulter DU 800) before proceeding to subsequent steps. Five-hundred nanograms of total RNA were reverse-transcribed using PrimeScriptTM RT Master Mix  Kit (Takara) according to the manufacturer’s instructions. Real-Time PCR was performed on an Opticon MONITORTM Software (MJ Research) using SYBR® Premix Ex TaqTM II (TliRNaseH Plus) (Takara). Expression levels for each target gene were calculated by the 2-ΔΔCT method (Total RNA from T method . All anat-test. All the curves and column diagrams were performed using Graphpad Prism 7 software.Survival analysis was done using logrank test. According to reference and previous study, we need 50 animals per group. Statistical analysis was done using SPSS Statistics software. Survival curves were analyzed by the Kaplan–Meier method. Chi-square was conducted in the daf16 translocation assay. We take Convenience Sampling. At least 50 animals were randomly selected in stress assay and 30 animals were randomly selected in daf16 translocation assay. Descriptive statistics of the results are using mean and SD. Results of Western and qRT-PCR were evaluated by using a SL and ZP conceived and coordinated the study and wrote the paper. SL, FS, and WG designed, performed, and analyzed the experiments shown in Figures 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.


Antibiotic resistance is a global health threat. Public knowledge is considered a prerequisite for appropriate use of antibiotics and limited spread of antibiotic resistance. Our aim was to examine the level of knowledge of antibiotics and antibiotic resistance among Norwegian pharmacy customers, and to assess to which degree beliefs, attitudes and sociodemographic factors are associated with this knowledge.general) , 3) attitudes toward antibiotic use (four statements), and 4) sociodemographic factors, life style and health. High knowledge level was defined as > 66% of maximum score. Factors associated with knowledge of antibiotics and antibiotic resistance were investigated through univariate and multiple linear regression. Hierarchical model regression was used to estimate a population average knowledge score weighted for age, gender and level of education.A questionnaire based, cross-sectional study was conducted among pharmacy customers in three Norwegian cities. The questionnaire covered 1) knowledge of antibiotics (13 statements) and antibiotic resistance (10 statements), 2) the general beliefs about medicines questionnaire .Despite a high level of knowledge of antibiotics and antibiotic resistance among Norwegian pharmacy customers, there are obvious knowledge gaps. We suggest that action is taken to increase the knowledge level, and particularly target people in vocational, male dominated occupations outside the health service, and primary/secondary school curricula.The online version of this article (10.1186/s12889-019-6409-x) contains supplementary material, which is available to authorized users. Increast today” . Parallet today” .Staphylococcus aureus (MRSA) since 2010, in addition to several major outbreaks of Vancomycin-resistant enterococci (VRE) [Inappropriate prescribing and/or overuse of antibiotics among patients are regarded among the main causes of the emergence of resistant bacteria . Althougci (VRE) .The Norwegian national strategy against antibiotic resistance 2015–2020) aims to reduce antibiotic use (measured in DDD/1000 inhabitants/day) by 30% from 2012 to 2020 , i.e. fr–2020 aim“communication tools targeting key audiences and contribute to the annual European Antibiotic Awareness Day (EAAD)” [The European strategy describes guidelines for the member states . It does (EAAD)” .Independent information and public education about medicines are two of the 12 key interventions that WHO advocates to promote more rational medicine use in general . When ithttps://www.facebook.com/helsedirektoratet/videos/1282170395192759/ and https://www.facebook.com/helsedirektoratet/videos/1249111068498692/).Information campaigns have been launched in several countries, with varying effects on antibiotic use . As a paIndependently of official information strategies, media may have covered various aspects of the antibiotic resistance challenge , 16. In Awareness about antibiotic resistance has been shown to be lower in countries where antibiotic resistance is high . Within The primary aim was to examine the level of knowledge of antibiotics and antibiotic resistance in the Norwegian population, explored among pharmacy customers. The secondary aim was to assess to which degree beliefs, attitudes and sociodemographic factors are associated with this knowledge.We carried out a questionnaire based, cross-sectional study among pharmacy customers from 20 pharmacies in three Norwegian cities representing different parts of the country: Tromsø in the north , Bergen in the southwest  and Skien in the southeast ). These cities were selected based on the location of the collaborating university departments represented in the project team, as well as their proximity to the area of residency of the master students performing the data collection. The pharmacies were chosen either based on their large client base (six in Bergen and two in Skien), or all pharmacies in the municipality were chosen . Four pharmacy students  year) from the Universities of Tromsø, Bergen and Oslo collaborated on the data collection, which took place from October 2016 to February 2017. The inclusion criteria were ≥ 18 years of age and ability to understand Norwegian. The master students were present in the pharmacy at all times during data collection to facilitate questionnaire completion, e.g. show them to the pharmacy information room or provide postage paid envelopes if they were in a hurry, and to prevent misunderstandings if they had difficulties understanding some of the questions. All individuals who entered the pharmacy during the appointed time for data collection were invited to participate.N = 29). Based on the results from this study, the questionnaire was shortened and some of the questions were rephrased. The revised version, tested among an additional six persons in Tromsø, showed that mean completion time at this study site was reduced from 41 min for the original questionnaire, to 26 min for the revised version. Participants from the face validity study were excluded from the main study. The complete questionnaire was not validated, but included some validated questions, i.e. the Beliefs about Medicines Questionnaire (BMQ) [A structured questionnaire in Norwegian was developed by the project team consisting of researchers with extensive experience in conducting questionnaire-based studies as well as clinical experience and expertise in pharmacy practice. The master students conducted a face validity study for assessment of comprehensibility and completion time among persons without health professional background (re (BMQ) . An EnglThe questionnaire was organised in four parts: 1) knowledge of antibiotics and antibiotic resistance, 2) general beliefs about medicines, 3) attitudes toward antibiotic use and 4) sociodemographic factors, lifestyle and health.The antibiotic knowledge part consisted of 23 statements, 13 regarding antibiotics in general and 10 regarding antibiotic resistance. Six items were sampled from the Eurobarometer , and thegeneral) [Beliefs about medicines were measured by the beliefs about medicines questionnaire  . The insgeneral) , but notTo measure attitudes toward antibiotics, we used four statements from a web survey commissioned by the Norwegian Directorate of Health : “I onlyWe included questions on age, gender, work situation, level of education, health professional background, marital status, health status, chronic disease, regular medication use, antibiotic use in the previous 12 months and smoking. Variables were categorized as presented in Table https://www.R-project.org/) for the statistical analyses. We present descriptive statistics as means with standard deviations (SD) or frequencies with proportions (%). Factors associated with knowledge of antibiotics and antibiotic resistance were investigated through univariate and multiple linear regression. Inspection of diagnostic plots of residuals  did not reveal serious violation of test assumptions. We performed hierarchical model regression to estimate an average knowledge score for the general population, weighted for age, gender and education.We used R version 3.4.1, R Foundation for Statistical Computing, Vienna, Austria . For other participants, missing values were imputed by setting missing to 3 on the Likert scale (uncertain) .For the BMQ and the attitude scores, participants with missing on more than one item within a subdomain were excluded from the analyses . Mean age was 49.6 (SD 18.4) years. One third of the participants were ≥ 60 years and two thirds were women. Seventy-five percent agreed that they had good health, 56% reported higher education, 43% had a chronic disease, 57% used medication regularly and 30% had used antibiotics during the last 12 months.Overall, the proportion with high knowledge (> 66% of maximum score) was 57% for antibiotics in general and 71% for antibiotic resistance. Fifty percent had high knowledge level on both domains, while 21% had low knowledge level on both domains.n = 1), to around 60%).Table Figure The mean overall antibiotic knowledge score was 15.6 (67.8% of maximum score), relatively higher on antibiotic resistance compared with general knowledge Table . The corThe BMQ scores show that the study population had an overall positive view of the value of medications with a mean per subdomain score of 4.1 for benefit, 2.6 for harm and 3.2 for overuse (Table p = 0.001).Results from the multiple linear regression analysis Table  showed tKnowledge of antibiotics and antibiotic resistance differed according to some of the background characteristics. Men were more likely to have lower general knowledge of antibiotics than women, but there were no gender difference in knowledge of antibiotic resistance. Higher age and higher education was associated with higher knowledge of antibiotic resistance but not of antibiotics in general. For education, this might have been a question of statistical power, as the trend in the coefficients were similar for both scores. The knowledge scores according to age category Table  suggesteAccording to the univariate regression . Combined with imputation of missing values on BMQ and attitude score variables, this ensured that only 6.9% of the population were excluded in the multiple linear regression analysis. The facilitation included help for participants who had difficulties understanding some of the questions. This implies a potential for overestimation of knowledge if the students were too helpful, but also underestimation if help had not been given. The students were conscious not to influence the participants’ answering, so we assume low influence from the facilitation.The cross-sectional design prevents us from drawing conclusions regarding causal relationships. For instance, high knowledge of antibiotics may just as likely be a prerequisite for restrictive attitudes toward antibiotics as vice versa.The study population is not representative for the Norwegian general population ≥ 18 years. Our sample is somewhat older with 36% ≥60 years compared with 28% in the general population , women wThere are no official statistics that demographically describe Norwegian pharmacy customers, i.e. our source population. Compared with the population who redeemed at least one prescription  in 2016, our study sample includes more women (68% versus 55% in NorPD), but the proportion ≥ 60 years is comparable (36% versus 37% in NorPD). However, the pharmacy customers in our study also include people merely buying over-the-counter medication and various products marketed for health and well-being. This may account for our higher proportion of younger people, and particularly women.Our study sample is relatively large, but with a moderate response rate. The motivation to participate is probably higher among people with an interest in, or experience with, antibiotics, which may contribute to an overestimation of knowledge level. The proportion reporting antibiotic use during the previous 12 months is fairly high (30%) compared to the one-year prevalence from NorPD for 2016 (23%) , and comThe knowledge and attitude statements in our questionnaire were not validated, but were mainly based on questionnaires used in previous studies , 26, andLack of scientific studies measuring the public’s knowledge about antibiotics and antibiotic resistance is worrying given the global call to fight antibiotic resistance. This study contributes to fill this gap. Moreover, it adds to standard surveys, like the Eurobarometer, by including measures of beliefs about medicines, attitudes toward antibiotics as well as background characteristics, thereby addressing risk factors for low level of knowledge. The study reveals knowledge gaps that should be filled and groups that should be targeted for efforts to increase the antibiotic knowledge. However, a large-scale nation-wide study would be desirable.We have identified a high level of knowledge of antibiotics, particularly antibiotic resistance, among Norwegian pharmacy customers. This suggests that Norwegians are aware that antibiotic resistance poses a health threat, and that unnecessary use of antibiotics is driving this threat. Nevertheless, there seems to be a knowledge gap when it comes to understanding the rationale behind the resistance problem. We suggest that action is taken to increase public knowledge of antibiotics, particularly among people without a health professional background, in less academic  and/or male dominated occupations. We also suggest that fathers, as well as mothers, are targeted in campaigns directed towards families with young children. Additionally, the upcoming revision of the national curriculum for primary and secondary school should include learning objectives regarding antibiotics and antibiotic resistance.Additional file 1:Results from univariate linear regression analyses (PDF 506 kb)


The effect of vehicle loads on reinforced concrete plate-girders was evaluated using the current Chinese specifications. Repeated loading performance tests with loading amplitudes of 77 kN, 97 kN, and 121 kN, which correspond to the standard vehicle load, 1.25 times overload, and 1.6 times overload proportions effect were carried out on three full-scale simply-supported reinforced concrete plate-girders. Our research results indicate that the development of cracks in reinforced concrete beams can be divided into three stages: rapid development, stability, and failure. During the entire process, the strain of steel and concrete did not reach their yield strain. The most severe damage done to the concrete beams was the brittle fractures caused by the fatigue fracturing of the rebar. When in a stable condition, the extent to which the vehicle was overloaded had a significant effect on the fatigue performance of the beam, and the corresponding residual deflection and residual strain increased with the rise in the overload proportion. In addition, as the overload proportion increased, the stiffness degradation and the cumulative damage that occurred under the same loading cycle was more significant. The test beam reached failure after being subjected to 350,000 and 670,000 repeated loading cycles, when the load was 1.6 times and 1.25 times of the standard load effect. With a standard vehicle load effect, the test beam was able to endure 2,000,000 repeated load cycles with no significant degradation in stiffness and bearing capacity. Concrete plate-girders with small or medium spans have been widely used in the construction of highway bridges due to their simple structure, low price, and the convenience of their fabrication. According to on-site inspections and the testing data obtained from existing bridges, damage gradually accumulates within concrete plate-girders, with strength degradation occurring due to the erosion caused by the external environment and the repeated effects of vehicle load. Moreover, the damage done to bridges which have endured overload for a long period of time tends to accumulate far more rapidly than the damage done to a bridge which has not been overloaded. Previous data has also verified that when the damage that has accumulated in the beam reaches a certain degree, sudden brittle failure of the bridge may occur, compromising the safety of the bridge and the surrounding traffic networks .In order to analyze the damage caused by fatigue, a series of theoretical analyses and fatigue tests were carried out on ordinary reinforced concrete, high strength reinforced concrete, pre-stressed reinforced concrete, corroded reinforced concrete, and different forms of reinforced concrete subjected to a range of environmental impact factors. The fatigue development law of the reinforced concrete members , the infThe purpose of this study is to analyze the mechanical behavior of small and medium span concrete bridges under a series of cyclic vehicle loads in different overload proportions. Three full-scale prototype concrete plate-girders were designed. A series of cyclic loading tests were conducted with loading amplitudes of 77 kN, 97 kN, and 121 kN, which correspond to the standard vehicle load, 1.25 times overload, and 1.6 times overload proportion effect. Through the tests, we analyze the failure law, deflection and strain variation law, cumulative damage evolution law, and the stiffness degradation law of the concrete plate-girders in different overload proportions, and the relationship between fatigue life of reinforced concrete plate-girders to overload proportions is fitted.Three full-scale prototype concrete plate-girders with a length of 6 m were designed according to the general principles presented in Highway Bridge and Culvert Standard Drawings (1973–1993), named as Beams-1–3. The dimensions of the test specimens are shown in γ was determined asI and It were the bending moment of inertia and torsion moment of inertia of the beams; b and l were the width and height of the rectangular beam section.Transverse joints were used for the beam connections, and the stiffness parameter The influence curve of the transverse distribution coefficient of the 1#–4# beam specimens is shown in According to provisions stated in the General Specifications for the Design of Highway Bridges and Culverts (JTG D60-2015), the transverse and longitudinal arrangements of highway grade I vehicle load is shown in In order to achieve a pure curved section in the middle portion of the beam specimens, and to simulate the actual axle action of a vehicle equivalent to a highway grade I vehicle load, the forces were loaded using distributive beams with a spacing of 1.4 m. Ordinary plate rubber bearings were set at both ends of the test specimens, as shown in A pre-loading not exceeding 70% of the normal service load was applied to the specimens to ensure that the instruments were functioning correctly. Monotonic static loading and unloading behaviors were conducted before the cyclic loading tests, and the process of both behaviors were divided into four stages. The cyclic loading tests were conducted at a frequency of 4 Hz. The residual deflection, the distribution, and the width of the cracks appearing on the beam specimens were measured after every 100,000 load cycles. Then a monotonic static loading of four stages was repeated to measure the strain on both the concrete and rebar, the crack width, and the deflection of the beam specimens at each stage. The distribution of cracks and the failure characteristics of the specimens were also recorded.In order to measure deflection at different positions along the beam span, displacement meters were installed at the middle span, the loading point, at the one-sixth span position, and at both ends of the beams. Additionally, concrete strain gauges were arranged along the perimeter of the beam sections, at both the middle span and the loading point of the test beam, in order to measure the concrete strain. Strain gauges were also arranged on seven of the lengths of longitudinal rebar in order to measure the strain experienced by the rebar in different positions, as shown in Under cyclic loading, brittle fractures occurred along main cracks near Beam-2 and Beam-3 but there was no obvious indication that failure was about to occur. During the failure process of Beam-2 and Beam-3, the amount of cracks and their width and height along the length of the beams experienced three stages: the early rapid development stage, the middle stability stage, and the final failure stage. Initially, when the loading of Beam-2 had reached the 100,000th cycle, all of the cracks in the specimen were half the height of the beam height. The cracks were distributed symmetrically along the span direction and on both sides of the cross section. Both the number of cracks and their width remained constant when the number of loading cycles increased from 100,000 times to 600,000 times, with the beam continuing to remain in the crack stability stage. Finally, a dominant crack in the beam span suddenly extended and widened when the 650,147th cycle was achieved, resulting in a fracture in the beam specimen. At this point, the beam underwent a sudden, major deformation and lost its bearing capability due to the ruptures of the five rebar steels in the bottom of the beam. The concrete in the compression zone was raised, accompanied by shedding. Similar behaviors occurred when Beam-3 reached the 10,000th load cycle but in this case the height of the cracks that developed along the side faces were larger. Beam-3 reached the crack stability stage between loadings of 100,000 times to 300,000 times. However, when the load cycles reached 377,240 an abrupt fracture occurred due to the rupture of the rebar in the bottom of the specimen. This rupture occurred without obvious precursors. Easily identifiable fatigue failure characteristics can usually be found on the fracture surface of the rebar, but in this case no necking phenomenon was observed. The limit failure modes of Beam-2 and Beam-3 are shown in No fractures occurred in Beam-1 after 2,000,000 loading cycles with a maximum loading force of 77 kN. At the base of the beam, the penetrating cracks were distributed uniformly along the length of the beam. The cracks also extended up to half of the height of the web, with a spacing of 20 cm. The cracks tended to close up following the unloading stage, with a maximum crack width of 0.1 mm. When adjacent to the middle part of the beam, residual crack widths were generally less than 0.08 mm. Throughout the cyclic loading process, the crack development of Beam-1 could be divided into the rapid development stage, the slow development stage, and the stable stage. The final condition of Beam-1 and its crack distribution is shown in P of 268 kN, which was defined as the residual bearing capacity of the test beam. This demonstrates that the difference between the theoretically calculated value of the ultimate bearing capacity of the beam and the residual bearing capacity was very small. Additionally, according to the test phenomena, a linear relationship exists between the force and the deflection of the mid span before the force exceeded 260 kN, indicating that the beam was basically in an elastic stage. A displacement-control loading scheme was used when the force was loaded to 260 kN, and the bearing capacity of the beam was able to remain stable for a long time during the increase in deflection that occurred when the ultimate load was reached. The ductile failure of the beam, with an ultimate deflection in the mid-span of 58 mm, was caused by the tensile yield of the bottom rebar. The residual bearing capacity of the beam was more than 90% of the theoretical ultimate bearing capacity, indicating that the ultimate bearing capacity of the reinforced concrete beams was largely provided by the tensile strength of the rebar. Under 2,000,000 standard load cycles, there was no obvious decrease in the bearing capacity of reinforced concrete beams, indicating that the residual bearing capacity of Beam-1 meets bearing capacity design requirements. However, the service life of Beam-2 and Beam-3 decreased rapidly when subjected to the overloaded cycles.A destructive test was conducted on Beam-1 after it had been subjected to 2,000,000 cyclic loadings. Finally, an abrupt fracture occurred in the Beam-1 along the cracks in the middle span with a bearing force The deflection curves of the different measuring points along the span of Beam-1, Beam-2, and Beam-3 under different cyclic loads of varying amplitudes are shown in Beam-3 displayed similar variation in vertical deflection to that of Beam-2. The mid-span deflection under peak loading was 13.6% higher than the deflection under the initial static load when the loading cycle had been repeated 10,000 times. The vertical deflection of the mid-span increased suddenly and obviously after 50,000 cyclic loadings, and the maximum deflection of the mid-span increased by about 23.8% compared with the initial value. The mid-span deflection was 19.75 mm, which is 30.8% higher than the initial static load after 300,000 loading cycles, and the deflection–span ratio of the beam was about 1/300. Finally, a brittle fracture failure occurred in the span of Beam-3 after to 370,000 loading cycles. During the entire cyclic loading process, the deflection of Beam-2 and Beam-3 increased nonlinearly with the increase in loading cycles, indicating that the damage to the beam accumulated gradually under repeated vehicle loads, while the stiffness of the members lessened.The vertical deflection of the different beams’ mid-span under a range of loading cycles is shown in Bending stiffness is one of the most important indicators used characterize the performance of reinforced concrete beams. The deflection analysis shows that the stiffness of the test beams decreased gradually with the increase of load cycles. In the test, the bending stiffness of the beam can be reflected by the load–deflection ratio of the mid-span section under different loading cycles, as shown in Our results indicate that the bending stiffness of the beam did not experience a gradual, stable degradation process under the cyclic vehicle loads; its change trend was related to the values of vehicle loads. Under the repeated action of a standard load, the stiffness of the beams did not degenerate significantly, and the members remained in an elastic stage. However, under 1.6 times the standard load, the bending stiffness of the beam decreased significantly with an increase of loading cycles, indicating that the beam was irreversibly damaged. Therefore, the ratio of overloaded vehicles and the number of passages should be strictly controlled in order to improve the mechanical performance of beams and extend the service life of the overall structure. The development of the internal damage done to the beams directly corresponds with residual deflection. The residual deflection curves along the key sections of Beam-1, Beam-2, and Beam-3 after different loading cycles are shown in The comparison of mid-span maximum residual deflection for the different test beams under a range of loading cycles is shown in According to our analysis of deflection and residual deflection, the absolute vertical deflection of each section along the span of Beam-1 changed slightly with the increase in cyclic loading times, and the corresponding residual deflection also experienced a slow increase. The deflection of Beam-1 was basically stable after 1,000,000 cycles, and the stiffness of the beam ceased to degenerate. For Beam-2 and Beam-3 the vertical absolute deflection of different sections increased with the increase in cyclic loading times, and the residual deflection also experienced a significant increase. The residual deformation of Beam-3 was notably larger than that of Beam-2 after experiencing the same number of loading cycles. This indicates that the plastic cumulative damage done to the beams increases gradually with an increase in repeated vehicle loads.During the cyclic loading process, the average strain on the rebar and concrete in a section of the beam was taken to be representative of the corresponding strain in that section. The variation trend displayed by the average longitudinal reinforcement tensile strain and the average concrete compressive strain along the different sections of the beam span are shown in According to our observations, the sudden increase in the strain on the bottom tensile rebar corresponds with the generation and rapid development of the cracks in the concrete beam. The stress is gradually transferred to the rebar as the concrete slowly develops cracks. The rebar was forced to bear the cyclic loads when the cracks developed completely. At this stage, the strain on the rebar entered the stable development stage under static load with a stable peak load. The strain on the rebar underwent a smaller increase under the same load because in this case the damage to the rebar had not yet developed. Until the fatigue fracture of the rebar occurred, the maximum strain in the rebar did not reach yield strain.Our results indicate that during the cyclic loading, when the simulated vehicle loads were 1.6 times larger than the standard load, the rebar was constantly in a high-stress state. This resulted in a rapid increase in fatigue and cumulative damage to the plasticity of the rebar. The ultimate failure of the beam was caused by the rebar’s development of a fatigue crack, not due to the yield failure of the rebar itself.During the initial loading stage, when the number of cycles was below 50,000, the concrete’s strain grows rapidly. As the cracks develop it gradually enters the stable stage. In the second stage, when the number of cycles was between 50,000 and 90% of the total number of loading cycles, the compressive strain growth of the concrete was small and the cracks also entered the stable development stage. In the third stage, when the number of cycles was more than 90% of the total number of loading cycles, the compressive strain of the concrete entered an ascending stage, ultimately leading to the failure of the specimens. The strain of the concrete at the compressive edge of the top of the beam did not reach yield strain during the entire loading process. The mechanical behavior of the concrete beams under cyclic loading is largely determined by the presence of tensile steel bars. According to our strain analysis of the rebar and concrete, the compressive strain of concrete and the tensile strain of the rebar fluctuated slightly within a certain range but generally the strain curves developed smoothly. The maximum tensile strain on the rebar and the maximum compressive strain on the concrete did not reach their ultimate strain values. Even though fractures occurred during the testing of Beam-2 and Beam-3, the rebar still did not reach yielding strain. Our results indicate that the failure of the beams was caused by the fatigue fracture of the tensile rebar, which is in accordance with the fatigue failure characteristics of reinforced concrete beams. In order to obtain the law of material strain distribution in the critical stress section, the top concrete compressive strain, web concrete compressive strain, tensile strain, and bottom longitudinal reinforcement tensile strain in the same section were measured, and their distribution along the section height of the beams is shown in It can be observed that the strain distribution of the concrete across different sections of the test beams possessed linear characteristics, which conforms to the plane section assumption. After 10,000 loading cycles the position of the neutral axis increased compared with the initial static load, and the position of the neutral axis was seen to increase slowly as the number of cycles rose. Because Beam-1 was in an elastic state during the loading process, the rebar’s strain under different cyclic cycles remained stable. However, due to a gradual accumulation of residual strain in the rebar during the testing process, the performance of Beam-2 and Beam-3 were gradually degraded, and the strain on the rebar increased gradually during different loading cycles. Our results indicate that the fatigue damage experienced by the steel bars was small and the functional performance of the beams remained stable under the repeated action of standard vehicle loads. However, when subjected to the repeated action of overloaded vehicles the damage to the rebar accumulated quickly and their performances degenerated significantly, leading to the early fatigue failure of the beams. S) can be calculated as,N is the number of cycles. Therefore, the relationship between the vehicle loads and the number of cycles can also be deduced from the results of this study. For example, when the vehicle load was 1.25–1.6 times the standard load, the number of cyclic loads experienced by the concrete beams was between 200,000–750,000.Based on our analysis of the fatigue test results for reinforced concrete beams, data with similar conditions were selected. The fatigue life data of different specimens under different stress levels as stated in reference ,18,19,20This paper investigated the mechanical performance of reinforced concrete plate-girders when subject to long-term cyclic vehicle loading. Our results indicate that the failure process of reinforced concrete beams under cyclic loading can be divided into three stages: the rapid development stage, the stable stage, and the failure stage. The mechanical behavior of reinforced concrete plate-girders under cyclic loading proved to be largely determined by the rebar. Although many cracks appeared in the concrete Beam-1 that was subjected to the standard loads, the overall bearing capacity and stiffness of the beams under cyclic loading showed almost no obvious degradation due to the small amount of accumulated damage in the rebar. With an increase in the overload ratio, the mid-span deflection and residual deflection increased gradually, and the bending stiffness of the beam decreased significantly, while the cumulative damage increased gradually. During the entire cyclic loading process, the tensile strain of the rebar and the compressive strain of the beams’ concrete was less than their ultimate strain, and the ultimate failure of the beam was caused by the sudden failure generated by the fatigue fracture of the tensile reinforcing bar. The overload proportion had a strong influence on the failure state and performance degradation law of the beams. The beams were not damaged after 2,000,000 cycles of a standard vehicle load, whereas fatigue fracture occurred when the vehicles were overloaded. The cyclic loadings with overload ratios of 25% and 60% had cyclic times of 670,000 and 350,000, respectively. Therefore, the overload ratio of vehicle loads should be strictly controlled in order to avoid or reduce the possibility of highway bridges experiencing structural brittle failure.